Biochemical & phytochemical insights of geographically diverse Acacia nilotica through HPLC–MS-based untargeted metabolomic studies

Biochemical & phytochemical insights of geographically diverse Acacia nilotica through HPLC–MS-based untargeted metabolomic studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biochemical & phytochemical insights of geographically diverse Acacia nilotica through HPLC–MS-based untargeted metabolomic studies Prabhjit Kaur, Satwinderjeet Kaur, Rajbir Singh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8084390/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acacia nilotica is tropical tree with great nutritional values, being consumed for their promising health benefits and their aerial parts is used as fodder for animals. The current report is undertaken to analyze phytochemistry and in vitro biological activities of leaf extracts of Acacia nilotica (ANL samples) of diverse geographical locations i.e. Bikaner, Rajasthan (RJ), Amritsar, Punjab (PB) & Palampur, Himachal Pradesh (HP). The methanolic extract were prepared from composite samples of different locations and further checked for their qualitative biochemical, photosynthetic investigation and HPLC-MS based untargeted metabolomics. The ANL extracts were also checked for their biological potentials. It was observed that, though the quality of phytochemicals amongst various leaf extracts remained same but quantitatively, these extracts showed differences as observed through biochemical and metabolomics studies. The significant differences were observed in the various biological activities of the leaf extracts. The results showed a positive correlation establishment between climatic variation and phenolic enrichment of Acacia nilotica plant which further affects the biological activities. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Chemical biology Biological sciences/Plant sciences Acacia nilotica bioactivities phenolic fingerprint geographical variations Metabolomics chemometrics studies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction A new generation of health and disease management is ensured by the medical revolutions brought about by the intensive study in free radical biology. They have both positive and negative effects and are the main byproducts of numerous cellular redox activities. They are helpful for immune response and cellular responses at low to moderate levels, but at high concentrations, they cause oxidative stress, which damages cell structures and functions [ 1 , 2 ]. With their diverse range of chemical components, plants are a prospective source of molecules with therapeutic qualities. These plants continue to promote human health in addition to serving as the foundation of traditional medicine. Natural foods and components derived from plants have received a lot of attention lately because they are thought to be safer and healthier than their synthetic counterparts [ 3 ]. Geographical variances and agroclimatic conditions can significantly impact the chemical makeup and therapeutic qualities of medicinal plants, as evidenced by Ayurvedic research. According to earlier research, the differences in the chemical composition and antioxidant capacities of plants from various geographic regions can be attributed to a variety of environmental conditions, including temperature and soil type. Significant phytochemical changes in maple leaf trees were caused by habitat, climate, harvest timing, and processing [ 4 ]. Numerous factors, including variety, age, plant maturity, collection season, geo-agro-climatic conditions, agronomical procedures, postharvest management, storage, processing, etc., affect the medicinal plants' highly variable phytochemical composition. As a result, the active principle can vary greatly, which would impact the bio-potency. For the plants to generate consistently high-quality products, chemical profiling is crucial. In order to choose and formulate plant-based dietary supplements and develop food-based therapeutic compounds, it is essential to confirm the bioconstituents and biological activities of plants across a variety of agro-ecologies. The goal of the current study was to compare the biological activities of A. nilotica leaf populations collected from three northern Indian states: Rajasthan (Bikaner), Punjab (Amritsar), and Himachal Pradesh (Palampur). India is a country with a variety of soil types, water levels, temperatures, and other environmental factors throughout the year. The current study has two scientific goals. Establishing a chemical variation among the A. nilotica population at various collecting sites was the first phase's goal. The second was to examine the significant biological activities based on the phenolic variance across plants from various geographic locations. Thus, this study provides more proof in favor of the widely accepted link between biological activity and phytochemical differences dependent on geoclimate. The bio-efficacy of the plant variations is significantly influenced by the active principle or specific polyphenols that may be dys-regulated due to geographical changes. The concept that there is a correlation between the phytochemistry of plants and the climatic variance of various geographical regions, which further influences the biological activities of medicinal plants, was further supported by this study. 2. Materials and Methods 2.1. Chemicals Sigma Chemical Co. (St. Louis, MO, USA) provided the 2-thiobarbituric acid and 2'-2' diphenyl-1-picrylhydrazyl (DPPH). Lancaster Synthesis Inc. (Windham, USA) provided the 2-deoxyribose, and Abcam, USA, provided the Caspase-3 kit. The following items were acquired from Sigma-Aldrich USA: gentamycin, penicillin, fetal calf serum (FCS), acetic acid, trichloroacetic acid (TCA), mitomycin-C, trypsin, RPMI-1640 medium, gentamycin, 5-fluorouracil (5-FU), and 2-thiobarbituric acid. The National Cancer Research Institute in the United States provided the human cancer cell lines. Tris EDTA from Hi Media, phosphate buffer saline (PBS) from Merck (Germany), and sulforhodamine B from Fluka. H 2 O that had been double distilled was used to make all stock solutions. Sigma Aldrich's HPLC-grade standards were utilized for HPLC screening. Cayman Chemicals (Michigan, USA) supplied the anti-inflammatory bioassay kit. All other chemicals were of analytical grade and procured from Ranbaxy Fine Chemicals Ltd. (New Delhi, India). 2.2 Plant Material In March 2020, nine randomized samples of Acacia nilotica leaves (ANL) were gathered from Bikaner, Rajasthan; Amritsar, Punjab; and Palampur, Himachal Pradesh, India, three from each site as per the institutional policy of the Khalsa College Amritsar. A single composite sample of each tree was created by first selecting three to five Acacia trees of comparable age and good growth state from each sampling locality, then taking an equal number of leaves from each of the four sides (NEWS) at the same height. In Khalsa College Amritsar, Punjab, India, the samples were recognized & identified by subject experts of P.G Department of Botany. The vouchers of the same were placed in the herbarium (A/C # 1051, dated 12-05-2020). Table 1 displays the precise geographic position, collection time, and area (km³) of the collection place and and each sample was given an accession code “RJB”, “PBB, & “HPB”. All biochemical experiments were performed in accordance with the approval of Chemical & Bioethical Committee (CBE) of Khalsa College Amritsar, Punjab, India. Table 1 The collection point with time and geographical coordinates of Northern Indian states. State District Geographical coordinates Time of Collection Area (sq kms.) Plant part Abbreviated name Punjab Amritsar 31.63° N, 74.87° E August 2023 10.36 Bark PBB Leaf PBL Flower PBF Himachal Pradesh Palampur 32.11° N, 76.53° E August 2023 5.78 Bark HPB Leaf HPL Flower HPF Jammu & Kashmir Kathua 32.37°N 75.52°E August 2023 10.93 Bark JKB Leaf JKL Flower JKF 2.3 Agro-climatic & Meteorological data The Ministry of Earth Sciences' Climate Data Service Portal Meteorological Department provided the comprehensive meteorological data, which included both geographic and climatic information ( https://cdsp.imdpune.gov.in/ ). Longitude, latitude, and altitude were the averaged geographic data (for the two weeks before sample collection) and temperature, humidity, sunshine duration, and rainfall were the averaged climatic data used in this study. The information reflected the geographical traits thought to be the most important determinants of the accumulation of metabolites and the biological activity of Acacia nilotica leaves. 2.4 Sample Preparation and Extraction All composite leaf samples of Acacia nilotica were cleaned and washed with tap water. They were then allowed to air dry and ground into a fine powder before being sieved through a 40-mesh screen in order to extract organic solvents. First, 450 milliliters of methanol were used to macerate 100 grams of fine powdered leaf material for 72 hrs. while being shaken intermittently. The supernatant was filtered after a day, and the powder that remained was macerated twice using new methanol solvent. For additional phenolic profiling and biochemical analysis, all three supernatants were combined, dried in a rotary evaporator (BUCHI R-300, SWITZERLAND), moved into vials, and stored at -20°C (Fig. 1). 2.5 Biochemical analysis Using the methods of Kujala et al. and Park et al., the total phenolic content (TPC), total flavonoid content (TFC), and total anthocyanidin content (TAC) of all methanolic ANL extracts were measured as gallic acid, quercetin equivalent, and cyanidin equivalent in mg/g DW, respectively [ 5 , 6 ]. 2.6 HPLC-MS based phenolic fingerprinting & Amino acid analysis of ANL extracts A Shimadzu Prominence HPLC system equipped with Luna C18 (2) column (250 mm × 4.6 mm, 5µm particle size) and Shimadzu LC solution (ver. 1.21 SP1) software was used for HPLC analysis. Using solvent A (water) and solvent B (0.02% trifluroacetic acid (TFA) in acetonitrile) at a column temperature of 27°C and a flow rate of 0.75 ml/min, a linear gradient elution was performed at λ 280 nm: 70% A (5 min), 15–35% (7 min), 35–45% (11 min), 45–35% (16 min), and 35–15% (20 min). Z-Spray ESI ion (positive & negative) modes were used to acquire mass data while adhering to the optimal settings: : capillary voltage 2.5 kV; sample cone voltage 30 V; desolvation temperature 400 ◦C; source temperature 110 ◦C; desolvation gas flow 800 L/h; cone gas flow 50 L/h; scanning time 2 s; and scan mass range 100–1600 Da. Solvent blanks (70:30 v/v MS-grade water:acetonitrile), 27 ANL samples (triplicates of nine samples), and nine QCs were added to the sequence for LC-MS data capture. In order to check and stabilize the machine, three blank injections were made first. Three QCs were then injected after every nine ANL samples. At the conclusion of the sample sequence, three blank injections were carried out. For calibration, leucine-enkephalin (1 µg/mL) was employed as a lock-mass solution at a flow rate of 20 µL/min, yielding reference ions of m/z 556.2771 [M + H] + and 554.2615 [M–H] − . For the purpose of creating calibration curves, the analyte-containing stock solution was made as 1 mg/1 ml in HPLC-grade methanol: water (90:10) and then further diluted to the proper concentrations of theses analyte were injected thrice for the quantitative analysis and the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The selectivity of the method was determined by analyzing standards and methanol extracts. The peaks of reference compounds were identified by comparing their retention times & UV spectra with those of the standards. 2.7 Metabolite profiling and chemometric analysis: The Shimadzu LC solution software (version 1.21 SP1) was used to assess the alignment, normalization, peak picking, and deconvolution of the spectral data of 27 ANL samples. For its chemometric analysis, the deconvoluted data without any filtering were exported to MetaboAnalyst 6.0 ( https://www.metaboanalyst.ca ). In order to distinguish the comparison groups in PCA & OPLS-DA, the marker chemicals were further screened using an S-plot. In an S-plot, the ions that made the most contributions were displayed at the top right (1) and bottom left (-1). Relative quantitative differences between substances based on peak intensity were clarified using heatmap. Smart Metabolites Database™ Ver.2 was used to further identify the probable chemical ions that were filtered by ANOVA (p value 1.5). 2.8 Biological activities: 2.8.1 In vitro antioxidant testing assays Because plant extracts are complex, a battery of in-vitro antioxidant testing systems was used to assess the detailed antioxidant capability of several ANL extracts [ 7 ]. The hydrogen-donating capacity of ANL extracts was assessed using the stable DPPH radical, and the hydroxyl radical scavenging potential was assessed using the site- and non-site-specific deoxyribose degradation assay [ 8 – 10 ]. The modified Dinis et al. approach was used to determine the chelating effect on ferrous ions [ 11 ]. Oyaizu's approach was used to determine the reducing power of ANL extracts [ 12 ]. In the lipid peroxidation experiment, the TBA combines with malondialdehyde (MDA) to generate a diadduct, a pink chromogen that is detected at 532 nm [ 13 ]. 2.8.2 In vitro Cytotoxicity Assay The cytotoxicity potential of several methanolic extracts of Acacia nilotica was assessed using the Sulforhodamine B dye test for the cell lines BEAS-2B (non-cancerous), A-549 (lung), DU-145 & PC-3 (prostate), IGROV-1 (ovary), and MCF-7 (breast) [ 14 ]. All the cell lines were procured from Genetic Toxicology Laboratory, Department of Botanical & Environmental Sciences, Guru Nank Dev University, Amritsar, Punjab, India. 2.8.3 Antimicrobial studies 2.8.3.1 Culturing of microorganisms strains: ATCC-25923 & ATCC-19615 were grown on prepared blood agar (BA) while ATCC-27853 cultured on cooked blood agar (CBA) and ATCC-10231 cultured on Sabouraud dextrose 4% agar (SDA) to check the antimicrobial potential of ANL extracts. 2.8.3.2 Antimicrobial activities through Agar disk diffusion assay The 7 mm sterile and dried paper disks were soaked in 50 µl of different concentrations of ANL extract and allowed to dry for 24 hours at 37°C. On agar inoculated with microbial culture and strains, 2 mm round and dried paper disks were carefully inserted. The disks were then incubated at 37°C for 24 hours for P. aeruginosa, S. aureus , and S. pyogenes, and 48 hours for Candida albicans . The measurement of the inhibitory zones was in millimeters. 50 µl of extracting solvent served as the negative control, while the broad spectrum antibiotics ampicillin (for S. pyogenes and S. aureus ), ciprofloxacin (for P. aeruginosa ), and fluconazole (for C. albicans ) served as the positive controls [ 15 ]. 2.8.3.3 Minimum inhibitory concentration (MIC) The micro-titer plate method was followed to estimate the MIC of ANL extracts [ 16 ]. 2.8.4 Caspase-3 assay: Using the Abcam, USA, caspase-3 colorimetric test kit methodology, caspase-3 activity was measured. This test quantifies the quantity of free p-nitroanilide (pNA) in the cell that results from active caspase-3 cleaving the DEVD-pNA link during apoptosis. By comparing the absorbance of p-NA from an apoptotic sample with that of untreated control cells, the fold increase in caspase-3 activity was ascertained. 2.8.5 Anti-inflammatory Activity ANL extracts' in vitro COX-2 inhibitory properties have been assessed using 96-well plates and a "COX (ovine) inhibitor screening assay" kit. Included were the COX-1 and COX-2 enzymes from cows [ 17 ]. PGF2α generated by SnCl2 reduction of COX-derived PGH2 is directly measured by this screening assay. The 96-well plate's wells with low absorption at 405 nm are indicative of low prostaglandin levels and, thus, lower enzyme activity. Consequently, the absorption values of the various wells in the 96-well plate may be used to quantify the COX inhibitory activities of ANL extracts. 2.9. Statistical analysis All analyses were performed in triplicate (n = 3) and the data was presented as mean ± SD. The results obtained were analyzed using two-way ANOVA to determine the significant differences between the experimental batches by taking the raw samples as control. 3. Results and Discussion The production of phytochemicals is significantly regulated by environmental and genotypic variables, which in turn have an impact on biological activity. According to earlier research, environmental factors were mostly responsible for the phytochemical diversification across various geographic regions, with genotype having a minor influence on phytochemicals [ 18 ]. 3.1 Metrological Data (soil and agro-climatic analysis): Table 1 explains annotations of leaf extracts of Acacia nilotica collected from northern states of India i.e. RJB (Rajasthan) PBB (Punjab) and HPB (Himachal Pradesh). Amritsar is located at 31.63°N 74.87°E with an average elevation of 234 metres (768 ft) with 2,683 square km area. The climate in Amritsar is warm and temperate. The Bikaner is located at 28.02° N, 73.31° E at an elevation of 225.73 meters (740.58 feet) above sea level. The city has a Subtropical desert climate. Palampur is a famous hill station at 32.11° N, 76.53° E. It is located at height of around 1300 metres, above sea level with 609 area in square km. The city has a monsoonal-influenced humid subtropical climate with moderate summers and cool winters. The composite leaves sample (explained in material method section) from all the collection point were collected in the March 2020. The soil sample of Bikaner has sandy to clay loam texture with alkaline content (pH 8.3) and 0.29 EC (dSm − 1 ) while Amritsar soil has coarse loamy texture with pH 6.9 (almost neutral) and 0.95 EC (dSm − 1 ). The sample of Palampur showed pH 6.7 and loam to clay textured soil. The results showed that Amritsar soil has more NPK value (219, 816, 99.7) as compared to Rajasthan (188, 756, 83.6) and Palampur (192, 782, 86.4) as shown in Table 1 (Supplementary). Previous studies revealed that environmental variables tremendously affected the phytochemistry of the plant and a significant difference in important environmental variable viz. annual temperature, rainfall, sunlight & average relative humidity (ARH) was observed. The Palampur collection site showed highest annual rainfall of 1288.3 mm and ARH of 54% and Rajasthan site exhibited maximum annual temperature of 30.2°C and annual sunlight of 3842.77 hrs. In case of Amritsar, it was observed moderate annual temperature (23.4°C), rainfall (572.2 mm), sunlight (3653.84 hrs.) and ARH (53%) (Spl. Table 2 ). Table 2 Extract yield and biochemical analysis of different ANL extracts. Plant Location Plant Part Extract Yield (%) TPC TFC TAC District: Amritsar State: Punjab Bark 24.59 395.12 308.27 288.41 Leaf Flower District: Palampur State: Himachal Pradesh Bark 28.14 465.09 372.62 366.16 Leaf Flower District: Kathua State: Jammu & Kashmir Bark 29.18 409.62 321.29 365.26 Leaf Flower TPC : Total Phenolic content as mg GAE/g extract; TFC : Total Flavonoid content as mg QE/g extract; TAC : Total anthocyanidin as mg cyanidin/g extract. 3.2 Biochemical & phytochemical characterization of ANL extracts: The HPLC-MS based phenolic fingerprinting revealed that phytochemicals in all ANL extracts qualitatively remained same but varied quantitatively as indicated by their phenol and flavonoid contents, which were observed proportional to their biological activities. It was observed in the results that nature of the phytochemicals in all ANL samples did not differ but their quantity varied according to their geographical conditions. The results of the current study are further supported by earlier investigations, which showed that the chemical contents of Coleus forskohlii roots varied depending on the geographical location of India [ 19 ]. Cannabis sativa L.'s cannabinoid concentration was also impacted by the soil and agroclimatic conditions [ 20 ]. The research revealed that the geographical characteristics played a crucial part in determining the phytochemical makeup of the samples, which in turn explained the observed variation in their antioxidant capacities. The major phytochemical screening tests were carried out for the ANL extracts which revealed the presence of alkaloids, reducing sugars, quinones, saponins, coumarins, triterpenoids, resins, flavonoids and tannins with varying concentration of low to high range (Spl. Table 3 ). This phytochemical variation was confirmatory with low to high TPC (395.12, 409.62 & 465.09 mg as GA /g DW) and TFC (308.27, 321.29, 372.62 mg as RE /g DW) & TAC (288.41, 365.26, 366.16 mg as cyanidin/g DW) for RJB, HPB & PJB respectively. The results also showed the %age yield of extract for RJB (24.59), PJB (28.14) & HPB (29.18) (Table 2 ). Table 3 The photosynthetic pigments, carbohydrate and total amino acids content of leaves of ANL samples. Plant Extract Chl. a (mg g − 1 FW) Chl. b (mg g − 1 FW) Carotenoids (mg g − 1 FW) Total carbohydrate (mg g − 1 ) Total amino acids (%) RJB 0.95 0.35 1.05 19.6 62.4 PBB 1.29 0.78 1.73 25.8 86.1 HPB 2.36 1.08 2.14 32.5 97.2 In the present study, it was also observed that the environmental variables significantly affected the photosynthetic pigments, total carbohydrate and amino acids content amongst the leaves of Acacia nilotica collected from different geographical regions. The HPB extracts exhibited highest Chl. a & b, Carotenoids (2.36, 1.08, 2.14 mg g − 1 FW), total carbohydrates (32.5 mg/g) and amino acids (97.2%) content as compared to PBB (1.29, 0.78, 1.73, 25.8, 86.1) and RJB (0.95, 0.35, 1.05, 19.6, 62.4) for Chl. a & b, Carotenoids, total carbohydrates & amino acids respectively (Table 3 ). It was found direct correlation between the photosynthetic pigments and annual rainfall & ARH and inverse correlation between annual the photosynthetic pigments and annual temperature & sunlight. 3.3 HPLC based Phenolic fingerprinting and metabolite identification: The phenolic fingerprinting of ANL extracts by HPLC-MS and analyzed by Shimadzu LC solution (ver. 1.21 SP1) software resulted in 2681, 2715 & 2937 chemical features detected after peak picking from the mass data obtained in both positive & negative ionization modes for RJB, PBB & HPB respectively (Spl. Table 4 ). The Venn diagram depicted the correlation between the chemical features and ANL samples (Fig. 2). Total 112 common feature were detected between all ANL samples and further filtered by ANOVA ( p value ≤ 0.05 and fold change ≥ 2) to eliminate false ions and finally 49 biologically important & putatively features were selected and identified through Smart Metabolites Database™ Ver.2. Table 4 HPLC-MS data of dysregulated phytochemicals of different ANL extracts. RT Mass m/z Mass Error (ppm) Molecular formula Tentatively Identified Compound Pub Chem. ID Fold Change Experimental Mass Theoretical Mass RJB/HPB PBB/HPB Positive Mode [M + H] + 2.15 199.0298 199.0303 2.51 C 9 H 10 O 5 Syringic acid 10742 ↑ ↓ 2.94 169.0191 169.0197 3.54 C 8 H 8 O 4 Vanillic acid 8468 ↑ ↑ 3.46 149.0295 149.0299 2.68 C 9 H 8 O 2 Cinnamic acid 444539 ↓ ↓ 5.24 443.3581 443.3585 0.90 C 30 H 50 O 2 Betulin 72326 ↑ ↑ 5.47 449.0785 449.0780 -1.11 C 21 H 20 O 11 Isoorientin 114776 ↓ ↓ 5.67 595.1364 595.1359 -0.84 C 27 H 30 O 15 Vicenin 442664 ↑ ↑ 6.11 301.0411 301.0408 -0.99 C 16 H 12 O 6 Diosmetin 5281612 ↓ ↓ 6.23 302.9835 302.9837 0.66 C 14 H 6 O 8 Ellagic acid 5281855 ↑ ↑ 7.61 170.9993 170.9998 2.92 C 7 H 6 O 5 Gallic acid 370 ↑ ↑ 8.26 127.0097 127.0091 -4.72 C 6 H 6 O 3 Pyrogallol 1057 ↑ ↓ 8.75 155.1129 155.1132 1.93 C 10 H 18 O Linalool 6549 ↓ ↓ 9.25 197.1234 197.1238 2.02 C 12 H 20 O 2 Bornyl acetate 6448 ↑ ↓ 11.09 319.0156 319.0151 -1.56 C 15 H 10 O 8 Myricetin 5281672 ↑ ↑ 11.52 457.3371 457.3378 1.53 C 30 H 48 O 3 Oleanolic acid 10494 ↑ ↑ 11.83 239.0407 239.0404 -1.25 C 15 H 10 O 3 7-Hydroxyflavone 5281894 ↓ ↓ 12.62 291.0558 291.0565 2.40 C 15 H 14 O 6 Catechin 9064 ↑ ↑ 15.26 165.0619 165.0612 -4.24 C 10 H 12 O 2 Eugenol 3314 ↓ ↓ 15.42 149.0304 149.0299 -3.35 C 9 H 8 O 2 t -cinnamic acid 444539 ↓ ↑ 16.29 287.0248 287.0252 1.39 C 15 H 10 O 6 Kaempferol 5280863 ↓ ↑ 16.62 291.0563 291.0565 0.68 C 15 H 14 O 6 Epicatechin 72276 ↑ ↑ 19.68 165.0242 165.0248 3.63 C 9 H 8 O 3 o -coumaric 637540 ↑ ↑ 20.15 195.0351 195.0354 1.53 C 10 H 10 O 4 Ferulic acid 445858 ↑ ↑ 21.24 341.0565 341.0569 1.17 C 15 H 16 O 9 Aesculin 5281417 ↓ ↓ 24.16 205.1648 205.1653 2.43 C 15 H 24 Humulene 5281520 ↓ ↓ 26.83 303.0205 303.0201 -1.32 C 15 H 10 O 7 Quercetin 5280343 ↓ ↑ 29.14 271.0297 271.0303 2.21 C 15 H 10 O 5 Apigenin 5280443 ↑ ↑ Negative Mode [M - H] − 3.16 135.1169 135.1173 2.96 C 10 H 16 Terpiniolene 11463 ↑ ↑ 4.16 177.0183 177.0187 2.25 C 9 H 6 O 4 Esculectin 5281416 ↑ ↓ 6.28 455.3519 455.3524 1.09 C 30 H 48 O 3 Ursolic acid 64945 ↑ ↑ 6.29 147.0814 147.0809 -3.39 C 10 H 12 O Estragole 8815 ↑ ↓ 6.72 103.0398 103.0394 -3.88 C 4 H 8 O 3 3-Hydroxybutyric acid 441 ↑ ↑ 7.54 167.0209 167.0204 -2.99 C 5 H 4 N 4 O 3 Uric acid 1175 ↓ ↓ 8.24 285.0401 285.0398 -1.05 C 15 H 10 O 6 Luteolin 5280445 ↑ ↑ 9.58 161.0232 161.0237 3.10 C 9 H 6 O 3 Umbelliferone 5281426 ↑ ↑ 9.62 219.1752 219.1748 -1.82 C 15 H 24 O Spathulenol 92231 ↑ ↑ 11.90 191.0187 191.0191 2.09 C 6 H 8 O 7 Citric acid 311 ↑ ↑ 13.22 179.0348 179.0343 -2.79 C 9 H 8 O 4 Caffeic acid 689043 ↑ ↑ 14.39 353.0867 353.0871 1.13 C 16 H 18 O 9 Chlorogenic acid 1794427 ↑ ↑ 15.29 153.0183 153.0187 2.61 C 7 H 6 O 4 Protocatehuic acid 0072 ↑ ↑ 16.37 221.1901 221.1904 1.35 C 15 H 26 O Guaiol 227829 ↑ ↑ 19.24 447.0933 447.0926 -1.56 C 21 H 20 O 11 Luteolin-7-O-glucoside 5280637 ↓ ↑ 21.05 223.0611 223.0605 -2.68 C 11 H 12 O 5 Sinapic acid 637775 ↑ ↑ 23.21 609.1450 609.1454 0.65 C 27 H 30 O 16 Rutin 5280805 ↑ ↑ 23.84 447.0919 447.0926 1.56 C 21 H 20 O 11 Orientin 5281675 ↑ ↑ 24.62 149.0959 149.0965 4.02 C 10 H 14 O Carvacrol 10364 ↓ ↓ 26.04 431.0973 431.0977 0.92 C 21 H 20 O 10 Apigenin-7-O-glucoside 12304093 ↑ ↑ 26.12 359.0762 359.0766 1.11 C 18 H 16 O 8 Rosmarinic acid 5281792 ↑ ↑ 27.82 271.0610 271.0605 -1.84 C 15 H 12 O 5 Naringenin 439246 ↑ ↑ 28.16 313.0717 313.0711 -1.91 C 17 H 14 O 6 Cirsimaritin 188323 ↑ ↓ 28.74 303.2327 303.2323 -1.31 C 20 H 32 O 2 Arachidonic acid 444899 ↑ ↑ 3.4 Metabolite dysregulation amongst ANL of various geographical regions: Using MetaboAnalyst 6.0 software, chemometric models (PCA, OPLS-DA, and S-plot) were used to evaluate the HPLC-MS data of RJB, PBB, and HPB in order to comprehend the metabolite dysregulation in ANL samples from various geographic locations. A visual TIC of 49 dysregulated phytochemicals in three ANL samples is evident from the random forest-based heatmap. It was shown that, in comparison to PBB, 16 compounds were up-regulated in RJB and 33 in HPB samples (Table 4 ). A distinct phenolic compound with a distinct mass to charge and retention time value is shown by each row (Fig. 3; Panel A). To get a comprehensive picture of the metabolite changes between ANL samples, PCA, an unsupervised technique, was applied to the chosen chemical characteristics without any filtering. ANL samples from Rajasthan, Punjab, and Himachal Pradesh were found to have divided into three distinct groups in PCA plots, with no overlap. This suggests that the metabolic profiles of the ANL samples from these three regions varied greatly from one another. Each of the three samples exhibited a loose clustering pattern, indicating that the chemical profiles of ANL samples from distinct zones differ significantly (PC1 = 94.3% versus PC2 = 4.5%, Fig. 3; Panel B). In this plot, the X-axis (dimension 1) denotes the interclass separation while the Y-axis (dimension 2) displays the intra-class variability According to unsupervised analysis by PCA in this study, the main driving force involved in the separation of extract samples seems to be the environmental variables of the different geographical zones. Figure 3 (Panel C&D) exhibited the DPSC correlation pattern and significant p-value (0.05) of dysregulated phytochemicals based on One Way ANOVA (Turkey’s HSD). The difference in the environmental variables, soil and agro-climatic factors led to greatly influence the resulting profile of phytochemicals present in various ANL samples. Our findings about the impact of four zones' climate factors—temperature, sunshine duration, rainfall, and altitude on the phytochemical content of Acer truncatum leaves are supported by earlier studies [ 21 ]. Altitude has been found to be closely associated with EGCG and ECG in Xinyang Maojian tea ( Camellia sinenesis ), a popular green tea brand in China and it was also noted that climate conditions could have a major impact on the catechin contents in Camellia sinenesis [ 22 ] The heatmap visualization & PCA plots revealed non-overlapping & open clustering between samples of RJB versus HPB and PBB versus HPB. It was also observed that intra-class separation between samples of HPB is much higher than the RJB and PBB samples (Fig. 4–5; Panel A-B). Figure 4–5 also showed the t-test analysis and volcano plots of p-value (0.05) and fold change (2) of the 49 dysregulated phytochemicals of ANL samplers (Panel C-D). 3.5 Analysis of HPLC based amino acid dysregulation: Apart from phytochemicals, 18 amino acids were also found present in ANL samples of different geographical regions of Northern Indian states through HPLC-MS analysis and same dysregulation pattern of putative amino acids was observed between ANL samples. Thirteen amino acids viz. Phenylalanine, Threonine, Tyrosine, Arginine, Aspartic acid, Histidine, Leucine, Lysine, Methionine, Proline, Isoleucine, Alanine, Valine were found up-regulated in HPB sample. The another five amino acids, Cystine & Tryptophan were up-regulated in PBB sample while Glutamic acid, Serine & Glycine were up-regulated in RJB sample (Fig. 6; Panel A). The unsupervised PCA plot revealed that all three samples clustered loosely, signifying ANL samples from different zones have significant difference in their chemical profiles (PC1 = 67.6% versus PC2 = 29.8%, Fig. 6; Panel B). Figure 6, Panel C-D showed SOM and significant p-value (0.05) of all 18 dysregulated amino acids between ANL samples. The relative content of amino acid panel in ANL samples of different geographical regions showed a clear difference of amino acid in different ANL samples (Fig. 7). 3.6 Studies on biological activities of ANL extracts: 3.6.1 Antioxidative potential analysis of ANL extracts: The significant difference in TPC, TFC, TAC & TIC of phytochemicals could be responsible for difference in biological activities of ANL samples of Northern Indian states i.e Rajasthan, Punjab & Haryana. Due to complex nature of phytochemicals the biochemical and antioxidant activity of plant extracts cannot be measured by a single approach. The detailed picture of the free radical scavenging potential of ANL samples were analyzed through battery of In-vitro antioxidant testing system. The well accepted DPPH assay is based on scavenging of DPPH radical by antioxidant/extracts through the degree of colour change, which is directly proportional to the radical scavenging potential of compound/extract. The large decrease in the absorbance of the reaction mixture indicates higher radical scavenging potential of the compound which is under test [ 23 ]. HPB shows strong DPPH scavenging potential of 83.87 ± 5.17% (IC 50 , 17.22​) as compared to PPB (77.64%), & RJB (71.40%) at highest tested dose of 250µg/ml concentration with no further inhibitory activity beyond this (Spl. Figure 1). One of the strongest reactive oxygen species in the body, hydroxyl radicals are known to be harmful in pathophysiological processes. They can harm important biological molecules and cause cancer and a host of other terrible illnesses [ 24 ]. The reaction between H 2 O 2 and the ferrous that would react with 2-deoxyribose produced hydroxyl radicals. This reaction was halted by adding TBA reagent, which would turn red if malonaldehyde was formed as a result of the radical's reaction with 2-deoxyribose [ 25 ]. The ANL extracts of diverse geographical locations showed moderate to high scavenging potential of 57.13%, 60.23%, & 65.43% (in site specific) and 68.19%, 70.09% & 73.92% in non-site specific hydroxyl radical scavenging potential for RJB, PPB & HPB respectively at 250 µg/ml concentration (Spl. Figure 2–3). Spl. Figure 4 revealed the dose dependent reducing potential of various extracts of Acacia nilotica of diverse geographical locations i.e. Bikaner, Amritsar & Palampur. It was observed that in reducing power assay that PPB showed more reducing potential i.e. 70.42% as compared to RJB (61.29%) & HPB (68.16%) at highest tested concentration of 150 µg/ml. This reducing potential reflects the electron donor capacity associated with antioxidant activity. The presence of reducers (antioxidants/free radical scavengers) in the extracts/tested compounds results in the reduction of the ferric complex to iron form and this reductive potential of the extracts can be determined by the direct reduction of Fe [(CN) 6 ] 3 to Fe [(CN) 6 ] 2 . The reducing power results of present study proven that all extracts of A. nilotica are rich with compounds having reducing power, such as phenolic and polyphenolic compounds. Previous studies reported that the extract of endemic plant Anabasis aretioïdes (Chenopodiaceae) have a good in vitro reducing and antioxidant potential [ 26 ]. The reducing power of the mushroom extracts can be due to the ability of hydrogen donation which led to stabilizing the molecules [ 27 ]. In chelating power assay, again HPB extract of Acacia nilotica showed highest chelating potential of 89.26% with IC 50 value of 130.86µg/ml than RJB (68.16% & IC 50 , 166.12µg/ml), PPB (81.15% & IC 50 , 145.35µg/ml) extracts (Spl. Figure 5). Spl. Figure 6 showed the In-vitro lipid peroxidation inhibition potential of ANL extracts and it was observed that likewise other assay, again HPB extract showed maximum inhibitory potential (92.15% with 112.56 µg/ml IC 50 value) as compared to other extracts of Acacia nilotica i.e. RJB (78.19% & IC 50 166.24 µg/ml), PPB (86.62% & IC 50 , 138.62 µg/ml), at maximum highest tested concentration dose of 250 µg/ml. In Nutshell, it was observed from the results that HPB extract of Acacia nilotica showed strong antioxidant potential then RJB & PPB. The present results also showed the linkage between antioxidant potential of extract and its phenolic content [ 28 ]. 3.6.2 In vitro Cytotoxicity analysis of ANL extracts: Five different cancer cell lines and one non-tumorigenic epithelial cell line from human bronchial epithelium (BEAS-2B) were selected for cytotoxicity analysis. The cell lines were treated with 4 mg/mL leaf extracts of ANL samples of diverse geographical regions. It was noticed that ANL extracts showed toxicity between 59–91% on all various cell lines. The RJB extract exhibited more activity of 91% (MCF-7), 90% (A-549, 88% (PC-3), 86% (IGROV-1) & 77% (DU-145) as compared to PBB & HPB leaf extracts of Acacia nilotica (Table 5 ). To date, most chemotherapeutic agents do not distinguish between normal and cancer cell lines. Therefore it is important to discover plant extracts with selective cytotoxicity for cancer cells and having less damage to normal cells after cancer therapy [ 29 ]. Therefore, the cytotoxic effect of various extracts of ANL samples on cancer cells were compared with the control (without extract) and the non-tumorigenic normal cell line (BEAS-2B). The time and dose dependent cytotoxic potential of ANL samples were also investigated. The cytotoxic potential of ANL extracts of 100–500µg/ml concentration at time interval of 24, 48, and 72 hr were studied and it was observed that level of cytotoxicity was proportional with the duration of exposure and concentration of different ANL extracts (Data not shown). Gordanian et al. (2012) reported that the cytotoxic activity of methanol extract from different organs of A. absinthium at higher altitude was 20–30% higher as compared with those of lower altitudes [ 30 ]. Previous studies showed that leaf extract of A. absinthium from Iran at 0.1 mg/mL resulted in 30% cytotoxicity on MCF-7 cell lines, whereas we found 20% cytotoxicity on MCF-7 cells at the same concentration. However, this could be due to different geographic regions for plant origins and consequently an accumulation of different secondary metabolites. Table 5 Cytotoxic potential of different ANL samples of diverse geographical regions at 100ug/ml concentration. Extract BEAS-2B (Non-cancerous) A-549 (Lung) DU-145 (Prostate) PC-3 (Prostate) IGROV-1 (Ovary) MCF-7 (Breast) RJB 18 90 77 88 86 91 PBB 12 89 59 85 79 84 HPB 21 81 86 88 91 67 Adriamycin Not Tested 84 69 80 81 79 5-FU Not Tested 65 11 13 95 86 3.6.3 In-vitro antimicrobial potential ANL extracts: The ANL extracts of different locations of northern states of India revealed low to moderate range of antimicrobial potential against different microorganisms. At 500 µg/ml (maximum tested concentration), HPB exhibited highest antimicrobial potential against P. aeruginosa (2.61%), S. aureus (2.89%), S. pyogenes (2.97%), C. albicans (3.11%) than other RJB & PBB extracts of A. nilotica (Spl. Table 5 ). It is evident from the results that extract concentration was linearly proportional to the inhibitory potential of the extracts. These extracts were further quantitatively assessed by minimum inhibitory concentration. MIC of HPB extract was found relatively less than extracts of Acacia nilotica against P. aeruginosa , S. aureus, S. pyogenes, C. albicans i.e. 112.4, 103.9, 101.5, 161.8µg/ml respectively (Table 6 ). These results confirmed the presence of phenolic compounds having antimicrobial potential in ANL samples. Table 6 The minimum inhibitory concentration (µg/ml) of different ANL extracts. Microorganism Minimum Inhibitory Concentration (µg/ml) RJB PBB HPB P. aeruginosa 119.5 132.6 112.4 S. aureus 109.2 125.7 103.9 S. pyogenes 153.7 159.2 101.5 C. albicans 161.8 130.4 161.8 3.6.4 Analysis of apoptosis by caspase-3 assay: Apoptosis is brought on by a sequence of caspase cleavages, which are serine proteases. Both internal and external apoptotic mechanisms involve the caspase cascade, a serial cleavage, with caspase-3 being the final protease to cause cell death. One reliable sign of apoptosis is the cleavage of the apoptotic substrate by activated caspase-3 [ 31 ]. In this work, the amount of active caspase-3 in the cell lysate of A-549 cells was determined following treatment with 500µg/ml of different ANL extracts from different geographical locations (Bikaner, Amritsar, and Palampur). Following 36 hours of treatment, it was shown that the cells had activated caspase-3, which was 4–4.5 times greater in cells treated with ANL extracts than in cells that were not treated (data not shown). 3.6.5 Anti-inflammatory potential of ANL extracts: The enzyme COX-2 is necessary for the production of pro-inflammatory prostaglandins and thus has been a target for many cancer-preventive and anti-inflammatory drugs of rheumatoid arthritis, arteriosclerosis, myocarditis, infections, cancer, and other metabolic disorders. The several plant based natural products have been shown to transmit their anti-inflammatory activities through suppression of COX-2 [ 32 ]. It was observed that various extracts (RJB, PBB & HPB) of A. nilotica were found to be selective inhibitor of COX-2 (COX-2 selectivity > 10) and inhibit COX-1 by 42.15%, 72.63% & 79.15% at 10 µM concentration respectively whereas COX-2 was inhibited by 71.45%, 77.22% & 81.56% respectively at same concentration i.e. 10 µM and it was noted that in the presence of ANL extracts, the level of PGE 2 dropped down (Table 7 ). The presence of polyphenols in plant extracts has already reported to block cyclooxygenase activity induced by UVB radiation. Thus it might also be implicated in suppression of cyclooxygenase-mediated inflammatory pathway [ 33 ]. Table 7 Cyclooxygenase enzyme mediated anti-inflammatory activities (COX-1 & COX-2) of ANL samples. Extract Compound % Inhibition IC 50 (µM) COX-2 Selectivity* COX-2 COX-1 COX-2 COX-1 1µM 10µM 10µM RJB 43.61 71.45 42.15 10 > 10 PBB 51.26 77.22 72.63 10 > 10 HPB 54.92 81.56 79.15 10 > 10 Rofecoxib** 75 100 75 0.3 40 ˜133 Celecoxib ** 50 100 65 1.2 14 ˜10 *COX-2 selectivity = IC 50 (COX-1)/ IC 50 (COX-2) **Reported in literature (Kaur at el., 2009) 22 . 4. Conclusion The HPLC-MS results in this study show the relative amounts of the main phytochemicals in different Acacia nilotica extracts that were gathered from different parts of India's Rajasthan, Punjab, and Himachal Pradesh. In all A. nilotica extracts, the phytochemical concentration was found to be proportionate to their biochemical and biological activity. The geographical characteristics and environmental factors influencing plant growth and development were found to be responsible for the maximum phytochemical content and biological activity in the ANL sample "HPB." Additionally, it was found that while the phytochemicals in the different ANL samples were all members of the same species, their types differed in abundance depending on their varied geographic locations. The results suggested the fundamental role of the geographical properties and environmental variable in differentiating the phytochemical composition of the samples which was further accountable for the observed disparity in their biochemical and biological activities. Declarations Conflict of interest: Authors declare not conflict of interest. Funding Declaration No external funding was received for the present study. The research was carried out using available institutional resources. Author Contribution P.K. collected plant material, performed the experimentation and Data analysis. S.K. validated and drafted manuscript. R.S. conceived idea, designed experiment and paper writing. All authors reviewed the results and approved the final version of the manuscript. 5. Acknowledgements Authors are thankful to Khalsa College Charitable Society, Amritsar, Punjab, India for providing research grant (5666/10-11-2023) under seed money grant programme and other technical facilities to complete present study. Data Availability All data generated or analysed during this study are included in this published article (and its supplementary information files). References Priya, S. H., Prakasan, N. & Purushothaman, J. Antioxidant activity, phenolic flavonoid content and high performance liquid chromatography profiling of three different variants of Syzygium cumini seeds: A comparative study (Journal of Intercultural Ethnopharmacology, 2017). Zhang, Y. J. et al. Antioxidant phytochemicals for the prevention and treatment of chronic diseases. Molecules 20 , 21138–21156 (2015). Inbathamizh, L. & Padmini, E. Effect of geographical properties on the phytochemical composition and antioxidant potential of Moringa oleifera flowers. BioMedRx 2013,1(3),239–247. (2013). 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Cytotoxic Activities of Certain Medicinal Plants on Different Cancer Cell Lines. Turk. J. Pharm. Sci. 14 (3), 222–230 (2017). Krakauer, T. Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB. Curr. Drug Targets- Inflamm. Allergy . 3 , 317–324 (2004). López-Posadas, R. et al. Sánchez de Medina, F. Flavonoids exert distinct modulatory actions on cyclooxygenase 2 and NF-kappaB in an intestinal epithelial cell line (IEC18). Br. J. Pharmacol. 160 , 1714–1726 (2010). Additional Declarations No competing interests reported. Supplementary Files Fig.16Supplementary.docx TablesSupplementary15.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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3","display":"","copyAsset":false,"role":"figure","size":1313658,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.17MainResults3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/4a02daf9fce825c496456b69.jpg"},{"id":97349621,"identity":"91dab801-1c75-4f36-8569-bff2d7eb3300","added_by":"auto","created_at":"2025-12-03 12:12:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1411762,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.17MainResults5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/825ff097f6d14e7ed6ba19b3.jpg"},{"id":97349627,"identity":"10be9904-ebd6-415b-8f82-0584230ff6d9","added_by":"auto","created_at":"2025-12-03 12:12:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1413320,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.17MainResults7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/b2b4d46cde98efbed2897758.jpg"},{"id":97369971,"identity":"0f307998-d6b6-4c85-9d73-748ba0d8e997","added_by":"auto","created_at":"2025-12-03 16:26:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1144614,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.17MainResults9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/9560df5fe09c636770af0c19.jpg"},{"id":97371231,"identity":"68c5466a-b3a4-4704-9be7-09a0d34779e8","added_by":"auto","created_at":"2025-12-03 16:28:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":354573,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.17MainResults11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/dfad15637cb0151402a9997b.jpg"},{"id":98429811,"identity":"6b93fea4-bb78-4bdd-8bdc-a4bda38bf13e","added_by":"auto","created_at":"2025-12-17 16:44:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8392369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/88905100-d088-4c65-b58e-688d4908ba9a.pdf"},{"id":97349622,"identity":"b47e7c64-530f-4019-985c-de4226c45c74","added_by":"auto","created_at":"2025-12-03 12:12:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":158984,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.16Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/b27df764c536337690079ad7.docx"},{"id":97371382,"identity":"e86b65b6-8bd3-4385-affc-aedd18ff694d","added_by":"auto","created_at":"2025-12-03 16:28:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20807,"visible":true,"origin":"","legend":"","description":"","filename":"TablesSupplementary15.docx","url":"https://assets-eu.researchsquare.com/files/rs-8084390/v1/44fbb30d43138c5f59659b44.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biochemical \u0026 phytochemical insights of geographically diverse Acacia nilotica through HPLC–MS-based untargeted metabolomic studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA new generation of health and disease management is ensured by the medical revolutions brought about by the intensive study in free radical biology. They have both positive and negative effects and are the main byproducts of numerous cellular redox activities. They are helpful for immune response and cellular responses at low to moderate levels, but at high concentrations, they cause oxidative stress, which damages cell structures and functions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With their diverse range of chemical components, plants are a prospective source of molecules with therapeutic qualities. These plants continue to promote human health in addition to serving as the foundation of traditional medicine.\u003c/p\u003e\u003cp\u003eNatural foods and components derived from plants have received a lot of attention lately because they are thought to be safer and healthier than their synthetic counterparts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Geographical variances and agroclimatic conditions can significantly impact the chemical makeup and therapeutic qualities of medicinal plants, as evidenced by Ayurvedic research. According to earlier research, the differences in the chemical composition and antioxidant capacities of plants from various geographic regions can be attributed to a variety of environmental conditions, including temperature and soil type. Significant phytochemical changes in maple leaf trees were caused by habitat, climate, harvest timing, and processing [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Numerous factors, including variety, age, plant maturity, collection season, geo-agro-climatic conditions, agronomical procedures, postharvest management, storage, processing, etc., affect the medicinal plants' highly variable phytochemical composition. As a result, the active principle can vary greatly, which would impact the bio-potency. For the plants to generate consistently high-quality products, chemical profiling is crucial. In order to choose and formulate plant-based dietary supplements and develop food-based therapeutic compounds, it is essential to confirm the bioconstituents and biological activities of plants across a variety of agro-ecologies.\u003c/p\u003e\u003cp\u003eThe goal of the current study was to compare the biological activities of \u003cem\u003eA. nilotica\u003c/em\u003e leaf populations collected from three northern Indian states: Rajasthan (Bikaner), Punjab (Amritsar), and Himachal Pradesh (Palampur). India is a country with a variety of soil types, water levels, temperatures, and other environmental factors throughout the year. The current study has two scientific goals. Establishing a chemical variation among the \u003cem\u003eA. nilotica\u003c/em\u003e population at various collecting sites was the first phase's goal. The second was to examine the significant biological activities based on the phenolic variance across plants from various geographic locations. Thus, this study provides more proof in favor of the widely accepted link between biological activity and phytochemical differences dependent on geoclimate. The bio-efficacy of the plant variations is significantly influenced by the active principle or specific polyphenols that may be dys-regulated due to geographical changes. The concept that there is a correlation between the phytochemistry of plants and the climatic variance of various geographical regions, which further influences the biological activities of medicinal plants, was further supported by this study.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\u003cp\u003eSigma Chemical Co. (St. Louis, MO, USA) provided the 2-thiobarbituric acid and 2'-2' diphenyl-1-picrylhydrazyl (DPPH). Lancaster Synthesis Inc. (Windham, USA) provided the 2-deoxyribose, and Abcam, USA, provided the Caspase-3 kit. The following items were acquired from Sigma-Aldrich USA: gentamycin, penicillin, fetal calf serum (FCS), acetic acid, trichloroacetic acid (TCA), mitomycin-C, trypsin, RPMI-1640 medium, gentamycin, 5-fluorouracil (5-FU), and 2-thiobarbituric acid. The National Cancer Research Institute in the United States provided the human cancer cell lines. Tris EDTA from Hi Media, phosphate buffer saline (PBS) from Merck (Germany), and sulforhodamine B from Fluka. H\u003csub\u003e2\u003c/sub\u003eO that had been double distilled was used to make all stock solutions. Sigma Aldrich's HPLC-grade standards were utilized for HPLC screening. Cayman Chemicals (Michigan, USA) supplied the anti-inflammatory bioassay kit. All other chemicals were of analytical grade and procured from Ranbaxy Fine Chemicals Ltd. (New Delhi, India).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Plant Material\u003c/h2\u003e\u003cp\u003eIn March 2020, nine randomized samples of \u003cem\u003eAcacia nilotica\u003c/em\u003e leaves (ANL) were gathered from Bikaner, Rajasthan; Amritsar, Punjab; and Palampur, Himachal Pradesh, India, three from each site as per the institutional policy of the Khalsa College Amritsar. A single composite sample of each tree was created by first selecting three to five \u003cem\u003eAcacia\u003c/em\u003e trees of comparable age and good growth state from each sampling locality, then taking an equal number of leaves from each of the four sides (NEWS) at the same height. In Khalsa College Amritsar, Punjab, India, the samples were recognized \u0026amp; identified by subject experts of P.G Department of Botany. The vouchers of the same were placed in the herbarium (A/C # 1051, dated 12-05-2020). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the precise geographic position, collection time, and area (km\u0026sup3;) of the collection place and and each sample was given an accession code \u0026ldquo;RJB\u0026rdquo;, \u0026ldquo;PBB, \u0026amp; \u0026ldquo;HPB\u0026rdquo;. All biochemical experiments were performed in accordance with the approval of Chemical \u0026amp; Bioethical Committee (CBE) of Khalsa College Amritsar, Punjab, India.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe collection point with time and geographical coordinates of Northern Indian states.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eState\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDistrict\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGeographical\u003c/p\u003e\u003cp\u003ecoordinates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTime of Collection\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArea\u003c/p\u003e\u003cp\u003e(sq kms.)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePlant part\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAbbreviated name\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePunjab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAmritsar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e31.63\u0026deg; N, 74.87\u0026deg; E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAugust 2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePBB\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePBL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePBF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHimachal Pradesh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePalampur\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e32.11\u0026deg; N, 76.53\u0026deg; E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAugust 2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHPB\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHPL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHPF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eJammu \u0026amp; Kashmir\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eKathua\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e32.37\u0026deg;N 75.52\u0026deg;E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAugust 2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eJKB\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eJKL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eJKF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Agro-climatic \u0026amp; Meteorological data\u003c/h2\u003e\u003cp\u003eThe Ministry of Earth Sciences' Climate Data Service Portal Meteorological Department provided the comprehensive meteorological data, which included both geographic and climatic information (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cdsp.imdpune.gov.in/\u003c/span\u003e\u003cspan address=\"https://cdsp.imdpune.gov.in/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Longitude, latitude, and altitude were the averaged geographic data (for the two weeks before sample collection) and temperature, humidity, sunshine duration, and rainfall were the averaged climatic data used in this study. The information reflected the geographical traits thought to be the most important determinants of the accumulation of metabolites and the biological activity of \u003cem\u003eAcacia nilotica\u003c/em\u003e leaves.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Sample Preparation and Extraction\u003c/h2\u003e\u003cp\u003eAll composite leaf samples of \u003cem\u003eAcacia nilotica\u003c/em\u003e were cleaned and washed with tap water. They were then allowed to air dry and ground into a fine powder before being sieved through a 40-mesh screen in order to extract organic solvents. First, 450 milliliters of methanol were used to macerate 100 grams of fine powdered leaf material for 72 hrs. while being shaken intermittently. The supernatant was filtered after a day, and the powder that remained was macerated twice using new methanol solvent. For additional phenolic profiling and biochemical analysis, all three supernatants were combined, dried in a rotary evaporator (BUCHI R-300, SWITZERLAND), moved into vials, and stored at -20\u0026deg;C (Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Biochemical analysis\u003c/h2\u003e\u003cp\u003eUsing the methods of Kujala et al. and Park et al., the total phenolic content (TPC), total flavonoid content (TFC), and total anthocyanidin content (TAC) of all methanolic ANL extracts were measured as gallic acid, quercetin equivalent, and cyanidin equivalent in mg/g DW, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 HPLC-MS based phenolic fingerprinting \u0026amp; Amino acid analysis of ANL extracts\u003c/h2\u003e\u003cp\u003eA Shimadzu Prominence HPLC system equipped with Luna C18 (2) column (250 mm \u0026times; 4.6 mm, 5\u0026micro;m particle size) and Shimadzu LC solution (ver. 1.21 SP1) software was used for HPLC analysis. Using solvent A (water) and solvent B (0.02% trifluroacetic acid (TFA) in acetonitrile) at a column temperature of 27\u0026deg;C and a flow rate of 0.75 ml/min, a linear gradient elution was performed at λ 280 nm: 70% A (5 min), 15\u0026ndash;35% (7 min), 35\u0026ndash;45% (11 min), 45\u0026ndash;35% (16 min), and 35\u0026ndash;15% (20 min). Z-Spray ESI ion (positive \u0026amp; negative) modes were used to acquire mass data while adhering to the optimal settings: : capillary voltage 2.5 kV; sample cone voltage 30 V; desolvation temperature 400 ◦C; source temperature 110 ◦C; desolvation gas flow 800 L/h; cone gas flow 50 L/h; scanning time 2 s; and scan mass range 100\u0026ndash;1600 Da.\u003c/p\u003e\u003cp\u003eSolvent blanks (70:30 v/v MS-grade water:acetonitrile), 27 ANL samples (triplicates of nine samples), and nine QCs were added to the sequence for LC-MS data capture. In order to check and stabilize the machine, three blank injections were made first. Three QCs were then injected after every nine ANL samples. At the conclusion of the sample sequence, three blank injections were carried out. For calibration, leucine-enkephalin (1 \u0026micro;g/mL) was employed as a lock-mass solution at a flow rate of 20 \u0026micro;L/min, yielding reference ions of m/z 556.2771 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e and 554.2615 [M\u0026ndash;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e. For the purpose of creating calibration curves, the analyte-containing stock solution was made as 1 mg/1 ml in HPLC-grade methanol: water (90:10) and then further diluted to the proper concentrations of theses analyte were injected thrice for the quantitative analysis and the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The selectivity of the method was determined by analyzing standards and methanol extracts. The peaks of reference compounds were identified by comparing their retention times \u0026amp; UV spectra with those of the standards.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Metabolite profiling and chemometric analysis:\u003c/h2\u003e\u003cp\u003eThe Shimadzu LC solution software (version 1.21 SP1) was used to assess the alignment, normalization, peak picking, and deconvolution of the spectral data of 27 ANL samples. For its chemometric analysis, the deconvoluted data without any filtering were exported to MetaboAnalyst 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In order to distinguish the comparison groups in PCA \u0026amp; OPLS-DA, the marker chemicals were further screened using an S-plot. In an S-plot, the ions that made the most contributions were displayed at the top right (1) and bottom left (-1). Relative quantitative differences between substances based on peak intensity were clarified using heatmap. Smart Metabolites Database\u0026trade; Ver.2 was used to further identify the probable chemical ions that were filtered by ANOVA (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and max fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Biological activities:\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1 In vitro antioxidant testing assays\u003c/h2\u003e\u003cp\u003eBecause plant extracts are complex, a battery of \u003cem\u003ein-vitro\u003c/em\u003e antioxidant testing systems was used to assess the detailed antioxidant capability of several ANL extracts [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The hydrogen-donating capacity of ANL extracts was assessed using the stable DPPH radical, and the hydroxyl radical scavenging potential was assessed using the site- and non-site-specific deoxyribose degradation assay [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The modified Dinis \u003cem\u003eet al.\u003c/em\u003e approach was used to determine the chelating effect on ferrous ions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Oyaizu's approach was used to determine the reducing power of ANL extracts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the lipid peroxidation experiment, the TBA combines with malondialdehyde (MDA) to generate a diadduct, a pink chromogen that is detected at 532 nm [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2 In vitro Cytotoxicity Assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity potential of several methanolic extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e was assessed using the Sulforhodamine B dye test for the cell lines BEAS-2B (non-cancerous), A-549 (lung), DU-145 \u0026amp; PC-3 (prostate), IGROV-1 (ovary), and MCF-7 (breast) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. All the cell lines were procured from Genetic Toxicology Laboratory, Department of Botanical \u0026amp; Environmental Sciences, Guru Nank Dev University, Amritsar, Punjab, India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.8.3 Antimicrobial studies\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section4\"\u003e\u003ch2\u003e2.8.3.1 Culturing of microorganisms strains:\u003c/h2\u003e\u003cp\u003eATCC-25923 \u0026amp; ATCC-19615 were grown on prepared blood agar (BA) while ATCC-27853 cultured on cooked blood agar (CBA) and ATCC-10231 cultured on Sabouraud dextrose 4% agar (SDA) to check the antimicrobial potential of ANL extracts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section4\"\u003e\u003ch2\u003e2.8.3.2 Antimicrobial activities through Agar disk diffusion assay\u003c/h2\u003e\u003cp\u003eThe 7 mm sterile and dried paper disks were soaked in 50 \u0026micro;l of different concentrations of ANL extract and allowed to dry for 24 hours at 37\u0026deg;C. On agar inoculated with microbial culture and strains, 2 mm round and dried paper disks were carefully inserted. The disks were then incubated at 37\u0026deg;C for 24 hours for \u003cem\u003eP. aeruginosa, S. aureus\u003c/em\u003e, and S. pyogenes, and 48 hours for \u003cem\u003eCandida albicans\u003c/em\u003e. The measurement of the inhibitory zones was in millimeters. 50 \u0026micro;l of extracting solvent served as the negative control, while the broad spectrum antibiotics ampicillin (for \u003cem\u003eS. pyogenes\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e), ciprofloxacin (for \u003cem\u003eP. aeruginosa\u003c/em\u003e), and fluconazole (for \u003cem\u003eC. albicans\u003c/em\u003e) served as the positive controls [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section4\"\u003e\u003ch2\u003e2.8.3.3 Minimum inhibitory concentration (MIC)\u003c/h2\u003e\u003cp\u003eThe micro-titer plate method was followed to estimate the MIC of ANL extracts [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.8.4 Caspase-3 assay:\u003c/h2\u003e\u003cp\u003eUsing the Abcam, USA, caspase-3 colorimetric test kit methodology, caspase-3 activity was measured. This test quantifies the quantity of free p-nitroanilide (pNA) in the cell that results from active caspase-3 cleaving the DEVD-pNA link during apoptosis. By comparing the absorbance of p-NA from an apoptotic sample with that of untreated control cells, the fold increase in caspase-3 activity was ascertained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.8.5 Anti-inflammatory Activity\u003c/h2\u003e\u003cp\u003eANL extracts' \u003cem\u003ein vitro\u003c/em\u003e COX-2 inhibitory properties have been assessed using 96-well plates and a \"COX (ovine) inhibitor screening assay\" kit. Included were the COX-1 and COX-2 enzymes from cows [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. PGF2α generated by SnCl2 reduction of COX-derived PGH2 is directly measured by this screening assay. The 96-well plate's wells with low absorption at 405 nm are indicative of low prostaglandin levels and, thus, lower enzyme activity. Consequently, the absorption values of the various wells in the 96-well plate may be used to quantify the COX inhibitory activities of ANL extracts.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll analyses were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3) and the data was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The results obtained were analyzed using two-way ANOVA to determine the significant differences between the experimental batches by taking the raw samples as control.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe production of phytochemicals is significantly regulated by environmental and genotypic variables, which in turn have an impact on biological activity. According to earlier research, environmental factors were mostly responsible for the phytochemical diversification across various geographic regions, with genotype having a minor influence on phytochemicals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Metrological Data (soil and agro-climatic analysis):\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e explains annotations of leaf extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e collected from northern states of India i.e. RJB (Rajasthan) PBB (Punjab) and HPB (Himachal Pradesh). Amritsar is located at 31.63\u0026deg;N 74.87\u0026deg;E with an average elevation of 234 metres (768 ft) with 2,683 square km area. The climate in Amritsar is warm and temperate. The Bikaner is located at 28.02\u0026deg; N, 73.31\u0026deg; E at an elevation of 225.73 meters (740.58 feet) above sea level. The city has a Subtropical desert climate. Palampur is a famous hill station at 32.11\u0026deg; N, 76.53\u0026deg; E. It is located at height of around 1300 metres, above sea level with 609 area in square km. The city has a monsoonal-influenced humid subtropical climate with moderate summers and cool winters. The composite leaves sample (explained in material method section) from all the collection point were collected in the March 2020.\u003c/p\u003e\u003cp\u003eThe soil sample of Bikaner has sandy to clay loam texture with alkaline content (pH 8.3) and 0.29 EC (dSm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) while Amritsar soil has coarse loamy texture with pH 6.9 (almost neutral) and 0.95 EC (dSm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The sample of Palampur showed pH 6.7 and loam to clay textured soil. The results showed that Amritsar soil has more NPK value (219, 816, 99.7) as compared to Rajasthan (188, 756, 83.6) and Palampur (192, 782, 86.4) as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Supplementary).\u003c/p\u003e\u003cp\u003ePrevious studies revealed that environmental variables tremendously affected the phytochemistry of the plant and a significant difference in important environmental variable viz. annual temperature, rainfall, sunlight \u0026amp; average relative humidity (ARH) was observed. The Palampur collection site showed highest annual rainfall of 1288.3 mm and ARH of 54% and Rajasthan site exhibited maximum annual temperature of 30.2\u0026deg;C and annual sunlight of 3842.77 hrs. In case of Amritsar, it was observed moderate annual temperature (23.4\u0026deg;C), rainfall (572.2 mm), sunlight (3653.84 hrs.) and ARH (53%) (Spl. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExtract yield and biochemical analysis of different ANL extracts.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlant Location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePlant Part\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExtract Yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTPC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTAC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDistrict: Amritsar\u003c/p\u003e\u003cp\u003eState: Punjab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e395.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e308.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e288.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDistrict: Palampur\u003c/p\u003e\u003cp\u003eState: Himachal Pradesh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e465.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e372.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e366.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDistrict: Kathua\u003c/p\u003e\u003cp\u003eState: Jammu \u0026amp; Kashmir\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBark\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e409.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e321.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e365.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFlower\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eTPC\u003c/b\u003e: Total Phenolic content as mg GAE/g extract; \u003cb\u003eTFC\u003c/b\u003e: Total Flavonoid content as mg QE/g extract; \u003cb\u003eTAC\u003c/b\u003e: Total anthocyanidin as mg cyanidin/g extract.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Biochemical \u0026amp; phytochemical characterization of ANL extracts:\u003c/h2\u003e\u003cp\u003eThe HPLC-MS based phenolic fingerprinting revealed that phytochemicals in all ANL extracts qualitatively remained same but varied quantitatively as indicated by their phenol and flavonoid contents, which were observed proportional to their biological activities. It was observed in the results that nature of the phytochemicals in all ANL samples did not differ but their quantity varied according to their geographical conditions. The results of the current study are further supported by earlier investigations, which showed that the chemical contents of \u003cem\u003eColeus forskohlii\u003c/em\u003e roots varied depending on the geographical location of India [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eCannabis sativa\u003c/em\u003e L.'s cannabinoid concentration was also impacted by the soil and agroclimatic conditions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The research revealed that the geographical characteristics played a crucial part in determining the phytochemical makeup of the samples, which in turn explained the observed variation in their antioxidant capacities.\u003c/p\u003e\u003cp\u003eThe major phytochemical screening tests were carried out for the ANL extracts which revealed the presence of alkaloids, reducing sugars, quinones, saponins, coumarins, triterpenoids, resins, flavonoids and tannins with varying concentration of low to high range (Spl. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This phytochemical variation was confirmatory with low to high TPC (395.12, 409.62 \u0026amp; 465.09 mg as GA /g DW) and TFC (308.27, 321.29, 372.62 mg as RE /g DW) \u0026amp; TAC (288.41, 365.26, 366.16 mg as cyanidin/g DW) for RJB, HPB \u0026amp; PJB respectively. The results also showed the %age yield of extract for RJB (24.59), PJB (28.14) \u0026amp; HPB (29.18) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe photosynthetic pigments, carbohydrate and total amino acids content of leaves of ANL samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlant Extract\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChl. a\u003c/p\u003e\u003cp\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFW)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChl. b\u003c/p\u003e\u003cp\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFW)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCarotenoids\u003c/p\u003e\u003cp\u003e(mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFW)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal carbohydrate (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTotal amino acids (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRJB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e62.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePBB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e86.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHPB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e97.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the present study, it was also observed that the environmental variables significantly affected the photosynthetic pigments, total carbohydrate and amino acids content amongst the leaves of \u003cem\u003eAcacia nilotica\u003c/em\u003e collected from different geographical regions. The HPB extracts exhibited highest Chl. a \u0026amp; b, Carotenoids (2.36, 1.08, 2.14 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFW), total carbohydrates (32.5 mg/g) and amino acids (97.2%) content as compared to PBB (1.29, 0.78, 1.73, 25.8, 86.1) and RJB (0.95, 0.35, 1.05, 19.6, 62.4) for Chl. a \u0026amp; b, Carotenoids, total carbohydrates \u0026amp; amino acids respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It was found direct correlation between the photosynthetic pigments and annual rainfall \u0026amp; ARH and inverse correlation between annual the photosynthetic pigments and annual temperature \u0026amp; sunlight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3 HPLC based Phenolic fingerprinting and metabolite identification:\u003c/h2\u003e\u003cp\u003eThe phenolic fingerprinting of ANL extracts by HPLC-MS and analyzed by Shimadzu LC solution (ver. 1.21 SP1) software resulted in 2681, 2715 \u0026amp; 2937 chemical features detected after peak picking from the mass data obtained in both positive \u0026amp; negative ionization modes for RJB, PBB \u0026amp; HPB respectively (Spl. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The Venn diagram depicted the correlation between the chemical features and ANL samples (Fig.\u0026nbsp;2). Total 112 common feature were detected between all ANL samples and further filtered by ANOVA (\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;2) to eliminate false ions and finally 49 biologically important \u0026amp; putatively features were selected and identified through Smart Metabolites Database\u0026trade; Ver.2.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHPLC-MS data of dysregulated phytochemicals of different ANL extracts.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMass m/z\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMass Error\u003c/p\u003e\u003cp\u003e(ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMolecular formula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTentatively Identified Compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePub Chem. ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003eFold Change\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExperimental Mass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTheoretical Mass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eRJB/HPB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003ePBB/HPB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\u003cp\u003ePositive Mode [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e199.0298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e199.0303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSyringic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10742\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e169.0191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e169.0197\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVanillic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8468\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e149.0295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e149.0299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCinnamic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e444539\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e443.3581\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e443.3585\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBetulin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72326\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e449.0785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e449.0780\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIsoorientin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e114776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e595.1364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e595.1359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e15\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVicenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e442664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e301.0411\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e301.0408\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDiosmetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281612\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e302.9835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e302.9837\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEllagic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281855\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e170.9993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e170.9998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGallic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e370\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127.0097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e127.0091\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-4.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePyrogallol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155.1129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e155.1132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e197.1234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e197.1238\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBornyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e319.0156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e319.0151\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMyricetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e457.3371\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e457.3378\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOleanolic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10494\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e239.0407\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e239.0404\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7-Hydroxyflavone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281894\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e291.0558\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e291.0565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e165.0619\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e165.0612\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-4.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEugenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3314\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e149.0304\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e149.0299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-3.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003et\u003c/em\u003e-cinnamic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e444539\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e287.0248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e287.0252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKaempferol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280863\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e291.0563\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e291.0565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEpicatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e165.0242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e165.0248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eo\u003c/em\u003e-coumaric\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e637540\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e195.0351\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e195.0354\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFerulic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e445858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e341.0565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e341.0569\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAesculin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281417\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e205.1648\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e205.1653\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHumulene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281520\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e303.0205\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e303.0201\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eQuercetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e29.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e271.0297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e271.0303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eApigenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNegative Mode [M - H]\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e135.1169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e135.1173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTerpiniolene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11463\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e177.0183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e177.0187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEsculectin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281416\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e455.3519\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e455.3524\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUrsolic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e64945\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e147.0814\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e147.0809\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-3.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEstragole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8815\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e103.0398\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e103.0394\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-3.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3-Hydroxybutyric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e167.0209\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e167.0204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-2.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e285.0401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e285.0398\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLuteolin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280445\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e161.0232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e161.0237\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUmbelliferone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e219.1752\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e219.1748\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSpathulenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e92231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e191.0187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e191.0191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCitric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e311\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179.0348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e179.0343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-2.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCaffeic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e689043\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e353.0867\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e353.0871\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChlorogenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1794427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e153.0183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e153.0187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eProtocatehuic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0072\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e221.1901\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e221.1904\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGuaiol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e227829\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e447.0933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e447.0926\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLuteolin-7-O-glucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280637\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e223.0611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e223.0605\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSinapic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e637775\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e609.1450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e609.1454\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRutin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5280805\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e447.0919\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e447.0926\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOrientin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281675\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e149.0959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e149.0965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCarvacrol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e431.0973\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e431.0977\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eApigenin-7-O-glucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12304093\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e359.0762\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e359.0766\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRosmarinic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5281792\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e271.0610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e271.0605\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNaringenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e439246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e313.0717\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e313.0711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCirsimaritin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e188323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e303.2327\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e303.2323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eArachidonic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e444899\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Metabolite dysregulation amongst ANL of various geographical regions:\u003c/h2\u003e\u003cp\u003eUsing MetaboAnalyst 6.0 software, chemometric models (PCA, OPLS-DA, and S-plot) were used to evaluate the HPLC-MS data of RJB, PBB, and HPB in order to comprehend the metabolite dysregulation in ANL samples from various geographic locations. A visual TIC of 49 dysregulated phytochemicals in three ANL samples is evident from the random forest-based heatmap. It was shown that, in comparison to PBB, 16 compounds were up-regulated in RJB and 33 in HPB samples (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A distinct phenolic compound with a distinct mass to charge and retention time value is shown by each row (Fig.\u0026nbsp;3; Panel A).\u003c/p\u003e\u003cp\u003eTo get a comprehensive picture of the metabolite changes between ANL samples, PCA, an unsupervised technique, was applied to the chosen chemical characteristics without any filtering. ANL samples from Rajasthan, Punjab, and Himachal Pradesh were found to have divided into three distinct groups in PCA plots, with no overlap. This suggests that the metabolic profiles of the ANL samples from these three regions varied greatly from one another. Each of the three samples exhibited a loose clustering pattern, indicating that the chemical profiles of ANL samples from distinct zones differ significantly (PC1\u0026thinsp;=\u0026thinsp;94.3% versus PC2\u0026thinsp;=\u0026thinsp;4.5%, Fig.\u0026nbsp;3; Panel B). In this plot, the X-axis (dimension 1) denotes the interclass separation while the Y-axis (dimension 2) displays the intra-class variability According to unsupervised analysis by PCA in this study, the main driving force involved in the separation of extract samples seems to be the environmental variables of the different geographical zones. Figure\u0026nbsp;3 (Panel C\u0026amp;D) exhibited the DPSC correlation pattern and significant \u003cem\u003ep-value\u003c/em\u003e (0.05) of dysregulated phytochemicals based on One Way ANOVA (Turkey\u0026rsquo;s HSD). The difference in the environmental variables, soil and agro-climatic factors led to greatly influence the resulting profile of phytochemicals present in various ANL samples.\u003c/p\u003e\u003cp\u003eOur findings about the impact of four zones' climate factors\u0026mdash;temperature, sunshine duration, rainfall, and altitude on the phytochemical content of \u003cem\u003eAcer truncatum\u003c/em\u003e leaves are supported by earlier studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Altitude has been found to be closely associated with EGCG and ECG in \u003cem\u003eXinyang Maojian\u003c/em\u003e tea (\u003cem\u003eCamellia sinenesis\u003c/em\u003e), a popular green tea brand in China and it was also noted that climate conditions could have a major impact on the catechin contents in \u003cem\u003eCamellia sinenesis\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] The heatmap visualization \u0026amp; PCA plots revealed non-overlapping \u0026amp; open clustering between samples of RJB versus HPB and PBB versus HPB. It was also observed that intra-class separation between samples of HPB is much higher than the RJB and PBB samples (Fig.\u0026nbsp;4\u0026ndash;5; Panel A-B). Figure\u0026nbsp;4\u0026ndash;5 also showed the t-test analysis and volcano plots of \u003cem\u003ep-value\u003c/em\u003e (0.05) and fold change (2) of the 49 dysregulated phytochemicals of ANL samplers (Panel C-D).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Analysis of HPLC based amino acid dysregulation:\u003c/h2\u003e\u003cp\u003eApart from phytochemicals, 18 amino acids were also found present in ANL samples of different geographical regions of Northern Indian states through HPLC-MS analysis and same dysregulation pattern of putative amino acids was observed between ANL samples. Thirteen amino acids viz. Phenylalanine, Threonine, Tyrosine, Arginine, Aspartic acid, Histidine, Leucine, Lysine, Methionine, Proline, Isoleucine, Alanine, Valine were found up-regulated in HPB sample. The another five amino acids, Cystine \u0026amp; Tryptophan were up-regulated in PBB sample while Glutamic acid, Serine \u0026amp; Glycine were up-regulated in RJB sample (Fig.\u0026nbsp;6; Panel A). The unsupervised PCA plot revealed that all three samples clustered loosely, signifying ANL samples from different zones have significant difference in their chemical profiles (PC1\u0026thinsp;=\u0026thinsp;67.6% versus PC2\u0026thinsp;=\u0026thinsp;29.8%, Fig.\u0026nbsp;6; Panel B). Figure\u0026nbsp;6, Panel C-D showed SOM and significant p-value (0.05) of all 18 dysregulated amino acids between ANL samples. The relative content of amino acid panel in ANL samples of different geographical regions showed a clear difference of amino acid in different ANL samples (Fig.\u0026nbsp;7).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Studies on biological activities of ANL extracts:\u003c/h2\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.6.1 Antioxidative potential analysis of ANL extracts:\u003c/h2\u003e\u003cp\u003eThe significant difference in TPC, TFC, TAC \u0026amp; TIC of phytochemicals could be responsible for difference in biological activities of ANL samples of Northern Indian states i.e Rajasthan, Punjab \u0026amp; Haryana. Due to complex nature of phytochemicals the biochemical and antioxidant activity of plant extracts cannot be measured by a single approach. The detailed picture of the free radical scavenging potential of ANL samples were analyzed through battery of \u003cem\u003eIn-vitro\u003c/em\u003e antioxidant testing system. The well accepted DPPH assay is based on scavenging of DPPH radical by antioxidant/extracts through the degree of colour change, which is directly proportional to the radical scavenging potential of compound/extract. The large decrease in the absorbance of the reaction mixture indicates higher radical scavenging potential of the compound which is under test [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. HPB shows strong DPPH scavenging potential of 83.87\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17% (IC\u003csub\u003e50\u003c/sub\u003e, 17.22​) as compared to PPB (77.64%), \u0026amp; RJB (71.40%) at highest tested dose of 250\u0026micro;g/ml concentration with no further inhibitory activity beyond this (Spl. Figure\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eOne of the strongest reactive oxygen species in the body, hydroxyl radicals are known to be harmful in pathophysiological processes. They can harm important biological molecules and cause cancer and a host of other terrible illnesses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The reaction between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and the ferrous that would react with 2-deoxyribose produced hydroxyl radicals. This reaction was halted by adding TBA reagent, which would turn red if malonaldehyde was formed as a result of the radical's reaction with 2-deoxyribose [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The ANL extracts of diverse geographical locations showed moderate to high scavenging potential of 57.13%, 60.23%, \u0026amp; 65.43% (in site specific) and 68.19%, 70.09% \u0026amp; 73.92% in non-site specific hydroxyl radical scavenging potential for RJB, PPB \u0026amp; HPB respectively at 250 \u0026micro;g/ml concentration (Spl. Figure\u0026nbsp;2\u0026ndash;3).\u003c/p\u003e\u003cp\u003eSpl. Figure\u0026nbsp;4 revealed the dose dependent reducing potential of various extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e of diverse geographical locations i.e. Bikaner, Amritsar \u0026amp; Palampur. It was observed that in reducing power assay that PPB showed more reducing potential i.e. 70.42% as compared to RJB (61.29%) \u0026amp; HPB (68.16%) at highest tested concentration of 150 \u0026micro;g/ml. This reducing potential reflects the electron donor capacity associated with antioxidant activity. The presence of reducers (antioxidants/free radical scavengers) in the extracts/tested compounds results in the reduction of the ferric complex to iron form and this reductive potential of the extracts can be determined by the direct reduction of Fe [(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csub\u003e3\u003c/sub\u003e to Fe [(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csub\u003e2\u003c/sub\u003e. The reducing power results of present study proven that all extracts of \u003cem\u003eA. nilotica\u003c/em\u003e are rich with compounds having reducing power, such as phenolic and polyphenolic compounds. Previous studies reported that the extract of endemic plant \u003cem\u003eAnabasis aretio\u0026iuml;des\u003c/em\u003e (Chenopodiaceae) have a good \u003cem\u003ein vitro\u003c/em\u003e reducing and antioxidant potential [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The reducing power of the mushroom extracts can be due to the ability of hydrogen donation which led to stabilizing the molecules [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn chelating power assay, again HPB extract of \u003cem\u003eAcacia nilotica\u003c/em\u003e showed highest chelating potential of 89.26% with IC\u003csub\u003e50\u003c/sub\u003e value of 130.86\u0026micro;g/ml than RJB (68.16% \u0026amp; IC\u003csub\u003e50\u003c/sub\u003e, 166.12\u0026micro;g/ml), PPB (81.15% \u0026amp; IC\u003csub\u003e50\u003c/sub\u003e, 145.35\u0026micro;g/ml) extracts (Spl. Figure\u0026nbsp;5). Spl. Figure\u0026nbsp;6 showed the \u003cem\u003eIn-vitro\u003c/em\u003e lipid peroxidation inhibition potential of ANL extracts and it was observed that likewise other assay, again HPB extract showed maximum inhibitory potential (92.15% with 112.56 \u0026micro;g/ml IC\u003csub\u003e50\u003c/sub\u003e value) as compared to other extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e i.e. RJB (78.19% \u0026amp; IC\u003csub\u003e50\u003c/sub\u003e166.24 \u0026micro;g/ml), PPB (86.62% \u0026amp; IC\u003csub\u003e50\u003c/sub\u003e, 138.62 \u0026micro;g/ml), at maximum highest tested concentration dose of 250 \u0026micro;g/ml. In Nutshell, it was observed from the results that HPB extract of \u003cem\u003eAcacia nilotica\u003c/em\u003e showed strong antioxidant potential then RJB \u0026amp; PPB. The present results also showed the linkage between antioxidant potential of extract and its phenolic content [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.6.2 In vitro Cytotoxicity analysis of ANL extracts:\u003c/h2\u003e\u003cp\u003eFive different cancer cell lines and one non-tumorigenic epithelial cell line from human bronchial epithelium (BEAS-2B) were selected for cytotoxicity analysis. The cell lines were treated with 4 mg/mL leaf extracts of ANL samples of diverse geographical regions. It was noticed that ANL extracts showed toxicity between 59\u0026ndash;91% on all various cell lines. The RJB extract exhibited more activity of 91% (MCF-7), 90% (A-549, 88% (PC-3), 86% (IGROV-1) \u0026amp; 77% (DU-145) as compared to PBB \u0026amp; HPB leaf extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To date, most chemotherapeutic agents do not distinguish between normal and cancer cell lines. Therefore it is important to discover plant extracts with selective cytotoxicity for cancer cells and having less damage to normal cells after cancer therapy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the cytotoxic effect of various extracts of ANL samples on cancer cells were compared with the control (without extract) and the non-tumorigenic normal cell line (BEAS-2B). The time and dose dependent cytotoxic potential of ANL samples were also investigated. The cytotoxic potential of ANL extracts of 100\u0026ndash;500\u0026micro;g/ml concentration at time interval of 24, 48, and 72 hr were studied and it was observed that level of cytotoxicity was proportional with the duration of exposure and concentration of different ANL extracts (Data not shown). Gordanian et al. (2012) reported that the cytotoxic activity of methanol extract from different organs of \u003cem\u003eA. absinthium\u003c/em\u003e at higher altitude was 20\u0026ndash;30% higher as compared with those of lower altitudes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previous studies showed that leaf extract of \u003cem\u003eA. absinthium\u003c/em\u003e from Iran at 0.1 mg/mL resulted in 30% cytotoxicity on MCF-7 cell lines, whereas we found 20% cytotoxicity on MCF-7 cells at the same concentration. However, this could be due to different geographic regions for plant origins and consequently an accumulation of different secondary metabolites.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCytotoxic potential of different ANL samples of diverse geographical regions at 100ug/ml concentration.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtract\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBEAS-2B\u003c/p\u003e\u003cp\u003e(Non-cancerous)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA-549 (Lung)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDU-145 (Prostate)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePC-3 (Prostate)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIGROV-1 (Ovary)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003cp\u003e(Breast)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRJB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e90\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e88\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e86\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e91\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePBB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHPB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdriamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNot Tested\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e84\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e69\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e80\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e81\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e79\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-FU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNot Tested\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e95\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e86\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.6.3 In-vitro antimicrobial potential ANL extracts:\u003c/h2\u003e\u003cp\u003eThe ANL extracts of different locations of northern states of India revealed low to moderate range of antimicrobial potential against different microorganisms. At 500 \u0026micro;g/ml (maximum tested concentration), HPB exhibited highest antimicrobial potential against \u003cem\u003eP. aeruginosa\u003c/em\u003e (2.61%), \u003cem\u003eS. aureus\u003c/em\u003e (2.89%), \u003cem\u003eS. pyogenes\u003c/em\u003e (2.97%), \u003cem\u003eC. albicans\u003c/em\u003e (3.11%) than other RJB \u0026amp; PBB extracts of \u003cem\u003eA. nilotica\u003c/em\u003e (Spl. Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It is evident from the results that extract concentration was linearly proportional to the inhibitory potential of the extracts. These extracts were further quantitatively assessed by minimum inhibitory concentration. MIC of HPB extract was found relatively less than extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e against \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus, S. pyogenes, C. albicans\u003c/em\u003e i.e. 112.4, 103.9, 101.5, 161.8\u0026micro;g/ml respectively (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results confirmed the presence of phenolic compounds having antimicrobial potential in ANL samples.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe minimum inhibitory concentration (\u0026micro;g/ml) of different ANL extracts.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMicroorganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMinimum Inhibitory Concentration (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRJB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePBB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHPB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e119.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e132.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e112.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e109.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e103.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e153.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e159.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e101.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e161.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e130.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e161.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.6.4 Analysis of apoptosis by caspase-3 assay:\u003c/h2\u003e\u003cp\u003eApoptosis is brought on by a sequence of caspase cleavages, which are serine proteases. Both internal and external apoptotic mechanisms involve the caspase cascade, a serial cleavage, with caspase-3 being the final protease to cause cell death. One reliable sign of apoptosis is the cleavage of the apoptotic substrate by activated caspase-3 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this work, the amount of active caspase-3 in the cell lysate of A-549 cells was determined following treatment with 500\u0026micro;g/ml of different ANL extracts from different geographical locations (Bikaner, Amritsar, and Palampur). Following 36 hours of treatment, it was shown that the cells had activated caspase-3, which was 4\u0026ndash;4.5 times greater in cells treated with ANL extracts than in cells that were not treated (data not shown).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e3.6.5 Anti-inflammatory potential of ANL extracts:\u003c/h2\u003e\u003cp\u003eThe enzyme COX-2 is necessary for the production of pro-inflammatory prostaglandins and thus has been a target for many cancer-preventive and anti-inflammatory drugs of rheumatoid arthritis, arteriosclerosis, myocarditis, infections, cancer, and other metabolic disorders. The several plant based natural products have been shown to transmit their anti-inflammatory activities through suppression of COX-2 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It was observed that various extracts (RJB, PBB \u0026amp; HPB) of \u003cem\u003eA. nilotica\u003c/em\u003e were found to be selective inhibitor of COX-2 (COX-2 selectivity\u0026thinsp;\u0026gt;\u0026thinsp;10) and inhibit COX-1 by 42.15%, 72.63% \u0026amp; 79.15% at 10 \u0026micro;M concentration respectively whereas COX-2 was inhibited by 71.45%, 77.22% \u0026amp; 81.56% respectively at same concentration i.e. 10 \u0026micro;M and it was noted that in the presence of ANL extracts, the level of PGE\u003csub\u003e2\u003c/sub\u003e dropped down (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The presence of polyphenols in plant extracts has already reported to block cyclooxygenase activity induced by UVB radiation. Thus it might also be implicated in suppression of cyclooxygenase-mediated inflammatory pathway [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCyclooxygenase enzyme mediated anti-inflammatory activities (COX-1 \u0026amp; COX-2) of ANL samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eExtract\u003c/p\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e% Inhibition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCOX-2 Selectivity*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eCOX-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCOX-1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCOX-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCOX-1\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRJB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e71.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePBB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e77.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e72.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHPB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e81.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e79.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRofecoxib**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e˜133\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCelecoxib **\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e˜10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e*COX-2 selectivity\u0026thinsp;=\u0026thinsp;IC\u003csub\u003e50\u003c/sub\u003e (COX-1)/ IC\u003csub\u003e50\u003c/sub\u003e (COX-2)\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e**Reported in literature (Kaur at el., 2009)\u003csup\u003e22\u003c/sup\u003e.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe HPLC-MS results in this study show the relative amounts of the main phytochemicals in different \u003cem\u003eAcacia nilotica\u003c/em\u003e extracts that were gathered from different parts of India's Rajasthan, Punjab, and Himachal Pradesh. In all \u003cem\u003eA. nilotica\u003c/em\u003e extracts, the phytochemical concentration was found to be proportionate to their biochemical and biological activity. The geographical characteristics and environmental factors influencing plant growth and development were found to be responsible for the maximum phytochemical content and biological activity in the ANL sample \"HPB.\" Additionally, it was found that while the phytochemicals in the different ANL samples were all members of the same species, their types differed in abundance depending on their varied geographic locations. The results suggested the fundamental role of the geographical properties and environmental variable in differentiating the phytochemical composition of the samples which was further accountable for the observed disparity in their biochemical and biological activities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest:\u003c/h2\u003e\u003cp\u003eAuthors declare not conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eNo external funding was received for the present study. The research was carried out using available institutional resources.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.K. collected plant material, performed the experimentation and Data analysis. S.K. validated and drafted manuscript. R.S. conceived idea, designed experiment and paper writing. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003e5. Acknowledgements\u003c/h2\u003e\u003cp\u003eAuthors are thankful to Khalsa College Charitable Society, Amritsar, Punjab, India for providing research grant (5666/10-11-2023) under seed money grant programme and other technical facilities to complete present study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its supplementary information files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePriya, S. 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Pharmacol.\u003c/em\u003e \u003cb\u003e160\u003c/b\u003e, 1714\u0026ndash;1726 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acacia nilotica, bioactivities, phenolic fingerprint, geographical variations, Metabolomics, chemometrics studies","lastPublishedDoi":"10.21203/rs.3.rs-8084390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8084390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAcacia nilotica\u003c/em\u003e is tropical tree with great nutritional values, being consumed for their promising health benefits and their aerial parts is used as fodder for animals. The current report is undertaken to analyze phytochemistry and \u003cem\u003ein vitro\u003c/em\u003e biological activities of leaf extracts of \u003cem\u003eAcacia nilotica\u003c/em\u003e (ANL samples) of diverse geographical locations i.e. Bikaner, Rajasthan (RJ), Amritsar, Punjab (PB) \u0026amp; Palampur, Himachal Pradesh (HP). The methanolic extract were prepared from composite samples of different locations and further checked for their qualitative biochemical, photosynthetic investigation and HPLC-MS based untargeted metabolomics. The ANL extracts were also checked for their biological potentials. It was observed that, though the quality of phytochemicals amongst various leaf extracts remained same but quantitatively, these extracts showed differences as observed through biochemical and metabolomics studies. The significant differences were observed in the various biological activities of the leaf extracts. The results showed a positive correlation establishment between climatic variation and phenolic enrichment of \u003cem\u003eAcacia nilotica\u003c/em\u003e plant which further affects the biological activities.\u003c/p\u003e","manuscriptTitle":"Biochemical \u0026amp; phytochemical insights of geographically diverse Acacia nilotica through HPLC–MS-based untargeted metabolomic studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 12:12:36","doi":"10.21203/rs.3.rs-8084390/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16cdf029-3dc1-441e-a6a9-f1dc89a6b14b","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58949283,"name":"Biological sciences/Biochemistry"},{"id":58949284,"name":"Biological sciences/Biological techniques"},{"id":58949285,"name":"Biological sciences/Biotechnology"},{"id":58949286,"name":"Biological sciences/Chemical biology"},{"id":58949287,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-12-12T16:53:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 12:12:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8084390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8084390","identity":"rs-8084390","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}