In Vitro Anti-Inflammatory, Anticancer Effects and Phytochemical Characterization of Organic Extracts from Aristolochia olivieri Colleg. Ex Boiss

In Vitro Anti-Inflammatory, Anticancer Effects and Phytochemical Characterization of Organic Extracts from Aristolochia olivieri Colleg. 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Ex Boiss Fuad O. Abdullah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4295072/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 Organic extract plays a crucial role in our lives, providing food and pharmaceuticals. Extracts from plants showed anti-inflammatory potential in an in vitro macrophage model, and their anticancer activities were assessed using real-time PCR and MTT assays. Major compounds in the n-hexane (AR-H) and the ethyl acetate (AR-E) Aristolochia olivieri extracts was n-hexadecanoic acid, and the major component of the methanol extract (AR-M) was pentacosane. The AR-M extract had a strong ability to induce mRNA expression of an inflammatory cytokine, IL-6, as an M1-like macrophage subset compared to the negative control (DMSO-treated cells). In contrast, AR-E treatment showed strong anti-inflammatory activity against macrophages. The AR-H extract had a moderate inflammatory effect against macrophages. The IC50 results of the anticancer assays ranged from 58.29 to 451.03 µg/mL for the three extracts. The anticancer action of the AR-E extract against U-87MG cells was higher (58.29 µg/mL) than that of AR-H and AR-M (156.38 and 196.14 µg/mL, respectively). The greater cytotoxicity effect observed with the AR-E extract against U-87MG can be linked to its high content of hexadecanoic acid (32.49%) and linolenic acid (12.90%). In this study to fully understand the therapeutic potential and uses as food to confirm safety. Aristolochia Olivieri Anti-inflammatory Anticancer Organic extract GC-MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Recently, there has been a significant increase in research on plant products, due to the use of these substances in pharmaceuticals and foods. The growing need for natural cosmetics, nutraceuticals, and functional foods has pushed academics and industry to explore new and natural sources of bioactive compounds, which can not only supplement conventional synthetic ingredients but also offer new health benefits (El Omari et al., 2019 ). Natural products, including phytocomplexes, have attracted increasing interest among food, cosmetic, and pharmaceutical industries (Abdullah, Hussain, Cucca, & Vidari, 2018 ; O Abdullah, 2021 ). The use of herbal remedies has been practiced in Iraqi Kurdistan since time immemorial. Evidence for this is the discovery of pollens of different medicinal plants, still growing in Kurdistan, in the plant samples of an approximately 60,000-year-old Neanderthal burial site at Shanidar IV cave in Northern Iraq (Solecki, 1975 ). The popularity of traditional medicines has increased in Kurdish society in the last 20 years, not only in rural areas but also in main towns where different medicinal plants are sold at local bazaars (Mati & de Boer, 2011 ). Indeed, the first choice for treating many diseases is the use of traditional plants (Abdullah et al., 2018 ; Abdullah, Hussain, Sardar, Vita-Finzi, & Vidari, 2016 ). This is due to medicinal use practices being a part of the country’s cultural heritage and the high cost of synthetic drugs, most of which are produced abroad and imported. However, despite the wide use of herbal remedies, a limited number of papers have been published in Kurdistan concerning the structures and bioactivities of certain specialized metabolites isolated from native plants that have been described in a recently published article (Abdullah, Hussain, Sardar, Gilardoni, Thu, et al., 2022). Aristolochia is a broad genus of over 500 species belonging to the Aristolochiaceae family. Plant extracts were investigated for their impact on cell proliferation and human embryonic fibroblasts to assess anticancer effects. Aristolochia is one of the best-known edible plants due to its long-established and widespread use. According to previous research on this plant genus, Aristolochia contains a rich variety of phytochemicals such as aristolochic acids, flavonoids, alkaloids, and lignans (Abdullah, Behrouzi, & Kaboudin, 2021 ; Micucci et al., 2023 ; Nandhini, Rajasekar, & Venmathi, 2017 ). Several members of the Aristolochia genus are utilized traditionally as anti-inflammatory, analgesic, anti-tumour, anti-obesity, and antitussive agents all over the world (Wiart, 2007 ). To the best of our knowledge, there are no GC-MS, anti-inflammatory, or anticancer activity studies on Aristolochia Olivieri. It is one of the medicinal plants used in Kurdistan and has traditionally been used to treat snake bites and inflammation. Therefore, we focused our attention on this plant species, performing GC-MS chemical characterization and biological activity assays of Aristolochia Olivieri organic extracts. The study will provide scientific validation for the roles of natural products (phytocomplexes) and their multiple potential applications in human health. 2. Materials and methods 2.1. Plant material collection The Aristolochia olivieri plant was collected in April 2023 from the Sakran Mountain, choman/Kurdistan region of northern Iraq. The voucher specimens, accession number ( 7705 ) were deposited in the Herbarium at Salahaddin University-Erbil, Iraq. The whole plants were air-dried separately under shade at room temperature (20–25°C). 2.2. Preparation of extracts using an ultrasonic method To prepare the AR-H extract, 50 g of the powder was suspended in n-hexane (3 X 200 mL). The suspension was placed into a conical flask and put in an ultrasonic device for 30 minutes with the temperature set to 40°C. The solution was then left for 30 minutes at room temperature. Subsequently, the mixture was filtered through filter paper. This procedure was repeated three times. The liquid was combined and the solvent was removed by a rotary evaporator to give a solid material (Ar-H, 0.418 g, 0.836% (w/w) dry material), which was stored in a vial at -5 ◦ C and protected from light. For the AR-E sample, the biomass was extracted with ethyl acetate (3 X 200 mL) in an ultrasonic bath for 30 minutes at 40°C and for 30 minutes at room temperature. Subsequently, the mixture was filtered. The process was repeated three times, each time using fresh solvent. The solvent was removed, leaving a solid residue (Ar-E, 0.480 g, 0.96% w/w dry material). Finally, the biomass was extracted three times with 200 mL methanol, following the procedure previously described. The AR-M extracts were then combined and, after methanol removal in a vacuum, the product was obtained (Ar-M, 2.986 g, 5.972% w/w dry material). The extract was stored in a bottle at -5 ◦ C until use (Abdullah, Hussain, Sardar, Gilardoni, Tosi, et al., 2022). 2.3. GC-MS analysis of phytochemical content The GC/GC-MS method was used to perform a qualitative analysis of the Aristolochia crude extracts AR-H, AR-E, and AR-M in order to identify their phytochemical components. Shimadzu Model QP-2010 GC and MS were used for the evaluation of the extracts. GC with a capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) and HP-5 MS (5% phenylmethyl siloxane) at a helium flow rate of 1.61 mL/minute at a temperature range of 60°C to 250°C for 10 minutes at a rate of 20°C/min were used. The electron energy and temperature of the ion source were kept at 70 eV and 250°C, respectively. Methanol was added to the extracts and 1 µL was injected through the column. By comparing the mass spectra of unknown compounds with the reference spectra in the Wiley GC/MS Library, Adams Library, and Mass Finder Library, the identity and exact molecular weight of each compound were determined (Jabbar, Abdullah, Abdulrahman, Galali, & Sardar, 2022 ; O Abdullah, K Abdulrahman, Galali, & Abdullah, 2022). 2.4. Anti-inflammatory activity 2.4.1. RNA extraction and cDNA synthesis Total populations of 2 × 10 6 J774.1A macrophages were pre-cultured in 6 well culture plates. Cells were treated for 24 hours with 100 ng/mL LPS (Sigma-Aldrich, USA) + 50 µg/mL IFN-γ (Biolegend, USA) to generate the M1 macrophages, or 100 µg/mL IL-4 (Biolegend, USA) to generate M2 macrophages. The next day, different concentrations of AR-H (15.62, 31.25, 62.5, 125, and 250 µg/mL), AR-E (7.81, 15.62, 31.25, 62.5, and 125 µg/mL), or AR-M (25, 50, 100, 200, and 400 µg/mL) extracts were introduced to the cells for an additional 24 hours. The LPS/IFN-γ and IL-4 treated cells were considered as M1 and M2 positive, respectively, while and 0.5% DMSO-treated J774.1A cells were considered as positive and negative controls, respectively. RNA extraction was performed using the phenol/chloroform method: Cells were re-suspended in 500 µL of TRIzol for 10 minutes followed by the addition of 200 µL of ice-cold chloroform. Then, cell debris was precipitated by centrifugation at 13,000 RPM for 12 min. 400 µL of aqueous supernatant was mixed with 400 µL of 70% ethanol followed by one-step centrifugation at 13000 RPM for 12 min, and washing with 70% ethanol. The air-dried purified total RNA was resuspended in DEPC-treated water followed by quantification with NanoDrop (Thermo Scientific, USA). A total RNA content of 2 µg was used for cDNA synthesis using a cDNA synthesis Kit (SMOBio Inc., Taiwan) within 60 minutes at 43 ºC using reverse transcriptase, RNase inhibitor, dNTP mix, and random hexamers (Rio, Ares, Hannon, & Nilsen, 2010 ). 2.4.2. Real-time PCR Real-time PCR was used to evaluate the expression levels of IL-6 and ARG1 as M1 and M2 biomarkers, respectively in comparison with the reference gene beta-actin. Accordingly, 5 pmol of each forward and reverse primer was mixed with no ROX 2x SYBR Green master mix (Amplicon Inc., Denmark) and 2 µL of cDNA sample in a total volume of 25 µL. The primer sequences were as follows: IL-6 F. primer: 5′-ACCTGTCTATACCACTTCAC-3′, R. primer: 5՜-GCATCATCGTTGTTCATAC-3′; ARG1 F. primer: 5′-AAGACAGGGCTCCTTTCAGG-3′, R. primer: 5′-AGCAAGCCAAGGTGAAAGCC-3′); and beta-actin (F. primer: 5′-CCAGGGTGTGATGGTGGGAATG-3′, R. primer: 5′-TGTAGAAGGTGTGGTGCCAGATC-3′). Real-time PCR was carried out using a Rotor-Gene Q real-time PCR cycler (Qiagen, USA) under the following program: one cycle at 95 ºC for 15 min, 40 cycles at 95 ºC for 30 s, 60 ºC for 30 s, and 72 ºC for 30 s. At the end of the amplification reaction, the melting curve was analysed from 65 to 95°C in 0.5°C increments at 5 s per step. The comparative Ct method (2 ΔΔCt method) was used to normalize the range of targeted mRNA level to that of the reference gene, and the relative expression of mRNA was evaluated (Orecchioni, Ghosheh, Pramod, & Ley, 2019 ). 2.5. Anticancer activity Cell viability tests were performed using five cell lines: U-87MG, hEF, HeLa, PC3, MCF-10A, and human embryonic fibroblast. The HeLa cells were grown in DMEM high glucose, while PC3, MCF-10A, and human embryonic fibroblasts were grown in RPMI1640, and U-87MG was grown in DMEM/HamsF12 medium supplemented with 10% fetal bovine serum (FBS), 1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO 2 . Cell survival was investigated by MTT assay (Jabbar et al., 2022 ). In summary, 1.0 × 10 4 cells were pre-cultured in each well of a 96-well plate and, after 16 h in the incubator, different concentrations of the complexes were introduced to the cells in fresh media within 72 h. After an appropriate time, fresh medium with MTT solution at a final concentration of 0.50 mg/mL was added to each well and incubated for an additional 4 h under the same conditions. Finally, a solvent buffer containing a culture medium was removed and 100 µL of 100% DMSO was used to dissolve the crystalline formazan. Then, the absorbance of the samples was read using a BMG Spectro Nano Elizabeth Reader at two wavelengths of 570 and 630 nm corresponding to the formazan and background absorbance, respectively. The following formula was used to calculate the percentage of live cells: Cell viability % = [A T (sample) / A T (control) ] × 100 Where the A T is defined as A 570 - A 630 . The IC 50 concentration was estimated as Mean ± Standard Deviation (STDEV) from three independent experiments using the GraphPad Prism 8 software. 3. Results and discussion 3.1. Phytochemical analysis The phytochemical qualitative study of extracts of Aristolochia n-hexane (AR-H), ethyl acetate (AR-E), and methanol (AR-M) extracts resulted in the identification of various chemicals as shown in Table 1 . The chemical profiling of the Aristolochia AR-H, AR-E, and AR-M extracts showed 26, 17, and 16 chemicals, respectively. The chemical profiling of AR-H extracts showed that n-hexadecanoic acid was the main compound in this extract (50.69%), while some compounds were detected in lower amounts, including eugenol (11.75%), oleic acid (5.18%), palmitaldehyde-diallyl acetal (4.98%), hexahydrofarnesyl acetone (4.51%), octadecanoic acid (4.34%) and neophytadiene (3.16%). The prevalent chemical compounds of AR-E extracts were n-hexadecanoic acid (32.49%), linolenic acid (12.90%), neophytadiene (10.8%), acetic acid-propyl ester (10.33%), isobutyl acetate (5.83%), eugenol (5.49%), and acetic acid-butyl ester (4.25%). The chemical profiling of AR-E extracts also revealed some phytochemicals in minor amounts including octadecanoic acid (3.14%), 4-vinylphenol (3.11%), docosane (3.02%), and a few others. The GC-MS investigation of AR-M extracts showed 16 compounds, including pentacosane (14.94%), n-hexadecanoic acid (14.77%), neophytadiene (14.61%), z-(13,14-epoxy) tetradec-11-en-1-ol acetate (10.35%), 5-(7-isopropyl-10-methyl-5-oxo-1,5-dithia-spiro [5.5] undec-2-yl)-pentanoic acid (9.18%), phytol (6.18%), and 3-eicosene (5.92%). Most of these compounds have been found to act as anticancer agents according to other studies (Karan & Erenler, 2018 ) and have been linked with antioxidant and anti-inflammatory activity; for example, hexadecanoic acids are saturated fatty acids that have been shown to act as effective antiradical and antitumour agents (J.-X. Zhou, Braun, Wetterauer, Wetterauer, & Wink, 2019 ). According to our data, the antitumour activity may be related to the presence of n-hexadecanoic acid (the major component of both AR-H and AR-E) and also the presence of pentacosane, a structurally related saturated hydrocarbon shown to be the main component of AR-M. Systematic Google and SciFinder searches did not find any earlier anticancer records for Aristolochia olivieri ; therefore, the present work is considered the first study to investigate the anticancer potential of Aristolochia olivieri . Table 1 Phytochemical profile of Aristolochia extracts using GC-MS PK RT (min) Components Name Similarity AR-H AR-E AR-M 1 6.116 Acetic acid, propyl ester 78 - 10.33 - 2 7.414 Isobutyl acetate 72 - 5.83 - 3 8.174 Hexanal 86 0.42 - - 4 8.539 Acetic acid, butyl ester 83 - 4.25 - 5 21.210 Nonanal 59 0.20 - - 6 27.589 4-vinylphenol 91 - 3.11 - 7 28.812 2-Decenal, (E)- 98 0.36 - - 8 33.058 Eugenol 98 11.75 5.49 - 9 37.453 2,6,10-Trimetyldecane 93 0.59 - - 10 40.122 γ-Muurolene 87 0.22 - - 11 40.768 Dihydroactinolide 97 - - 1.91 12 42.666 Spathulenol 99 0.37 13 42.874 Caryophyllene oxide 99 0.62 - 1.44 14 50.021 Loliolide 98 - 1.03 - 15 50.278 Tetradecyl pentafluoropropionate 94 - 0.72 - 16 51.784 Neophytadiene 99 3.16 10.8 14.61 17 51.976 Hexahydrofarnesyl acetone 99 4.51 - - 18 51.979 Benzyl (dideuterated) methyl ether 96 - 1.67 2.75 19 52.622 (Z)-1,3-Phytadiene 76 - - 3.41 20 52.639 Di-isobutyl phthalate 81 1.87 - - 21 54.016 2-Heptadecanone 81 0.45 - - 22 54.405 Cyclohexadecane 90 0.36 - - 23 54.599 Pentadecafluorooctanoic acid, octadecyl ester 38 1.71 - - 24 54.797 Hexadecanoic acid, methyl ester 98 0.66 - 3.01 25 56.268 n-Hexadecanoic acid 99 50.69 32.49 14.77 26 56.971 Phytol 99 1.92 1.93 6.18 27 57.022 Pentadecyl pentafluoropropionate 94 - 0.83 - 28 57.440 Z-(13,14-Epoxy) tetradec-11-en-1-ol acetate 46 - - 10.35 29 57.880 5-(7-Isopropyl-10-methyl-5-oxo-1,5-dithia-spiro[5.5]undec-2-yl)-pentanoic acid 41 - - 9.18 30 57.891 Celidoniol, deoxy 93 0.36 - - 31 58.034 8.beta.-(1'-Pentynyl)-3a,7a-diazatricyclo[7.7.3.3.0(3a,9a).0(4,7a)]dodecane 86 0.74 - - 32 59.789 Octatriacontyl pentafluoropropionate 87 1.08 - - 33 60.343 11-Octadecenoic acid, methyl ester 99 - - 3.15 34 60.366 9-(Tetrahydropyran-2-yloxy)-4,6-dioxatricyclo[5.3.1.0(3,8)]undecan-5-one 49 1.51 - - 35 60.537 Octadecane, 1-(ethenyloxy)- 64 - 0.52 - 36 60.686 tert-Butyl 8-Methyl-10-azabicyclo[4.3.1]deca-3,7-diene-10-carboxylate 90 0.45 - - 37 60.692 Octadecyl vinyl ether 64 - - 2.70 38 61.138 Octadecanoic acid, methyl ester 96 0.87 - - 39 61.138 Methyl stearate 95 - - 2.31 40 61.175 Oleic acid 99 5.18 - 3.37 41 61.606 Palmitaldehyde, diallyl acetal 53 4.98 - - 42 61.606 3-Eicosene, (E) 64 - - 5.92 43 61.720 Linolenic acid 99 - 12.90 - 44 62.378 Octadecanoic acid 99 4.34 3.14 - 45 62.606 Pentacosane 97 - - 14.94 46 62.618 Docosane 99 - 3.02 - 47 63.172 Cycloeicosane 95 - 0.40 - AR-H: Aristolochia Olivieri -hexane extract; AR-E: Aristolochia Olivieri- ethyl acetate extract; AR-M: Aristolochia Olivieri methanol extract. 3.2. Anti-inflammatory activity Inflammation is a natural immune system response that safeguards the body against injury or infection. However, prolonged inflammation can contribute to various health issues. Anti-inflammatory agents are substances designed to reduce inflammation within the body. The primary function of anti-inflammatory agents like nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and herbal remedies is to hinder the production or activity of pro-inflammatory molecules in the body, such as cytokines and prostaglandins. They also assist in regulating the immune response to prevent excessive inflammation. NSAIDs like ibuprofen and aspirin work by blocking enzymes such as cyclooxygenase (COX) involved in the production of inflammatory molecules. Corticosteroids mimic the effects of naturally occurring hormones to suppress inflammation. Herbal remedies such as curcumin in turmeric and 6-shogaol in ginger are known for their anti-inflammatory properties (Chen et al., 2018 ; Dinarello, 2010 ; X. Zhou et al., 2022 ). Using real-time PCR, the expression levels of IL-6 (M1 biomarker) and ARG1 (M2 biomarker) were assessed in relation to the reference gene beta-actin in an in vitro macrophage model. The results (Fig. 1 ) showed that the AR-M extract had a strong ability to induce mRNA expression of the inflammatory cytokine IL-6 (producing an M1-like macrophage phenotype) compared to the negative control. Also, the expression level of this pro-inflammatory cytokine was much greater than the LPS/IFN-γ induced group. However, the elevation was only statistically significant at a high concentration (400 µg/mL, p = 0.024). The low concentrations of the AR-H extract (15.62 and 31.25 µg/mL) had a significant suppressive effect on IL-6 mRNA expression compared to the M1 stimulated macrophage, indicating a shift from away from an M1-like macrophage phenotype towards that of the untreated control. The moderate concentration of AR-H extract shifted the macrophage population to an M1/M2-like mixture, while high dosage significantly induced IL-6. In contrast with other extracts, the AR-E extract showed strong anti-inflammatory activity. At concentrations greater than 15.62 µg/mL, the high expression levels of ARG1 (M2-like phenotype) increased the production of highly active M2-like anti-inflammatory macrophage populations (31.25, 62.5, and 125.0 µg/mL AR-E, with p values of 0.041, 0.026, and 0.0086, respectively). 3.3. Anticancer activity To investigate the potential anticancer activities of the phytochemicals identified in the extracts, the effect of AR-M, AR-H, and AR-E on the proliferation of U-87MG, HeLa, PC3, MCF-10A and hEF cell lines, and human embryonic fibroblast was measured and expressed as IC 50 values. All the analysed plant extracts had a dose-dependent anti-proliferative effect. The ells were cultured for 72 hours in the presence of each plant extract at various concentrations, and cell viability was measured by an MTT assay. Among the three extracts tested (Table 2 and Figs. 2 – 4 ), AR-M was the least effective at inhibiting the growth of HeLa, hEF, MCF-10A, PC3, and U87-MG cancer cells, with IC 50 values of 451.03, 423.76, 412.75, 386.57 and 196.14 µg/mL, respectively. This was followed by AR-H with IC 50 values of 403.73, 290.19, 274.64, 253.92, and 156.38 µg/mL in MCF-10A, HeLa, PC3, hEF, and U87-MG cells, respectively. Finally, AR-E most effectively inhibited cell growth with IC 50 values of 189.68, 127.95, 126.91, 96.26, and 58.29 µg/mL in HeLa, MCF-10A, hEF, PC3, and U87-MG cells, respectively. The IC 50 ranged between 58.29 to 451.03 µg/mL. Anticancer chemotherapeutic agents are measured by IC 50 which is the half-maximal (50%) inhibitory concentration. The most extensively used method for IC 50 measurements is the MTT assay (He et al., 2016 ). The anticancer action of AR-E extracts of Aristolochia olivieri against U87-MG was higher (58.29 µg/mL) than that for AR-H and AR-M (156.38 µg/mL and 196.14 µg/mL, respectively). This greater anticancer effect of AR-E extract against U87-MG may be linked to its high contents of certain constituents, namely n-hexadecanoic acid (32.49%) and linolenic acid (12.90%). The anticancer activities are probably also due to other active compounds available in the AR-H and AR-E extracts; other research studies have indicated that compounds like eugenol and other compounds identified in Aristolochia olivieri extracts demonstrate significant anticancer activity (Zari, Zari, & Hakeem, 2021 ). Table 2 Antiproliferation activities of Aristolochia olivieri extracts in five different cell lines (IC 50 , µg/mL) Samples U87-MG HeLa PC3 MCF-10A hEF AR-M 196.14 ± 7.53 451.03 ± 7.99 386.57 ± 23.12 412.75 ± 21.05 423.76 ± 23.44 AR-E 58.29 ± 5.62 189.68 ± 19.47 96.26 ± 9.35 127.95 ± 6.77 126.91 ± 7.21 AR-H 156.38 ± 12.01 290.19 ± 21.02 274.64 ± 18.94 403.73 ± 26.14 253.92 ± 13.98 Cisplatin 15.76 ± 3.44 13.94 ± 2.26 18.07 ± 1.97 22.57 ± 2.83 29.57 ± 1.26 AR-H: Aristolochia olivieri hexane extract; AR-E: Aristolochia olivieri ethyl acetate extract; AR-M: Aristolochia olivieri methanol extract; Cisplatin: as a positive control. 4. Conclusions In conclusion, the extract mixtures prepared from Aristolochia olivieri possessed anti-inflammatory and anticancer activities. Anti-inflammatory analysis demonstrated that, in comparison to the negative control, the AR-M extract had a strong ability to stimulate mRNA expression of the inflammatory cytokine, IL-6, as an M1-like macrophage subset. Furthermore, IL-6, a pro-inflammatory cytokine, is expressed at significantly higher levels than in the LPS/IFN-γ driven group; however, this elevation is only statistically significant at high concentrations (400 µg/mL, p = 0.024). Our data revealed that the AR-H, AR-E, and AR-M extracts of the plant Aristolochia olivieri have significant potential to reduce the proliferation of U-87MG, HeLa, PC3, MCF-10A cell lines, and hEF. Further study is needed to determine the toxicity and drawbacks of Aristolochia olivieri as a potential treatment for various human diseases, but the current research provides a basic knowledge of the phytochemical profile and bioactivity of Aristolochia olivieri . This information could be used for future biomedical analysis and biological study programs. Declarations None of the authors of this paper has any conflicts of interest Author contribution: Fuad O. A. prepared the original manuscript and experimental and data analysis. Data availability statement : Original contributions reflected in this work can be obtained from the article. Intellectual property rights: We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property. Funding: No funding was received for this work. Acknowledgments: The author would like to thank the Chemistry Department, College of Science at Salahaddin University-Erbil for their support and encouragement. Supplementary materials Not applicable References Abdullah FO, Behrouzi L, Kaboudin B (2021) A novel synthesis of highly stable palladium nanoparticles and their application in the reduction of nitroaromatic compounds. Mater Res Express 8(9):095002 Abdullah FO, Hussain FH, Cucca LI, Vidari G (2018) Phytochemical investigation and antioxidant effects of different solvent extracts of Pterocephalus nestorianus Nab. growing in Kurdistan Region-Iraq. Sci J Univ Zakho 6(1):21–25 Abdullah FO, Hussain FH, Sardar AS, Gilardoni G, Thu ZM, Vidari G (2022) Bio-Active compounds from Teucrium plants used in the traditional medicine of Kurdistan Region, Iraq. 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Molecules 26(23):7407 Zhou J-X, Braun MS, Wetterauer P, Wetterauer B, Wink M (2019) Antioxidant, cytotoxic, and antimicrobial activities of Glycyrrhiza glabra L., Paeonia lactiflora Pall., and Eriobotrya japonica (Thunb.) Lindl. extracts. Medicines 6(2):43 Zhou X, Afzal S, Wohlmuth H, Münch G, Leach D, Low M, Li CG (2022) Synergistic anti-inflammatory activity of ginger and turmeric extracts in inhibiting lipopolysaccharide and interferon-γ-induced proinflammatory mediators. Molecules 27(12):3877 Additional Declarations No competing interests reported. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4295072","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294119391,"identity":"07feb759-f4d7-44c5-9f44-20f1ea73f6d4","order_by":0,"name":"Fuad O. Abdullah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBAC9gYGBgkGhgOMDezMDcwMFWBBA6AAA2MDDi08B2BamIGI4QzJWhjbiNEikfzwBkPNHdkNhxkbPxfOq0tsYG/eJsFw5p4sbi1pxhYMx54ZA7U0S8/cdjixgedYmQTDjWJjXFrsJRLMJBjYDicCtTRI8247kNggkQMU+ZCQiNuW9G8SDP/AWpp/884BOkz+DSEtQDMZ28Ba2qR5G5iBtvAAtdzAo4XnTbFFYt8z45lALdY8xw4bt/GkFVsknEnA6Rce9vSNNz58uyPbd7z58G2emjrZfvbDQJFjCThDDAwSkDlsGCKjYBSMglEwCkgGAG3rX5grZE4dAAAAAElFTkSuQmCC","orcid":"","institution":"Salahaddin University-Erbil","correspondingAuthor":true,"prefix":"","firstName":"Fuad","middleName":"O.","lastName":"Abdullah","suffix":""}],"badges":[],"createdAt":"2024-04-19 21:16:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4295072/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4295072/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55286245,"identity":"5a1d231c-ba51-408a-b11d-9de067669af8","added_by":"auto","created_at":"2024-04-25 08:06:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74834,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of AR-M, AR-H, and AR-E extracts on IL-6 and ARG1 mRNA expression in J774.1A cells. J774.1A cells were induced with LPS/IFN-ɣ or IL-4 as positive controls for IL-6 and ARG1 expression, respectively, or with DMSO for the negative control. The Kruskal–Wallis one-way ANOVA statistical analysis was performed with the following significant levels: * pV\u0026lt;0.05; ** pV\u0026lt;0.01; *** pV\u0026lt;0.001, and **** pV\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/6c2e04dcb3d65921f89baabd.png"},{"id":55286249,"identity":"659689b3-c0ad-44b2-b274-e61be8c9e60b","added_by":"auto","created_at":"2024-04-25 08:06:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5636,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of AR-M extracts against cancer cells (MTT assay)\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/f735cb1744e0f39292e66501.png"},{"id":55286248,"identity":"82f74af2-0996-4327-94eb-314aa9ec6301","added_by":"auto","created_at":"2024-04-25 08:06:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5552,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of AR-E extracts against cancer cells (MTT assay)\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/a77d6413e7cb1d3c010f7a1d.png"},{"id":55286247,"identity":"2265f7ad-1a01-4915-848b-ffa0b7514fe7","added_by":"auto","created_at":"2024-04-25 08:06:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5554,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of AR-H extracts against cancer cells (MTT assay)\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/8781d78836dad963c43dfd63.png"},{"id":55286819,"identity":"40b15751-5a64-4244-9325-d44ebb8294d1","added_by":"auto","created_at":"2024-04-25 08:14:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5908,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of Cisplatin standard against cancer cells (MTT assay)\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/154be4d179d9b829e66ab48f.png"},{"id":55320037,"identity":"7b86f00b-dd41-428b-bc01-e7a5fb47ebda","added_by":"auto","created_at":"2024-04-25 16:04:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":801629,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4295072/v1/98faac7e-55c1-43fb-9cd2-604e519ab6c6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro Anti-Inflammatory, Anticancer Effects and Phytochemical Characterization of Organic Extracts from Aristolochia olivieri Colleg. Ex Boiss","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRecently, there has been a significant increase in research on plant products, due to the use of these substances in pharmaceuticals and foods. The growing need for natural cosmetics, nutraceuticals, and functional foods has pushed academics and industry to explore new and natural sources of bioactive compounds, which can not only supplement conventional synthetic ingredients but also offer new health benefits (El Omari et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Natural products, including phytocomplexes, have attracted increasing interest among food, cosmetic, and pharmaceutical industries (Abdullah, Hussain, Cucca, \u0026amp; Vidari, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; O Abdullah, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The use of herbal remedies has been practiced in Iraqi Kurdistan since time immemorial. Evidence for this is the discovery of pollens of different medicinal plants, still growing in Kurdistan, in the plant samples of an approximately 60,000-year-old Neanderthal burial site at Shanidar IV cave in Northern Iraq (Solecki, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). The popularity of traditional medicines has increased in Kurdish society in the last 20 years, not only in rural areas but also in main towns where different medicinal plants are sold at local bazaars (Mati \u0026amp; de Boer, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Indeed, the first choice for treating many diseases is the use of traditional plants (Abdullah et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Abdullah, Hussain, Sardar, Vita-Finzi, \u0026amp; Vidari, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This is due to medicinal use practices being a part of the country\u0026rsquo;s cultural heritage and the high cost of synthetic drugs, most of which are produced abroad and imported. However, despite the wide use of herbal remedies, a limited number of papers have been published in Kurdistan concerning the structures and bioactivities of certain specialized metabolites isolated from native plants that have been described in a recently published article (Abdullah, Hussain, Sardar, Gilardoni, Thu, et al., 2022). \u003cem\u003eAristolochia\u003c/em\u003e is a broad genus of over 500 species belonging to the Aristolochiaceae family. Plant extracts were investigated for their impact on cell proliferation and human embryonic fibroblasts to assess anticancer effects. \u003cem\u003eAristolochia\u003c/em\u003e is one of the best-known edible plants due to its long-established and widespread use. According to previous research on this plant genus, \u003cem\u003eAristolochia\u003c/em\u003e contains a rich variety of phytochemicals such as aristolochic acids, flavonoids, alkaloids, and lignans (Abdullah, Behrouzi, \u0026amp; Kaboudin, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Micucci et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nandhini, Rajasekar, \u0026amp; Venmathi, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Several members of the \u003cem\u003eAristolochia\u003c/em\u003e genus are utilized traditionally as anti-inflammatory, analgesic, anti-tumour, anti-obesity, and antitussive agents all over the world (Wiart, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, there are no GC-MS, anti-inflammatory, or anticancer activity studies on \u003cem\u003eAristolochia Olivieri.\u003c/em\u003e It is one of the medicinal plants used in Kurdistan and has traditionally been used to treat snake bites and inflammation. Therefore, we focused our attention on this plant species, performing GC-MS chemical characterization and biological activity assays of \u003cem\u003eAristolochia Olivieri\u003c/em\u003e organic extracts. The study will provide scientific validation for the roles of natural products (phytocomplexes) and their multiple potential applications in human health.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant material collection\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eAristolochia olivieri\u003c/em\u003e plant was collected in April 2023 from the Sakran Mountain, choman/Kurdistan region of northern Iraq. The voucher specimens, accession number (\u003cb\u003e7705\u003c/b\u003e) were deposited in the Herbarium at Salahaddin University-Erbil, Iraq. The whole plants were air-dried separately under shade at room temperature (20\u0026ndash;25\u0026deg;C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of extracts using an ultrasonic method\u003c/h2\u003e \u003cp\u003eTo prepare the AR-H extract, 50 g of the powder was suspended in n-hexane (3 X 200 mL). The suspension was placed into a conical flask and put in an ultrasonic device for 30 minutes with the temperature set to 40\u0026deg;C. The solution was then left for 30 minutes at room temperature. Subsequently, the mixture was filtered through filter paper. This procedure was repeated three times. The liquid was combined and the solvent was removed by a rotary evaporator to give a solid material (Ar-H, 0.418 g, 0.836% (w/w) dry material), which was stored in a vial at -5 \u003csup\u003e◦\u003c/sup\u003eC and protected from light. For the AR-E sample, the biomass was extracted with ethyl acetate (3 X 200 mL) in an ultrasonic bath for 30 minutes at 40\u0026deg;C and for 30 minutes at room temperature. Subsequently, the mixture was filtered. The process was repeated three times, each time using fresh solvent. The solvent was removed, leaving a solid residue (Ar-E, 0.480 g, 0.96% w/w dry material). Finally, the biomass was extracted three times with 200 mL methanol, following the procedure previously described. The AR-M extracts were then combined and, after methanol removal in a vacuum, the product was obtained (Ar-M, 2.986 g, 5.972% w/w dry material). The extract was stored in a bottle at -5 \u003csup\u003e◦\u003c/sup\u003eC until use (Abdullah, Hussain, Sardar, Gilardoni, Tosi, et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. GC-MS analysis of phytochemical content\u003c/h2\u003e \u003cp\u003eThe GC/GC-MS method was used to perform a qualitative analysis of the \u003cem\u003eAristolochia\u003c/em\u003e crude extracts AR-H, AR-E, and AR-M in order to identify their phytochemical components. Shimadzu Model QP-2010 GC and MS were used for the evaluation of the extracts. GC with a capillary column (30 m \u0026times; 0.25 mm i.d., film thickness 0.25 \u0026micro;m) and HP-5 MS (5% phenylmethyl siloxane) at a helium flow rate of 1.61 mL/minute at a temperature range of 60\u0026deg;C to 250\u0026deg;C for 10 minutes at a rate of 20\u0026deg;C/min were used. The electron energy and temperature of the ion source were kept at 70 eV and 250\u0026deg;C, respectively. Methanol was added to the extracts and 1 \u0026micro;L was injected through the column. By comparing the mass spectra of unknown compounds with the reference spectra in the Wiley GC/MS Library, Adams Library, and Mass Finder Library, the identity and exact molecular weight of each compound were determined (Jabbar, Abdullah, Abdulrahman, Galali, \u0026amp; Sardar, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; O Abdullah, K Abdulrahman, Galali, \u0026amp; Abdullah, 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Anti-inflammatory activity\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. RNA extraction and cDNA synthesis\u003c/h2\u003e \u003cp\u003eTotal populations of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e J774.1A macrophages were pre-cultured in 6 well culture plates. Cells were treated for 24 hours with 100 ng/mL LPS (Sigma-Aldrich, USA)\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL IFN-γ (Biolegend, USA) to generate the M1 macrophages, or 100 \u0026micro;g/mL IL-4 (Biolegend, USA) to generate M2 macrophages. The next day, different concentrations of AR-H (15.62, 31.25, 62.5, 125, and 250 \u0026micro;g/mL), AR-E (7.81, 15.62, 31.25, 62.5, and 125 \u0026micro;g/mL), or AR-M (25, 50, 100, 200, and 400 \u0026micro;g/mL) extracts were introduced to the cells for an additional 24 hours. The LPS/IFN-γ and IL-4 treated cells were considered as M1 and M2 positive, respectively, while and 0.5% DMSO-treated J774.1A cells were considered as positive and negative controls, respectively. RNA extraction was performed using the phenol/chloroform method: Cells were re-suspended in 500 \u0026micro;L of TRIzol for 10 minutes followed by the addition of 200 \u0026micro;L of ice-cold chloroform. Then, cell debris was precipitated by centrifugation at 13,000 RPM for 12 min. 400 \u0026micro;L of aqueous supernatant was mixed with 400 \u0026micro;L of 70% ethanol followed by one-step centrifugation at 13000 RPM for 12 min, and washing with 70% ethanol. The air-dried purified total RNA was resuspended in DEPC-treated water followed by quantification with NanoDrop (Thermo Scientific, USA). A total RNA content of 2 \u0026micro;g was used for cDNA synthesis using a cDNA synthesis Kit (SMOBio Inc., Taiwan) within 60 minutes at 43 \u0026ordm;C using reverse transcriptase, RNase inhibitor, dNTP mix, and random hexamers (Rio, Ares, Hannon, \u0026amp; Nilsen, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Real-time PCR\u003c/h2\u003e \u003cp\u003eReal-time PCR was used to evaluate the expression levels of IL-6 and ARG1 as M1 and M2 biomarkers, respectively in comparison with the reference gene beta-actin. Accordingly, 5 pmol of each forward and reverse primer was mixed with no ROX 2x SYBR Green master mix (Amplicon Inc., Denmark) and 2 \u0026micro;L of cDNA sample in a total volume of 25 \u0026micro;L. The primer sequences were as follows: IL-6 F. primer: 5\u0026prime;-ACCTGTCTATACCACTTCAC-3\u0026prime;, R. primer: 5՜-GCATCATCGTTGTTCATAC-3\u0026prime;; ARG1 F. primer: 5\u0026prime;-AAGACAGGGCTCCTTTCAGG-3\u0026prime;, R. primer: 5\u0026prime;-AGCAAGCCAAGGTGAAAGCC-3\u0026prime;); and beta-actin (F. primer: 5\u0026prime;-CCAGGGTGTGATGGTGGGAATG-3\u0026prime;, R. primer: 5\u0026prime;-TGTAGAAGGTGTGGTGCCAGATC-3\u0026prime;). Real-time PCR was carried out using a Rotor-Gene Q real-time PCR cycler (Qiagen, USA) under the following program: one cycle at 95 \u0026ordm;C for 15 min, 40 cycles at 95 \u0026ordm;C for 30 s, 60 \u0026ordm;C for 30 s, and 72 \u0026ordm;C for 30 s. At the end of the amplification reaction, the melting curve was analysed from 65 to 95\u0026deg;C in 0.5\u0026deg;C increments at 5 s per step. The comparative Ct method (2 \u003csup\u003eΔΔCt\u003c/sup\u003e method) was used to normalize the range of targeted mRNA level to that of the reference gene, and the relative expression of mRNA was evaluated (Orecchioni, Ghosheh, Pramod, \u0026amp; Ley, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Anticancer activity\u003c/h2\u003e \u003cp\u003eCell viability tests were performed using five cell lines: U-87MG, hEF, HeLa, PC3, MCF-10A, and human embryonic fibroblast. The HeLa cells were grown in DMEM high glucose, while PC3, MCF-10A, and human embryonic fibroblasts were grown in RPMI1640, and U-87MG was grown in DMEM/HamsF12 medium supplemented with 10% fetal bovine serum (FBS), 1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cell survival was investigated by MTT assay (Jabbar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In summary, 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were pre-cultured in each well of a 96-well plate and, after 16 h in the incubator, different concentrations of the complexes were introduced to the cells in fresh media within 72 h. After an appropriate time, fresh medium with MTT solution at a final concentration of 0.50 mg/mL was added to each well and incubated for an additional 4 h under the same conditions. Finally, a solvent buffer containing a culture medium was removed and 100 \u0026micro;L of 100% DMSO was used to dissolve the crystalline formazan. Then, the absorbance of the samples was read using a BMG Spectro Nano Elizabeth Reader at two wavelengths of 570 and 630 nm corresponding to the formazan and background absorbance, respectively. The following formula was used to calculate the percentage of live cells:\u003c/p\u003e \u003cp\u003eCell viability % = [A\u003csub\u003eT (sample)\u003c/sub\u003e / A\u003csub\u003eT (control)\u003c/sub\u003e] \u0026times; 100\u003c/p\u003e \u003cp\u003eWhere the A\u003csub\u003eT\u003c/sub\u003e is defined as A\u003csub\u003e570\u003c/sub\u003e - A\u003csub\u003e630\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e concentration was estimated as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation (STDEV) from three independent experiments using the GraphPad Prism 8 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Phytochemical analysis\u003c/h2\u003e \u003cp\u003eThe phytochemical qualitative study of extracts of \u003cem\u003eAristolochia\u003c/em\u003e n-hexane (AR-H), ethyl acetate (AR-E), and methanol (AR-M) extracts resulted in the identification of various chemicals as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The chemical profiling of the \u003cem\u003eAristolochia\u003c/em\u003e AR-H, AR-E, and AR-M extracts showed 26, 17, and 16 chemicals, respectively. The chemical profiling of AR-H extracts showed that n-hexadecanoic acid was the main compound in this extract (50.69%), while some compounds were detected in lower amounts, including eugenol (11.75%), oleic acid (5.18%), palmitaldehyde-diallyl acetal (4.98%), hexahydrofarnesyl acetone (4.51%), octadecanoic acid (4.34%) and neophytadiene (3.16%). The prevalent chemical compounds of AR-E extracts were n-hexadecanoic acid (32.49%), linolenic acid (12.90%), neophytadiene (10.8%), acetic acid-propyl ester (10.33%), isobutyl acetate (5.83%), eugenol (5.49%), and acetic acid-butyl ester (4.25%). The chemical profiling of AR-E extracts also revealed some phytochemicals in minor amounts including octadecanoic acid (3.14%), 4-vinylphenol (3.11%), docosane (3.02%), and a few others. The GC-MS investigation of AR-M extracts showed 16 compounds, including pentacosane (14.94%), n-hexadecanoic acid (14.77%), neophytadiene (14.61%), z-(13,14-epoxy) tetradec-11-en-1-ol acetate (10.35%), 5-(7-isopropyl-10-methyl-5-oxo-1,5-dithia-spiro [5.5] undec-2-yl)-pentanoic acid (9.18%), phytol (6.18%), and 3-eicosene (5.92%). Most of these compounds have been found to act as anticancer agents according to other studies (Karan \u0026amp; Erenler, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and have been linked with antioxidant and anti-inflammatory activity; for example, hexadecanoic acids are saturated fatty acids that have been shown to act as effective antiradical and antitumour agents (J.-X. Zhou, Braun, Wetterauer, Wetterauer, \u0026amp; Wink, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). According to our data, the antitumour activity may be related to the presence of n-hexadecanoic acid (the major component of both AR-H and AR-E) and also the presence of pentacosane, a structurally related saturated hydrocarbon shown to be the main component of AR-M. Systematic Google and SciFinder searches did not find any earlier anticancer records for \u003cem\u003eAristolochia olivieri\u003c/em\u003e; therefore, the present work is considered the first study to investigate the anticancer potential of \u003cem\u003eAristolochia olivieri\u003c/em\u003e.\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\u003ePhytochemical profile of \u003cem\u003eAristolochia\u003c/em\u003e extracts using GC-MS\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=\"char\" char=\".\" 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=\"char\" char=\".\" 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\u003ePK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComponents Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSimilarity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAR-H\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAR-E\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAR-M\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcetic acid, propyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsobutyl acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHexanal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcetic acid, butyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNonanal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-vinylphenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-Decenal, (E)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEugenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37.453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,6,10-Trimetyldecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eγ-Muurolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDihydroactinolide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpathulenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaryophyllene oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLoliolide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50.278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetradecyl pentafluoropropionate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51.784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeophytadiene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHexahydrofarnesyl acetone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51.979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBenzyl (dideuterated) methyl ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52.622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(Z)-1,3-Phytadiene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52.639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDi-isobutyl phthalate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-Heptadecanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyclohexadecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePentadecafluorooctanoic acid, octadecyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHexadecanoic acid, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56.268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en-Hexadecanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56.971\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhytol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePentadecyl pentafluoropropionate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57.440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZ-(13,14-Epoxy) tetradec-11-en-1-ol acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57.880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5-(7-Isopropyl-10-methyl-5-oxo-1,5-dithia-spiro[5.5]undec-2-yl)-pentanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57.891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCelidoniol, deoxy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.beta.-(1'-Pentynyl)-3a,7a-diazatricyclo[7.7.3.3.0(3a,9a).0(4,7a)]dodecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59.789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctatriacontyl pentafluoropropionate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11-Octadecenoic acid, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9-(Tetrahydropyran-2-yloxy)-4,6-dioxatricyclo[5.3.1.0(3,8)]undecan-5-one\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctadecane, 1-(ethenyloxy)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etert-Butyl 8-Methyl-10-azabicyclo[4.3.1]deca-3,7-diene-10-carboxylate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctadecyl vinyl ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctadecanoic acid, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethyl stearate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOleic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePalmitaldehyde, diallyl acetal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Eicosene, (E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinolenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctadecanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePentacosane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDocosane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63.172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCycloeicosane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\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 \u003cp\u003eAR-H: \u003cem\u003eAristolochia Olivieri\u003c/em\u003e-hexane extract; AR-E: \u003cem\u003eAristolochia Olivieri-\u003c/em\u003eethyl acetate extract; AR-M: \u003cem\u003eAristolochia Olivieri\u003c/em\u003e methanol extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Anti-inflammatory activity\u003c/h2\u003e \u003cp\u003eInflammation is a natural immune system response that safeguards the body against injury or infection. However, prolonged inflammation can contribute to various health issues. Anti-inflammatory agents are substances designed to reduce inflammation within the body. The primary function of anti-inflammatory agents like nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and herbal remedies is to hinder the production or activity of pro-inflammatory molecules in the body, such as cytokines and prostaglandins. They also assist in regulating the immune response to prevent excessive inflammation. NSAIDs like ibuprofen and aspirin work by blocking enzymes such as cyclooxygenase (COX) involved in the production of inflammatory molecules. Corticosteroids mimic the effects of naturally occurring hormones to suppress inflammation. Herbal remedies such as curcumin in turmeric and 6-shogaol in ginger are known for their anti-inflammatory properties (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dinarello, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; X. Zhou et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Using real-time PCR, the expression levels of \u003cem\u003eIL-6\u003c/em\u003e (M1 biomarker) and \u003cem\u003eARG1\u003c/em\u003e (M2 biomarker) were assessed in relation to the reference gene beta-actin in an in vitro macrophage model. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that the AR-M extract had a strong ability to induce mRNA expression of the inflammatory cytokine \u003cem\u003eIL-6\u003c/em\u003e (producing an M1-like macrophage phenotype) compared to the negative control. Also, the expression level of this pro-inflammatory cytokine was much greater than the LPS/IFN-γ induced group. However, the elevation was only statistically significant at a high concentration (400 \u0026micro;g/mL, p\u0026thinsp;=\u0026thinsp;0.024). The low concentrations of the AR-H extract (15.62 and 31.25 \u0026micro;g/mL) had a significant suppressive effect on \u003cem\u003eIL-6\u003c/em\u003e mRNA expression compared to the M1 stimulated macrophage, indicating a shift from away from an M1-like macrophage phenotype towards that of the untreated control. The moderate concentration of AR-H extract shifted the macrophage population to an M1/M2-like mixture, while high dosage significantly induced \u003cem\u003eIL-6.\u003c/em\u003e In contrast with other extracts, the AR-E extract showed strong anti-inflammatory activity. At concentrations greater than 15.62 \u0026micro;g/mL, the high expression levels of \u003cem\u003eARG1\u003c/em\u003e (M2-like phenotype) increased the production of highly active M2-like anti-inflammatory macrophage populations (31.25, 62.5, and 125.0 \u0026micro;g/mL AR-E, with p values of 0.041, 0.026, and 0.0086, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Anticancer activity\u003c/h2\u003e \u003cp\u003eTo investigate the potential anticancer activities of the phytochemicals identified in the extracts, the effect of AR-M, AR-H, and AR-E on the proliferation of U-87MG, HeLa, PC3, MCF-10A and hEF cell lines, and human embryonic fibroblast was measured and expressed as IC\u003csub\u003e50\u003c/sub\u003e values. All the analysed plant extracts had a dose-dependent anti-proliferative effect. The ells were cultured for 72 hours in the presence of each plant extract at various concentrations, and cell viability was measured by an MTT assay. Among the three extracts tested (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), AR-M was the least effective at inhibiting the growth of HeLa, hEF, MCF-10A, PC3, and U87-MG cancer cells, with IC\u003csub\u003e50\u003c/sub\u003e values of 451.03, 423.76, 412.75, 386.57 and 196.14 \u0026micro;g/mL, respectively. This was followed by AR-H with IC\u003csub\u003e50\u003c/sub\u003e values of 403.73, 290.19, 274.64, 253.92, and 156.38 \u0026micro;g/mL in MCF-10A, HeLa, PC3, hEF, and U87-MG cells, respectively. Finally, AR-E most effectively inhibited cell growth with IC\u003csub\u003e50\u003c/sub\u003e values of 189.68, 127.95, 126.91, 96.26, and 58.29 \u0026micro;g/mL in HeLa, MCF-10A, hEF, PC3, and U87-MG cells, respectively. The IC\u003csub\u003e50\u003c/sub\u003e ranged between 58.29 to 451.03 \u0026micro;g/mL. Anticancer chemotherapeutic agents are measured by IC\u003csub\u003e50\u003c/sub\u003e which is the half-maximal (50%) inhibitory concentration. The most extensively used method for IC\u003csub\u003e50\u003c/sub\u003e measurements is the MTT assay (He et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The anticancer action of AR-E extracts of \u003cem\u003eAristolochia olivieri\u003c/em\u003e against U87-MG was higher (58.29 \u0026micro;g/mL) than that for AR-H and AR-M (156.38 \u0026micro;g/mL and 196.14 \u0026micro;g/mL, respectively). This greater anticancer effect of AR-E extract against U87-MG may be linked to its high contents of certain constituents, namely n-hexadecanoic acid (32.49%) and linolenic acid (12.90%). The anticancer activities are probably also due to other active compounds available in the AR-H and AR-E extracts; other research studies have indicated that compounds like eugenol and other compounds identified in \u003cem\u003eAristolochia olivieri\u003c/em\u003e extracts demonstrate significant anticancer activity (Zari, Zari, \u0026amp; Hakeem, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\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\u003eAntiproliferation activities of \u003cem\u003eAristolochia olivieri\u003c/em\u003e extracts in five different cell lines (IC\u003csub\u003e50\u003c/sub\u003e, \u0026micro;g/mL)\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=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eU87-MG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHeLa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePC3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMCF-10A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ehEF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAR-M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e196.14\u0026thinsp;\u0026plusmn;\u0026thinsp;7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e451.03\u0026thinsp;\u0026plusmn;\u0026thinsp;7.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e386.57\u0026thinsp;\u0026plusmn;\u0026thinsp;23.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e412.75\u0026thinsp;\u0026plusmn;\u0026thinsp;21.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e423.76\u0026thinsp;\u0026plusmn;\u0026thinsp;23.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAR-E\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e58.29\u0026thinsp;\u0026plusmn;\u0026thinsp;5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e189.68\u0026thinsp;\u0026plusmn;\u0026thinsp;19.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e96.26\u0026thinsp;\u0026plusmn;\u0026thinsp;9.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e127.95\u0026thinsp;\u0026plusmn;\u0026thinsp;6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e126.91\u0026thinsp;\u0026plusmn;\u0026thinsp;7.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAR-H\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e156.38\u0026thinsp;\u0026plusmn;\u0026thinsp;12.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e290.19\u0026thinsp;\u0026plusmn;\u0026thinsp;21.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e274.64\u0026thinsp;\u0026plusmn;\u0026thinsp;18.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e403.73\u0026thinsp;\u0026plusmn;\u0026thinsp;26.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e253.92\u0026thinsp;\u0026plusmn;\u0026thinsp;13.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCisplatin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e22.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\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\u003eAR-H: \u003cem\u003eAristolochia olivieri\u003c/em\u003e hexane extract; AR-E: \u003cem\u003eAristolochia olivieri\u003c/em\u003e ethyl acetate extract; AR-M: \u003cem\u003eAristolochia olivieri\u003c/em\u003e methanol extract; Cisplatin: as a positive control.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn conclusion, the extract mixtures prepared from \u003cem\u003eAristolochia olivieri\u003c/em\u003e possessed anti-inflammatory and anticancer activities. Anti-inflammatory analysis demonstrated that, in comparison to the negative control, the AR-M extract had a strong ability to stimulate mRNA expression of the inflammatory cytokine, IL-6, as an M1-like macrophage subset. Furthermore, IL-6, a pro-inflammatory cytokine, is expressed at significantly higher levels than in the LPS/IFN-γ driven group; however, this elevation is only statistically significant at high concentrations (400 \u0026micro;g/mL, p\u0026thinsp;=\u0026thinsp;0.024). Our data revealed that the AR-H, AR-E, and AR-M extracts of the plant \u003cem\u003eAristolochia olivieri\u003c/em\u003e have significant potential to reduce the proliferation of U-87MG, HeLa, PC3, MCF-10A cell lines, and hEF. Further study is needed to determine the toxicity and drawbacks of \u003cem\u003eAristolochia olivieri\u003c/em\u003e as a potential treatment for various human diseases, but the current research provides a basic knowledge of the phytochemical profile and bioactivity of \u003cem\u003eAristolochia olivieri\u003c/em\u003e. This information could be used for future biomedical analysis and biological study programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eNone of the authors of this paper has any conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e Fuad O. A. prepared the original manuscript and experimental and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e: Original contributions reflected in this work can be obtained from the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntellectual property rights:\u003c/strong\u003e We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e No funding was received for this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The author would like to thank the Chemistry Department, College of Science at Salahaddin University-Erbil for their support and encouragement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdullah FO, Behrouzi L, Kaboudin B (2021) A novel synthesis of highly stable palladium nanoparticles and their application in the reduction of nitroaromatic compounds. 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Molecules 27(12):3877\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":"Aristolochia Olivieri, Anti-inflammatory, Anticancer, Organic extract, GC-MS","lastPublishedDoi":"10.21203/rs.3.rs-4295072/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4295072/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrganic extract plays a crucial role in our lives, providing food and pharmaceuticals. Extracts from plants showed anti-inflammatory potential in an in vitro macrophage model, and their anticancer activities were assessed using real-time PCR and MTT assays. Major compounds in the n-hexane (AR-H) and the ethyl acetate (AR-E) \u003cem\u003eAristolochia olivieri\u003c/em\u003e extracts was n-hexadecanoic acid, and the major component of the methanol extract (AR-M) was pentacosane. The AR-M extract had a strong ability to induce mRNA expression of an inflammatory cytokine, IL-6, as an M1-like macrophage subset compared to the negative control (DMSO-treated cells). In contrast, AR-E treatment showed strong anti-inflammatory activity against macrophages. The AR-H extract had a moderate inflammatory effect against macrophages. The IC50 results of the anticancer assays ranged from 58.29 to 451.03 \u0026micro;g/mL for the three extracts. The anticancer action of the AR-E extract against U-87MG cells was higher (58.29 \u0026micro;g/mL) than that of AR-H and AR-M (156.38 and 196.14 \u0026micro;g/mL, respectively). The greater cytotoxicity effect observed with the AR-E extract against U-87MG can be linked to its high content of hexadecanoic acid (32.49%) and linolenic acid (12.90%). In this study to fully understand the therapeutic potential and uses as food to confirm safety.\u003c/p\u003e","manuscriptTitle":"In Vitro Anti-Inflammatory, Anticancer Effects and Phytochemical Characterization of Organic Extracts from Aristolochia olivieri Colleg. Ex Boiss","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 08:06:07","doi":"10.21203/rs.3.rs-4295072/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":"fde1cbef-4f34-4d06-a02b-eb7848dce40e","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-25T15:57:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 08:06:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4295072","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4295072","identity":"rs-4295072","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}