Antibacterial and Anticancer Efficacy of Different Parts of Pistacia Integerrima Plant Extracts

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Bhatti, Jamshed Iqbal, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-396639/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 This study explored the antibacterial efficacy of Pistacia integerrima gall, leaf and bark in different solvents. Extracts of these parts prepared in ethanol, methanol and distilled water by using rotary evaporator. To check the antibacterial potential of this plant, minimal inhibitory concentrations of each extract analysed by agar well diffusion method against Staphylococcus aureus, E. coli, Proteus vulgaris, Bacillus subtilis and Pseudomonas aeruginosa . For anticancer activity, hexane, chloroform and ethyl acetate fractions of P. integerrima gall, leaf and bark used against human cervical cancer (HeLa) and baby hamster kidney (BHK–21) cell lines. Results showed that maximum zone of inhibition 25mm formed with ethanolic gall extract in 200µL concentration against B. subtilis. P. vulgaris shows resistance to methanol and aqueous extracts but inhibited with ethanolic leaf extract. Among different parts of P. integerrima , n-hexane leaf fraction shown to be most effective against HeLa cell lines with IC 50 of 7.45 µg/mL. In case of BHK-21, highest cell inhibition of 46.8% observed with crude leaf extract than ethyl-acetate bark extract (44.9%) of P. integerrima . It is concluded that the effective inhibitor was hexane leaf fraction against HeLa cell lines in which Heneicosane was found (39.7%) which might be responsible for anticancer activity. Cancer Biology General Microbiology Pistacia integerrima antibacterial activity gall HeLa cell lines GC-MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Medicinal plants have been found effective against many human pathogens, recently natural products are gaining attentions due to antibiotic resistance of different bacteria and more side effects of synthetic antibiotics. World Health Organization has considered natural herbs as the best source of medicine formulation 1 . P. integerrima an important medicinal plant of family Anacardiaceaee, native in Asia and commonly present in East Afghanistan, Pakistan and North-West and West Himalaya to Kumaon growing at a height of 800-1900m 2 . The common name of this medicinally important plant are crab’s claws and zebrawood, beside other parts of this plant, gall (Fig. 1) is extensively used for the treatment of cough, liver problem, allergy and snake bite 3 . P. integerrima is well distinct due to presence of galls on leaves and petioles. Galls are the store houses of secondary metabolites which has significance in Indian old medicine 4 . The galls of P. integerrima are utilized for the cure of lungs diseases, allergy, dysentery, hiccough, stomach problems, fever, problem associated with nervous system, skin diseases, leucorrhoea, and general debility. One of the good sources of antioxidant in P. integerrima is the presence of its phenolic compounds. For the treatment of jaundice, dysentery, chronic injuries and for the treatment of breast cancer cell line MCF-7, ethnobotanically significant species P. integerrima stew.ex Brand was traditionally used. The crude extract and different segment of P. integerrima were tested for cytotoxic activity in Michigan Cancer Foundation, against 7 human breast cancer cell line. The crude methanol extract exhibited week antifungal activity but good antitumor (IC 50 125ppm) activity and could be a source of novel biologically active compounds 5 . Antibacterial, antioxidant and cytotoxicity of different parts of this plant is due to its chemical compounds mainly terpenoids, saponin, phenols and sterols which are released by the plants as secondary metabolites 3 . Secondary metabolites are produce as defensive mechanism against disease and environmental stress. Theses metabolites are found in the different parts stem, leaves, roots and bark of the plant 6 . In the present study the potential of P. integerrima plant extract was analyzed to confirm its ethnomedicinal importance against HeLa and BHK-21 cell lines. Some important bacteria of human concern are used in this study including P. aeruginosa which is a worldwide public health threat due to its resistant strains and hospital acquired infections 7 . E. coli and Proteus vulgaris are used which is present in the human gastrointestinal tract and responsible for urinary tract infections 8 . Beside these bacteria, one spore former B. subtilis was also used which is non-pathogenic but its spore are highly resistant and involved in food spoilage. Materials And Methods Extraction of plant materials for antibacterial activity. P. integerrima leaves, barks, gall were collected from local market and first shade dried and then grinded in Mortar and pestle. Plant material was collected, stored and processed following standard protocols by WHO and institutional, national, and international guidelines and legislations, therefore no risks were associated in any steps of handling to disposal. For extraction purpose cold maceration technique was used 16 . Powdered plant material 10gm was individually macerated in 100mL of distilled H 2 O, methanol and ethanol and kept for 24 hours on a rotary shaker. These extracts then filtered 4-5 times through Whatman filter paper till all soluble compounds were completely extracted. Then the evaporation of extract was carried out by using rotary evaporator (Buchi Rotavapor R-300, Japan) and dried residues were used to check the antibacterial efficacy. Bacterial culture collection. Different bacterial pathogens were obtained from Ayub Medical Complex lab Abbottabad including Staphylococcus aureus, E. coli, Proteus vulgaris, Bacillus subtilis, Pseudomonas aeruginosa. Liquid inoculums were prepared by adding pure bacterial strains in 10mL normal saline and used further. Agar well diffusion method used to evaluate the antibacterial efficacy of the different parts of plant. Mueller Hinton agar (Oxoid) plate surface was inoculated by spreading 50 µL inoculums, McFarland 0.5 BaSO 4 standard used for balancing the turbidity of used inoculum as 10 8 CFU mL -1 17 . Then 8mm borer used to make 5 holes aseptically on agar plate. In 4 wells 50 µL of leaf, bark and gall extracts (in concentration of 200, 150, 100, and 50µL) poured. For negative control pure dimethyl sulfoxide (DMSO) poured in one agar well and disc of Ciprofloxacin taken as positive control in the center of plate and all agar plates were incubated for 24 hours at temperature 37 o C 18 . The inhibitory effects measured by the zones of inhibition in mm around the wells. Extraction and fractionation of plant materials for anticancer activity. Shade dried leaves, galls and barks of P. integerrima soaked with methanol for one week at room temperature. By using rotary evaporator, methanolic extract of each portion of plant was evaporated at 40 o C temperature. Then for the process of liquid-liquid partitioned in separatory funnel, the crude methanolic extract of each portion of plant was suspended in water and fractionated with n-hexane, chloroform, ethyl acetate in sequenced manner, to get their respective fractions 13 . All standard procedures were adopted and experiment performed in fume hood 16 . Amount of 10mg of each crude and fractionated extracts of P. integerrima bark, gall and leaves in hexane, chloroform and ethyl acetate carefully weighed in 1.5ml Eppendorf tubes and dissolved in DMSO. After vortex mixing, 1 mg of each of the crude extract and fraction was used in cancer cell culture. Anticancer activity through cell viability assay. Plant extracts along with their subsequent fractions evaluated for their anti-proliferative activity against HeLa and BHK-21 by utilizing the dimethyl–2–thiazolyl–2,5–diphenyl–2 H –tetrazolium bromide (MTT) based cell metabolic assay 19, 20 . After sub-culturing, grown cell colonies were harvested using proteolytic trypsin enzyme. The cellular content along with sufficient culture media was transferred to a 15 ml conical tube and centrifuged for 5 minutes at 150 speed and 37°C. After careful removal of supernatant, cell pellet dissolved in 1ml culture media. Cells counted by pipetting out a volume of 10 µL from cell suspension and dropped onto Neubauer counting chamber to count cells under microscope. Cell suspension diluted accordingly to make 2.5x10 4 cells mL -1 , from this suspension with the help of a multi-channel micro-pipette 90 μL added in each well of 96-wells microliter plate and left for 24 hours incubation in a cell culture incubator set at 5% CO 2 and 37˚C. Each plant extracted sample poured into these wells at 100 µg/mL of final concentration and 10 μL of media was used in control well. Carboplatin at a final concentration of 100 µM was used as a standard for cell lines. For IC 50 determination, three-fold dilutions used and 10 μL of MTT (5mg/mL PBS) after 24 hours placed in all wells. After incubation of 4 hours in a cell culture incubator 100 µL of stopping reagent (isopropanol and 10% sodium dodecyl sulfate) poured to solubilize formazan crystals and kept at room temperature with subsequent agitation for half an hour. Absorbance recorded using a 96–well microtiter plate reader (Bio–TekELx 800 TM ) at 570 nm, background signal (690 nm) was subtracted and results were evaluated as inhibition values percentage. Analysis of active ingredients by GC-MS. Identification and separation of various extracts of plant were carried out on Perkin Elmer Clarus 600 gas chromatograph (GC) connected with mass spectrometer (MS). Plants extracts dissolved in respective organic solvents with concentration of 5mg.mL -1 and 1µL was injected to GC-MS and protocol was adopted as described by Shah et al ., (2016) 21 . Different separated compounds identified by comparing their mass spectra to the Finnigan NIST-05 (National Institute of Standard and Technology, USA) Mass spectrum library and available literature. Relative concentration of each identified compound found in injected sample was determined by comparing peak area of the compound to the sum of all peaks’ area in a total ion current (TIC) chromatogram. Statistical Analysis. Two factor ANOVA with replication applied by using Data analysis Tools of MS Excel 2010. It was used for the analysis of significance difference (at p > 0.05) among zone of inhibition by using different doses and different types of solvent extracts. PRISM 5 software used for the determination of IC 50 value. Results The present research work conducted to determine the antimicrobial and cytotoxic potential of medicinal plant P. integerrima (leaf, gall and bark) extracts. The antibacterial activity of P. integerrima extracts were applied on five important bacteria of human concern with different extracts concentrations. Antibacterial activity of Pistacia integerrima in different solvents In case of P. integerrima gall extract it was observed that with increase in different extracts concentration, inhibition zone was also increased. All solvent’s extracts showed effectiveness against all bacterial species, except for aqueous gall extract which was ineffective against E. coli, P. aeruginosa and Proteus vulgaris. Gall extract was less effective against Proteus (Gram-negative) and more against Bacillus (Gram-positive). Maximum zone of inhibition 25mm was formed with ethanolic gall extract in 200µL concentration against B. subtilis (Table 1). In the present study, the probability values by using two factor ANOVA with replication was less than α value of 0.05 (p < 0.05) which shows that significance difference exist between zone of inhibition of different bacterial species and different solvents extract of various concentrations. Methanol leaf extract was found more effective than ethanolic leaf extract, maximum zone of inhibition 22mm against Bacillus subtilis was observed with 200 µL concentration (Table 2). P. vulgaris shows resistance to methanol and aqueous extracts but inhibited with ethanolic leaf extract with 10mm maximum zone of inhibition. Aqueous leaf extracts found ineffective against all except Bacillus subtilis . P. integerrima methanolic bark extract was found more effective against all 4 bacterial species than ethanolic and aqueous extract. Highest and similar zone of inhibition of 19mm were found against Bacillus subtilis and Proteus vulgaris with 200 µL concentration. Ethanolic bark extract was effective against E.coli, P. aeruginosa and Proteus vulgaris while B. subtilis and S. aureus showed resistant (Table 3) . Overall gall extracts has more antibacterial potential than other parts of this plant. While Bacillus subtilis was found more susceptible than other bacteria with different parts of plant extract except ethanolic and aqueous bark extracts which showed less effectiveness. Anticancer activity When cytotoxic assay of fractions and extracts of different parts (gall, leaf, bark) of P. integerrima were carried out it showed profound cytotoxic effect against HeLA and BHK-21 cell line. Among different parts of P. integerrima , n-hexane leaf fraction was shown to be most effective against HeLa cell lines with IC 50 of 7.45 µg/mL than chloroform (IC 50 of 4.82 µg/mL) and other fractions (Fig. 2). In this n-hexane leaf fraction, Heneicosane was found (39.7%) as an active compound (Table 4) and responsible for anticancer activity, Crude extract of P. integerrima bark part has showed IC 50 of 4.77 µg/mL almost similar with chloroform leaf extract. In case of BHK-21, highest cell inhibition of 46.8% was observed with crude leaf extract than ethyl-acetate bark extract (44.9%) of P. integerrima (Fig. 3). Overall leaf extract has showed more potential against cancer cell than other parts. Gas chromatography-mass spectrometry (GC-MS) analysis of P. integerrima GC-MS analysis of different parts of plant extracts showed that leaf extract of P. integerrima contained greater number of compounds compared to other parts of the plant (Table 4). In hexane gall extract Heneicosane found 39.7% (Fig.4). Main component in chloroform gall extract was 55% D-Limonene and hexane was 1-Pentacontanol 74%. Chloroform leaf extract contain Heptacosane,1-chlor 50.5% an active ingredient (Fig. 5). Chloroform bark extract contain Nonadecane 42.5% and Dotriacontane, 1-iodo- 41.7% as active compound (Fig. 6) (Table 4). Discussion In the present study P. integerrima showed more effectiveness against Gram positive bacteria than Gram negative. Similarly one study evaluated P. integerrima activity of gall extracts (aqueous and ethanol) against S. aureus, E. coli, B. cereus, P. aeruginosa and K. pneumoniae . Among these bacterial species, both ethanolic and aqueous extract inhibited the Gram-positive bacteria better than the Gram-negative bacteria 9 . Bacillus subtilis is Gram-positive spore forming bacteria causes food spoilage and its spores are very hard to kill but P. integerrima has successfully inhibited its growth. With 200 µL concentration of methanolic bark extract, highest and similar zone of inhibition found against Bacillus subtilis and Proteus vulgaris . Previously Proteus mirablis was found to be less susceptible than E. coli, Bacillus subtilis and Staphylococcus aureus 10 . Proteus vulgaris has clinically importance and responsible for urinary tract infection, bacteremia and brain abscesses and enter in the human body through intestinal tract 11 and resistant to most of antibiotics 8 . In the present study n-hexane leaf fraction of P. integerrima found more lethal against HeLa cell lines than other parts of the plant, similar finding was observed by Ahmad et al., (2006) 12 . While in contrast with Uddin et al., 2013 who also conducted study to determine the anticancer efficacy of different parts of P. integerrima fractions against brine shrimp Artemia salina and found gall of this plant has more cytotoxic potential than other parts of the plant 13 . Heneicosane found in higher concentration in n-hexane leaf fraction which might be associated with higher anticancer activity with this extract, previously this compound was found as active component in the flowers of Carthamus tinctorius which showed antioxidant property to protect human bone cells 14 . Overall chloroform leaf extract showed highest anticancer activity, in which Heptacosane, 1-chlor was found as an active ingredient and this compound showed anticancer activity in Achyranthes aspera L weed 15 . Conclusion The results showed that solvents extract has profound antibacterial activity than aqueous extract against tested pathogens except Bacillus subtilis and Staphylococcus aureus . Methanolic and ethanolic extracts of P. integerrima gall has showed more significant inhibition effect than leaf and bark. The most potent inhibitor was hexane leaf fraction against HeLa cell lines, in this Heneicosane was found (39.7%) which might be responsible for anticancer activity. Declarations Author contributions W.R. performed all experiments, F.M. has supervised and planned research design, Z.A.B. helped in manuscript writing, J.I. helped and provided lab facility for anticancer activity, M.F.S. did statistical analysis, S.P. helped in making graphs and figures, U.K. helped in GC-MS experiment and interpretation, I.K. helped in anticancer activity experiment. All authors reviewed the manuscript before submission. Funding No funding is available for open access. Competing interests The authors declare no competing interests. References Mohammad, G. J., Jawad, M. & Hameed, I. H. Proteus species: Characterization and herbal antibacterial: A Review. Inter. J. of Pharmaco. Phytochem. Res. 8 (11), 1844–1854 (2016). Pant, S. & Samant, S. S. Ethnobotanical observations in the Mornaula reserve forest of Komoun, West Himalaya, India. Ethnobot. Leaflet. 14 , 193–217 (2010). Bibi, Y., Zia, M. & Qayyum, A. An overview of Pistacia integerrima a medicinal plant species: Ethnobotany, biological activities and phytochemistry. Pak. J. Pharm. Sci. 28 (3), 1009–1013 (2015). Chopra, R., Chopra, I., Handa, K. & Kapoor, L. Indigenous Drugs of India 2(nd Edn (Academic Publishers, India, 2006). Bibi, Y., Nisa, S., Chaudhary, F. M. & Zia, M. Antibacterial activity of some selected medicinal plants of Pakistan. BMC Complement. Altern. Med. 11, 52 (2011). Delgoda, R. & Murray, J. E. Evolutionary perspectives on the role of plant secondary metabolites. In: Badal S, Delgoda R, editor(s). Pharmacognosy Fundamentals, Applications and Strategies, Academic Press 93–100(2017). Fata, M. M., Shirin, G. & Rehm, B. H. A. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front. Cell. Infec. Micro. 7 , 39 (2017). Armbruster, C. E. & Mobley, H. L. T. Proteus species (Website: http://www.antimicrobe.org/b226.asp ), accessed on January 3, 2020. Ramachandra, Y. L., Ravi, S. B. E., Sujan, G. S. & Sundar, R. S. In-vitro antimicrobial activity of Pistaacia integerrima leaf gall extracts. Pharmacopho. 1 (2), 149–154 (2010). Ahmad, R., Pieters, L., Rehman, N. & Riaz, M. Antimicrobial and antioxidant activity of crude extracts of two medicinal plants P. Integerrima and Debregeasia salicifolia. Int. J. Pharm. Sci. Rev. Res. 18 (1), 13–17 (2013). Bloch, J. et al. Brain abscesses during Proteus vulgaris bacteremia. Neurol. Sci. 32 , 661–663 (2011). Ahmad, N., Farman, M., Najmi, M., Mian, K. & Hasan, A. Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase. J. Basic Appl. Sci. 2 , 1–6 (2006). Uddin, G., Rauf, A., Siddique, B. S. & Khan, H. Cytotoxic activity of extracts/fractions of various parts of P. integerrima. Transl. Med. 3 , 118 (2013). Choi, E. M., Kim, G. H. & Lee, Y. S. Carthamus tinctorius flower extract prevents H2O2-induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells. Phytother. Res. 24 (7), 1037–1041 (2010). Mary, A. P. F. & Giri, R. S. GC-MS analysis of bioactive compunds Achyranthes aspera L. Wor. J. Pharmac. Res. 7 (1), 1045–1056 (2017). WHO Technical Report 52, Annex 1, WHO guidelines on good herbal processing practices for herbal medicines. Geneva (2018). Donay, J. L., Fernandes, P., Lagrange, P. H. & Herrmann, J. L Evaluation of the inoculation procedure using a 0.25 McFarland standard for the BD Phoenix automated microbiology system. J. Clin. Micro. 45 (12), 4088–4089 (2007). Balouiri, M., Sadiki, M. & Ibnsouda, S. K. Methods for in vitro evaluating antimicrobial activity: A review. J Pharmace. Anal. 6 (2), 71–79 (2016). Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immuno. Meth. 65 (1–2), 55–63 (1983). Niks, M. & Otto, M. Towards an optimized MTT assay. J. Immuno. Meth. 130 (1), 149–151 (1990). Shah, R. M. et al. Effects of different animal manures on attraction and reproductive behaviors of common house fly, Musca domestica L. Parasitol Res. 115 , 3585–3598 (2016). Tables Table 1. Antibacterial activities of P. integerrima gall extract in different (200, 150, 100, 50µl) concentration against different bacteria. Bacterial species Ethanolic gall extract Methanolic gall extract Aqueous gall extract 200 150 100 50 200 150 100 50 200 150 100 50 B. subtilis 25 20 17 8 22 17 14 13 21 17 14 10 E. coli 14 10 8 5 18 12 10 7 0 0 0 0 P. aeruginosa 18 15 12 10 19 16 13 11 0 0 0 0 Staph. aureus 16 10 6 3 14 11 8 4 17 14 10 7 Proteus vulgaris 7 4 2 1 10 8 5 3 0 0 0 0 Table 2. Antibacterial activities of P. integerrima leaf extract in different (200, 150, 100, 50µl) concentration against different bacteria. Bacterial species Ethanolic leaf extract Methanolic leaf extract Aqueous leaf extract 200 150 100 50 200 150 100 50 200 150 100 50 B. subtilis 18 17 14 13 22 18 15 11 21 18 15 10 E. coli 18 10 7 3 20 16 14 10 0 0 0 0 P. aeruginosa 14 0 0 0 15 13 11 9 0 0 0 0 Staph. aureus 16 13 10 9 17 15 12 10 0 0 0 0 Proteus vulgaris 10 7 4 3 0 0 0 0 0 0 0 0 Table 3. Antibacterial activities of P. integerrima bark extract in different (200, 150, 100, 50µl) concentration against different bacteria. Bacterial species Ethanolic bark extract Methanolic bark extract Aqueous bark extract 200 150 100 50 200 150 100 50 200 150 100 50 B. subtilis 0 0 0 0 19 16 13 11 0 0 0 0 E. coli 12 10 7 4 17 15 12 10 0 0 0 0 P. aeruginosa 12 10 7 4 17 15 12 10 0 0 0 0 Staph. aureus 0 0 0 0 18 15 12 10 0 0 0 0 Proteus vulgaris 12 10 7 4 19 17 15 8 0 0 0 0 Table 4. Active compounds found in different extracts of P. integerrima presented as the relative % amounts based on peak area using GC-MS chromatograms. Extracts of gall Compounds Retention Time % composition Chloroform D-Limonene 10.952 55.26787 - 1,6:3,4-Dianhydro-2 14.548 9.574625 - Endo-2,3-o-Ethyliden 21.086 1.337547 Ethyl acetate Caryophyllene 21.94 14.78013 Hexane 1-Pentacontanol 18.5 74.87532 - Octatriacontane,1,3 39.914 8.967928 Extracts of leaf Compounds Retention Time % Composition Chloroform Phenol-3,5-bis(1,1dimethylet) 24.973 21.08146 - 2-Methylhexacosane 29.59 25.9933 - Heptacosane,1-chlor 33.076 50.4986 Ethyl acetate Nonadecane 17.95 22.42712 - Caryophyllene 21.98 20.00786 - Neophytadiene 32.20 16.80024 - 7,hexadecenal,(z)- 40.83 16.92081 Hexane 2,4-Dimethyl-1-hepte 5.575 0.539882 - Dodecane,4,6-dimeth 11.592 5.148011 - Undecane,3,8-dimeth 18.085 26.44024 - Heneicosane 24.062 39.70888 - 2-Methyloctacosane 30.93 9.112275 Extracts of bark Compounds Retention Time % Composition Chloroform Bicyclo[3.1.0]hexa-2- 7.80 3.496445 - Nonadecane 17.93 42.59506 - 2,4-Di-tert-butylphe 24.68 11.62349 - Dotriacontane,1-iodo- 28.94 41.84876 Ethyl acetate 1-Butanol,2-methyl- 4.33 0.051697 - 7-Hexadecenal,(z)- 38.34 17.01617 Hexane Tetrapentacontane,1,54-dibromo 53.344 0.013458 Additional Declarations No competing interests reported. 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B= bark, G= Gall, L=leaf, EA = Ethyl acetate, CF= Chloroform, HX= hexane, CP= Carboplatin ","description":"","filename":"Onlinedrawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/850eecd97dac066430252692.png"},{"id":8065139,"identity":"68c5685a-0d8a-4d3b-80c2-de5dfd234dd4","added_by":"auto","created_at":"2021-04-15 21:06:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3323,"visible":true,"origin":"","legend":"Inhibition percentage of bark, gall and leaf extracts of P. integerrima against BHK–21 cell line. B= bark, G= Gall, L=leaf, EA = Ethyl acetate, CF= Chloroform, HX= hexane, CP= Carboplatin.\n","description":"","filename":"Onlinedrawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/ae80cc671a1db1949f5841b3.png"},{"id":8065138,"identity":"aa45a58f-7eec-4f8d-82fa-e6ef2bbdf86c","added_by":"auto","created_at":"2021-04-15 21:06:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36423,"visible":true,"origin":"","legend":"Total ion chromatogram of hexane extract of P. integerrima gall","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/c327554e3f2dd790ef75cf33.png"},{"id":8065148,"identity":"d52773d9-6e42-4331-8781-8c788e82a0a5","added_by":"auto","created_at":"2021-04-15 21:06:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38704,"visible":true,"origin":"","legend":"Total ion chromatogram of chloroform extract of P. integerrima leaf","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/c533b8958d5e50d0d9b4a4a4.png"},{"id":8065143,"identity":"f0e3c9a3-b599-46a4-9146-534208b718f8","added_by":"auto","created_at":"2021-04-15 21:06:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48134,"visible":true,"origin":"","legend":"Total ion chromatogram of chloroform extract of P. integerrima bark","description":"","filename":"Fig06.png","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/56d26cccdf75a1cb1cce9f36.png"},{"id":13686586,"identity":"ffc3aaa4-d632-4bbd-abf7-619657da7022","added_by":"auto","created_at":"2021-09-17 12:16:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":772294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-396639/v1/c21fd08f-8c5e-454b-a5bf-d9808031650f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAntibacterial and Anticancer Efficacy of Different Parts of Pistacia Integerrima Plant Extracts\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedicinal plants have been found effective against many human pathogens, recently natural products are gaining attentions due to antibiotic resistance of different bacteria and more side effects of synthetic antibiotics. World Health Organization has considered natural herbs as the best source of medicine formulation\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eP. integerrima\u003c/em\u003e an important medicinal plant of family Anacardiaceaee, native in Asia and commonly present in East Afghanistan, Pakistan and North-West and West Himalaya to Kumaon growing at a height of 800-1900m\u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e. The common name of this medicinally important plant are crab\u0026rsquo;s claws and zebrawood, beside other parts of this plant, gall (Fig. 1) is extensively used for the treatment of cough, liver problem, allergy and snake bite\u003cstrong\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eP. integerrima\u003c/em\u003e is well distinct due to presence of galls on leaves and petioles. Galls are the store houses of secondary metabolites which has significance in Indian old medicine\u003cstrong\u003e\u003csup\u003e4\u003c/sup\u003e\u003c/strong\u003e. The galls of \u003cem\u003eP. integerrima\u003c/em\u003e are utilized for the cure of lungs diseases, allergy, dysentery, hiccough, stomach problems, fever, problem associated with nervous system, skin diseases, leucorrhoea, and general debility. One of the good sources of antioxidant in \u003cem\u003eP. integerrima\u003c/em\u003e is the presence of its phenolic compounds. For the treatment of jaundice, dysentery, chronic injuries and for the treatment of breast cancer cell line MCF-7, ethnobotanically significant species \u003cem\u003eP. integerrima stew.ex Brand \u003c/em\u003ewas traditionally used. The crude extract and different segment of \u003cem\u003eP. integerrima \u003c/em\u003ewere tested for cytotoxic activity in Michigan Cancer Foundation, against 7 human breast cancer cell line. The crude methanol extract exhibited week antifungal activity but good antitumor (IC\u003csub\u003e50\u003c/sub\u003e 125ppm) activity and could be a source of novel biologically active compounds\u003cstrong\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/strong\u003e. Antibacterial, antioxidant and cytotoxicity of different parts of this plant is due to its chemical compounds mainly terpenoids, saponin, phenols and sterols which are released by the plants as secondary metabolites\u003cstrong\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e. Secondary metabolites are produce as defensive mechanism against disease and environmental stress. Theses metabolites are found in the different parts stem, leaves, roots and bark of the plant\u003cstrong\u003e\u003csup\u003e6\u003c/sup\u003e\u003c/strong\u003e. In the present study the potential of \u003cem\u003eP. integerrima \u003c/em\u003eplant extract was analyzed to confirm its ethnomedicinal importance against HeLa and BHK-21 cell lines. Some important bacteria of human concern are used in this study including \u003cem\u003eP. aeruginosa\u003c/em\u003e which is a worldwide public health threat due to its resistant strains and hospital acquired infections\u003cstrong\u003e\u003csup\u003e7\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eE. coli \u003c/em\u003eand \u003cem\u003eProteus vulgaris\u003c/em\u003e are used which is present in the human gastrointestinal tract and responsible for urinary tract infections\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. Beside these bacteria, one spore former \u003cem\u003eB. subtilis\u003c/em\u003e was also used which is non-pathogenic but its spore are highly resistant and involved in food spoilage.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eExtraction of plant materials for antibacterial activity. \u003c/strong\u003e\u003cem\u003eP. integerrima \u003c/em\u003eleaves, barks, gall were collected from local market and first shade dried and then grinded in Mortar and pestle. Plant material was collected, stored and processed following standard protocols by WHO and institutional, national, and international guidelines and legislations, therefore no risks were associated in any steps of handling to disposal. For extraction purpose cold maceration technique was used\u003cstrong\u003e\u003csup\u003e16\u003c/sup\u003e\u003c/strong\u003e. Powdered plant material 10gm was individually macerated in 100mL of distilled H\u003csub\u003e2\u003c/sub\u003eO, methanol and ethanol and kept for 24 hours on a rotary shaker. These extracts then filtered 4-5 times through Whatman filter paper till all soluble compounds were completely extracted. Then the evaporation of extract was carried out by using rotary evaporator (Buchi Rotavapor R-300, Japan) and dried residues were used to check the antibacterial efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial culture collection. \u003c/strong\u003eDifferent bacterial pathogens were obtained from Ayub Medical Complex lab Abbottabad including \u003cem\u003eStaphylococcus aureus, E. coli, Proteus vulgaris, Bacillus subtilis, Pseudomonas aeruginosa. \u003c/em\u003eLiquid inoculums were prepared by adding pure bacterial strains in 10mL normal saline and used further.\u003c/p\u003e\n\u003cp\u003eAgar well diffusion method used to evaluate the antibacterial efficacy of the different parts of plant. Mueller Hinton agar (Oxoid) plate surface was inoculated by spreading 50 \u0026micro;L inoculums, McFarland 0.5 BaSO\u003csub\u003e4 \u003c/sub\u003estandard used for balancing the turbidity of used inoculum as 10\u003csup\u003e8 \u003c/sup\u003eCFU mL\u003csup\u003e-1\u003c/sup\u003e\u003cstrong\u003e\u003csup\u003e17\u003c/sup\u003e\u003c/strong\u003e. Then 8mm borer used to make 5 holes aseptically on agar plate. In 4 wells 50 \u0026micro;L of leaf, bark and gall extracts (in concentration of 200, 150, 100, and 50\u0026micro;L) poured. For negative control pure dimethyl sulfoxide (DMSO) poured in one agar well and disc of Ciprofloxacin taken as positive control in the center of plate and all agar plates were incubated for 24 hours at temperature 37\u003csup\u003eo\u003c/sup\u003eC\u003cstrong\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/strong\u003e. The inhibitory effects measured by the zones of inhibition in mm around the wells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction and fractionation of plant materials for anticancer activity. \u003c/strong\u003eShade dried leaves, galls and barks of \u003cem\u003eP. integerrima\u003c/em\u003e soaked with methanol for one week at room temperature. By using rotary evaporator, methanolic extract of each portion of plant was evaporated at 40\u003csup\u003eo\u003c/sup\u003eC temperature. Then for the process of liquid-liquid partitioned in separatory funnel, the crude methanolic extract of each portion of plant was suspended in water and fractionated with n-hexane, chloroform, ethyl acetate in sequenced manner, to get their respective fractions\u003cstrong\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/strong\u003e. All standard procedures were adopted and experiment performed in fume hood\u003cstrong\u003e\u003csup\u003e16\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAmount of 10mg of each crude and fractionated extracts of \u003cem\u003eP. integerrima\u003c/em\u003e bark, gall and leaves in hexane, chloroform and ethyl acetate carefully weighed in 1.5ml Eppendorf tubes and dissolved in DMSO. After vortex mixing, 1 mg of each of the crude extract and fraction was used in cancer cell culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnticancer activity through cell viability assay. \u003c/strong\u003ePlant extracts along with their subsequent fractions evaluated for their anti-proliferative activity against HeLa and BHK-21 by utilizing the dimethyl\u0026ndash;2\u0026ndash;thiazolyl\u0026ndash;2,5\u0026ndash;diphenyl\u0026ndash;2\u003cem\u003eH\u003c/em\u003e\u0026ndash;tetrazolium bromide (MTT) based cell metabolic assay\u003cstrong\u003e\u003csup\u003e19, 20\u003c/sup\u003e\u003c/strong\u003e. After sub-culturing, grown cell colonies were harvested using proteolytic trypsin enzyme. The cellular content along with sufficient culture media was transferred to a 15 ml conical tube and centrifuged for 5 minutes at 150 speed and 37\u0026deg;C. After careful removal of supernatant, cell pellet dissolved in 1ml culture media. Cells counted by pipetting out a volume of 10 \u0026micro;L from cell suspension and dropped onto Neubauer counting chamber to count cells under microscope. Cell suspension diluted accordingly to make 2.5x10\u003csup\u003e4 \u003c/sup\u003ecells mL\u003csup\u003e-1\u003c/sup\u003e, from this suspension with the help of a multi-channel micro-pipette 90 \u0026mu;L added in each well of 96-wells microliter plate and left for 24 hours incubation in a cell culture incubator set at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37˚C. Each plant extracted sample poured into these wells at 100 \u0026micro;g/mL of final concentration and 10 \u0026mu;L of media was used in control well. Carboplatin at a final concentration of 100 \u0026micro;M was used as a standard for cell lines. For IC\u003csub\u003e50\u003c/sub\u003e determination, three-fold dilutions used and 10 \u0026mu;L of MTT (5mg/mL PBS) after 24 hours placed in all wells. After incubation of 4 hours in a cell culture incubator 100 \u0026micro;L of stopping reagent (isopropanol and 10% sodium dodecyl sulfate) poured to solubilize formazan crystals and kept at room temperature with subsequent agitation for half an hour. Absorbance recorded using a 96\u0026ndash;well microtiter plate reader (Bio\u0026ndash;TekELx 800\u003csup\u003eTM\u003c/sup\u003e) at 570 nm, background signal (690 nm) was subtracted and results were evaluated as inhibition values percentage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of active ingredients by GC-MS. \u003c/strong\u003eIdentification and separation of various extracts of plant were carried out on Perkin Elmer Clarus 600 gas chromatograph (GC) connected with mass spectrometer (MS). Plants extracts dissolved in respective organic solvents with concentration of 5mg.mL\u003csup\u003e-1\u003c/sup\u003e and 1\u0026micro;L was injected to GC-MS and protocol was adopted as described by Shah \u003cem\u003eet al\u003c/em\u003e., (2016)\u003cstrong\u003e\u003csup\u003e21\u003c/sup\u003e\u003c/strong\u003e. Different separated compounds identified by comparing their mass spectra to the Finnigan NIST-05 (National Institute of Standard and Technology, USA) Mass spectrum library and available literature. Relative concentration of each identified compound found in injected sample was determined by comparing peak area of the compound to the sum of all peaks\u0026rsquo; area in a total ion current (TIC) chromatogram.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis. \u003c/strong\u003eTwo factor ANOVA with replication applied by using Data analysis Tools of MS Excel 2010. It was used for the analysis of significance difference (at p \u0026gt; 0.05) among zone of inhibition by using different doses and different types of solvent extracts. PRISM 5 software used for the determination of IC\u003csub\u003e50\u003c/sub\u003e value.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe present research work conducted to determine the antimicrobial and cytotoxic potential of medicinal plant \u003cem\u003eP. integerrima\u003c/em\u003e (leaf, gall and bark) extracts. The antibacterial activity of \u003cem\u003eP. integerrima\u003c/em\u003e extracts were applied on five important bacteria of human concern with different extracts concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial activity of \u003cem\u003ePistacia integerrima\u003c/em\u003e in different solvents \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn case of \u003cem\u003eP. integerrima \u003c/em\u003egall extract it was observed that with increase in different extracts concentration, inhibition zone was also increased. All solvent\u0026rsquo;s extracts showed effectiveness against all bacterial species, except for aqueous gall extract which was ineffective against \u003cem\u003eE. coli, P. aeruginosa \u003c/em\u003eand\u003cem\u003e Proteus vulgaris.\u003c/em\u003e Gall extract was less effective against Proteus (Gram-negative) and more against Bacillus (Gram-positive). Maximum zone of inhibition 25mm was formed with ethanolic gall extract in 200\u0026micro;L concentration against \u003cem\u003eB. subtilis\u003c/em\u003e (Table 1). In the present study, the probability values by using two factor ANOVA with replication was less than \u0026alpha; value of 0.05 (p \u0026lt; 0.05) which shows that significance difference exist between zone of inhibition of different bacterial species and different solvents extract of various concentrations.\u003c/p\u003e\n\u003cp\u003eMethanol leaf extract was found more effective than ethanolic leaf extract, maximum zone of inhibition 22mm against \u003cem\u003eBacillus subtilis \u003c/em\u003ewas observed with 200 \u0026micro;L concentration (Table 2). \u003cem\u003eP. vulgaris\u003c/em\u003e shows resistance to methanol and aqueous extracts but inhibited with ethanolic leaf extract with 10mm maximum zone of inhibition. Aqueous leaf extracts found ineffective against all except \u003cem\u003eBacillus subtilis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. integerrima \u003c/em\u003emethanolic bark extract was found more effective against all 4 bacterial species than ethanolic and aqueous extract. Highest and similar zone of inhibition of 19mm were found against \u003cem\u003eBacillus subtilis \u003c/em\u003eand \u003cem\u003eProteus vulgaris\u003c/em\u003e with 200 \u0026micro;L concentration. Ethanolic bark extract was effective against \u003cem\u003eE.coli,\u003c/em\u003e\u003cem\u003eP. aeruginosa \u003c/em\u003eand\u003cem\u003e Proteus vulgaris\u003c/em\u003e while \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e showed resistant (Table 3)\u003cem\u003e. \u003c/em\u003eOverall gall extracts has more antibacterial potential than other parts of this plant. While \u003cem\u003eBacillus subtilis \u003c/em\u003ewas found more susceptible than other bacteria with different parts of plant extract except ethanolic and aqueous bark extracts which showed less effectiveness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnticancer activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen cytotoxic assay of fractions and extracts of different parts (gall, leaf, bark) of \u003cem\u003eP. integerrima\u003c/em\u003e were carried out it showed profound cytotoxic effect against HeLA and BHK-21 cell line. Among different parts of \u003cem\u003eP.\u003c/em\u003e\u003cem\u003eintegerrima\u003c/em\u003e, n-hexane leaf fraction was shown to be most effective against HeLa cell lines with IC\u003csub\u003e50\u003c/sub\u003e of 7.45 \u0026micro;g/mL than chloroform (IC\u003csub\u003e50\u003c/sub\u003e of 4.82 \u0026micro;g/mL) and other fractions (Fig. 2). In this n-hexane leaf fraction, Heneicosane was found (39.7%) as an active compound (Table 4) and responsible for anticancer activity, Crude extract of \u003cem\u003eP. integerrima\u003c/em\u003e bark part has showed IC\u003csub\u003e50\u003c/sub\u003e of 4.77 \u0026micro;g/mL almost similar with chloroform leaf extract. In case of BHK-21, highest cell inhibition of 46.8% was observed with crude leaf extract than ethyl-acetate bark extract (44.9%) of \u003cem\u003eP. integerrima\u003c/em\u003e (Fig. 3). Overall leaf extract has showed more potential against cancer cell than other parts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas chromatography-mass spectrometry (GC-MS) analysis of \u003cem\u003eP. integerrima\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGC-MS analysis of different parts of plant extracts showed that leaf extract of \u003cem\u003eP. integerrima\u003c/em\u003e contained greater number of compounds compared to other parts of the plant (Table 4). In hexane gall extract Heneicosane found 39.7% (Fig.4). Main component in chloroform gall extract was 55% D-Limonene and hexane was 1-Pentacontanol 74%. Chloroform leaf extract contain Heptacosane,1-chlor 50.5% an active ingredient (Fig. 5). Chloroform bark extract contain Nonadecane 42.5% and Dotriacontane, 1-iodo- 41.7% as active compound (Fig. 6) (Table 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study \u003cem\u003eP. integerrima\u003c/em\u003e showed more effectiveness against Gram positive bacteria than Gram negative. Similarly one study evaluated \u003cem\u003eP. integerrima \u003c/em\u003eactivity of gall extracts (aqueous and ethanol) against \u003cem\u003eS. aureus,\u003c/em\u003e \u003cem\u003eE. coli, B. cereus, P. aeruginosa \u003c/em\u003eand \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e Among these bacterial species, both ethanolic and aqueous extract inhibited the Gram-positive bacteria better than the Gram-negative bacteria\u003cstrong\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eBacillus subtilis\u003c/em\u003e is Gram-positive spore forming bacteria causes food spoilage and its spores are very hard to kill but \u003cem\u003eP. integerrima\u003c/em\u003e has successfully inhibited its growth. With 200 \u0026micro;L concentration of methanolic bark extract, highest and similar zone of inhibition found against \u003cem\u003eBacillus subtilis \u003c/em\u003eand \u003cem\u003eProteus vulgaris\u003c/em\u003e. Previously \u003cem\u003eProteus\u003c/em\u003e \u003cem\u003emirablis\u003c/em\u003e was found to be less susceptible than \u003cem\u003eE. coli,\u003c/em\u003e \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eProteus vulgaris\u003c/em\u003e has clinically importance and responsible for urinary tract infection, bacteremia and brain abscesses and enter in the human body through intestinal tract\u003cstrong\u003e\u003csup\u003e11\u003c/sup\u003e\u003c/strong\u003e and resistant to most of antibiotics\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn the present study n-hexane leaf fraction of \u003cem\u003eP. integerrima\u003c/em\u003e found more lethal against HeLa cell lines than other parts of the plant, similar finding was observed by Ahmad \u003cem\u003eet al., \u003c/em\u003e(2006)\u003cstrong\u003e\u003csup\u003e12\u003c/sup\u003e\u003c/strong\u003e. While in contrast with Uddin \u003cem\u003eet al., \u003c/em\u003e2013 who also conducted study to determine the anticancer efficacy of different parts of \u003cem\u003eP. integerrima\u003c/em\u003e fractions against brine shrimp \u003cem\u003eArtemia salina\u003c/em\u003e and found gall of this plant has more cytotoxic potential than other parts of the plant\u003cstrong\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/strong\u003e. Heneicosane found in higher concentration in n-hexane leaf fraction which might be associated with higher anticancer activity with this extract, previously this compound was found as active component in the flowers of \u003cem\u003eCarthamus tinctorius\u003c/em\u003e which showed antioxidant property to protect human bone cells\u003cstrong\u003e\u003csup\u003e14\u003c/sup\u003e\u003c/strong\u003e. Overall chloroform leaf extract showed highest anticancer activity, in which Heptacosane, 1-chlor was found as an active ingredient and this compound showed anticancer activity in \u003cem\u003eAchyranthes aspera L\u003c/em\u003e weed\u003cstrong\u003e\u003csup\u003e15\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results showed that solvents extract has profound antibacterial activity than aqueous extract against tested pathogens except \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Methanolic and ethanolic extracts of \u003cem\u003eP. integerrima\u003c/em\u003e gall has showed more significant inhibition effect than leaf and bark. The most potent inhibitor was hexane leaf fraction against HeLa cell lines, in this Heneicosane was found (39.7%) which might be responsible for anticancer activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.R. performed all experiments, F.M. has supervised and planned research design, Z.A.B. helped in manuscript writing, J.I. helped and provided lab facility for anticancer activity, M.F.S. did statistical analysis, S.P. helped in making graphs and figures, U.K. helped in GC-MS experiment and interpretation, I.K. helped in anticancer activity experiment. All authors reviewed the manuscript before submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding is available for open access.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003cbr /\u003e\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMohammad, G. J., Jawad, M. \u0026amp; Hameed, I. H. Proteus species: Characterization and herbal antibacterial: A Review. \u003cem\u003eInter. J. of Pharmaco. Phytochem. Res.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (11), 1844\u0026ndash;1854 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePant, S. \u0026amp; Samant, S. S. Ethnobotanical observations in the Mornaula reserve forest of Komoun, West Himalaya, India. \u003cem\u003eEthnobot. Leaflet.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 193\u0026ndash;217 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBibi, Y., Zia, M. \u0026amp; Qayyum, A. An overview of Pistacia integerrima a medicinal plant species: Ethnobotany, biological activities and phytochemistry. \u003cem\u003ePak. J. Pharm. Sci.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (3), 1009\u0026ndash;1013 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChopra, R., Chopra, I., Handa, K. \u0026amp; Kapoor, L. \u003cem\u003eIndigenous Drugs of India\u003c/em\u003e 2(nd Edn (Academic Publishers, India, 2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBibi, Y., Nisa, S., Chaudhary, F. M. \u0026amp; Zia, M. Antibacterial activity of some selected medicinal plants of Pakistan.\u003cem\u003eBMC Complement. Altern. Med.\u003c/em\u003e11, \u003cb\u003e52\u003c/b\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelgoda, R. \u0026amp; Murray, J. E. Evolutionary perspectives on the role of plant secondary metabolites. In: Badal S, Delgoda R, editor(s). Pharmacognosy Fundamentals, Applications and Strategies, Academic Press 93\u0026ndash;100(2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFata, M. M., Shirin, G. \u0026amp; Rehm, B. H. A. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. \u003cem\u003eFront. Cell. Infec. Micro.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 39 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmbruster, C. E. \u0026amp; Mobley, H. L. T. Proteus species (Website: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.antimicrobe.org/b226.asp\u003c/span\u003e\u003c/span\u003e), accessed on January 3, 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamachandra, Y. L., Ravi, S. B. E., Sujan, G. S. \u0026amp; Sundar, R. S. In-vitro antimicrobial activity of Pistaacia integerrima leaf gall extracts. \u003cem\u003ePharmacopho.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e (2), 149\u0026ndash;154 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad, R., Pieters, L., Rehman, N. \u0026amp; Riaz, M. Antimicrobial and antioxidant activity of crude extracts of two medicinal plants P. Integerrima and Debregeasia salicifolia. \u003cem\u003eInt. J. Pharm. Sci. Rev. Res.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (1), 13\u0026ndash;17 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBloch, J. \u003cem\u003eet al.\u003c/em\u003e Brain abscesses during Proteus vulgaris bacteremia. \u003cem\u003eNeurol. Sci.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 661\u0026ndash;663 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad, N., Farman, M., Najmi, M., Mian, K. \u0026amp; Hasan, A. Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase. \u003cem\u003eJ. Basic Appl. Sci.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 1\u0026ndash;6 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUddin, G., Rauf, A., Siddique, B. S. \u0026amp; Khan, H. Cytotoxic activity of extracts/fractions of various parts of P. integerrima. \u003cem\u003eTransl. Med.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 118 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi, E. M., Kim, G. H. \u0026amp; Lee, Y. S. Carthamus tinctorius flower extract prevents H2O2-induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells. \u003cem\u003ePhytother. Res.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (7), 1037\u0026ndash;1041 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMary, A. P. F. \u0026amp; Giri, R. S. GC-MS analysis of bioactive compunds Achyranthes aspera L. \u003cem\u003eWor. J. Pharmac. Res.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (1), 1045\u0026ndash;1056 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO Technical Report 52, Annex 1, WHO guidelines on good herbal processing practices for herbal medicines. Geneva (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonay, J. L., Fernandes, P., Lagrange, P. H. \u0026amp; Herrmann, J. L Evaluation of the inoculation procedure using a 0.25 McFarland standard for the BD Phoenix automated microbiology system. \u003cem\u003eJ. Clin. Micro.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e (12), 4088\u0026ndash;4089 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalouiri, M., Sadiki, M. \u0026amp; Ibnsouda, S. K. Methods for in vitro evaluating antimicrobial activity: A review. \u003cem\u003eJ Pharmace. Anal.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (2), 71\u0026ndash;79 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. \u003cem\u003eJ. Immuno. Meth.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e (1\u0026ndash;2), 55\u0026ndash;63 (1983).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiks, M. \u0026amp; Otto, M. Towards an optimized MTT assay. \u003cem\u003eJ. Immuno. Meth.\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e (1), 149\u0026ndash;151 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah, R. M. \u003cem\u003eet al.\u003c/em\u003e Effects of different animal manures on attraction and reproductive behaviors of common house fly, Musca domestica L. \u003cem\u003eParasitol Res.\u003c/em\u003e \u003cb\u003e115\u003c/b\u003e, 3585\u0026ndash;3598 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eAntibacterial activities of \u003cem\u003eP. integerrima\u003c/em\u003e gall extract in different (200, 150, 100, 50\u0026micro;l) concentration against different bacteria.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"115\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBacterial species\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"157\"\u003e\n\u003cp\u003e\u003cstrong\u003eEthanolic gall extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eMethanolic gall extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eAqueous gall extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cem\u003eProteus vulgaris\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u003c/strong\u003eAntibacterial activities of \u003cem\u003eP. integerrima\u003c/em\u003e leaf extract in different (200, 150, 100, 50\u0026micro;l) concentration against different bacteria.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial species\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"157\"\u003e\n\u003cp\u003e\u003cstrong\u003eEthanolic leaf extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eMethanolic leaf extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eAqueous leaf extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cem\u003eProteus vulgaris\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Antibacterial activities of \u003cem\u003eP. integerrima\u003c/em\u003e bark extract in different (200, 150, 100, 50\u0026micro;l) concentration against different bacteria.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"109\"\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial species\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"157\"\u003e\n\u003cp\u003e\u003cstrong\u003eEthanolic bark extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eMethanolic bark extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003eAqueous bark extract\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"109\"\u003e\n\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"109\"\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"109\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"109\"\u003e\n\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"109\"\u003e\n\u003cp\u003e\u003cem\u003eProteus vulgaris\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"45\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. \u003c/strong\u003eActive compounds found in different extracts of \u003cem\u003eP. integerrima\u003c/em\u003e presented as the relative % amounts based on peak area using GC-MS chromatograms.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u003cstrong\u003eExtracts of gall\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e\u003cstrong\u003eRetention Time\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e\u003cstrong\u003e% composition\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eChloroform\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eD-Limonene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e10.952\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e55.26787\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e1,6:3,4-Dianhydro-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e14.548\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e9.574625\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eEndo-2,3-o-Ethyliden\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e21.086\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e1.337547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eEthyl acetate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eCaryophyllene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e21.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e14.78013\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eHexane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e1-Pentacontanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e18.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e74.87532\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eOctatriacontane,1,3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e39.914\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e8.967928\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u003cstrong\u003eExtracts of leaf\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e\u003cstrong\u003eRetention Time\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e\u003cstrong\u003e% Composition\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eChloroform\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003ePhenol-3,5-bis(1,1dimethylet)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e24.973\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e21.08146\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e2-Methylhexacosane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e29.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e25.9933\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eHeptacosane,1-chlor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e33.076\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e50.4986\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eEthyl acetate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eNonadecane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e17.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e22.42712\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eCaryophyllene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e21.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd 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width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eUndecane,3,8-dimeth\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e18.085\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e26.44024\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eHeneicosane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e24.062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e39.70888\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; 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width=\"216\"\u003e\n\u003cp\u003e2,4-Di-tert-butylphe\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e24.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e11.62349\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e\u0026nbsp; Dotriacontane,1-iodo-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e28.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e41.84876\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eEthyl acetate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e1-Butanol,2-methyl-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e4.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e0.051697\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; -\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003e7-Hexadecenal,(z)-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e38.34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e17.01617\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003eHexane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"216\"\u003e\n\u003cp\u003eTetrapentacontane,1,54-dibromo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"117\"\u003e\n\u003cp\u003e53.344\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"150\"\u003e\n\u003cp\u003e0.013458\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\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":"Pistacia integerrima, antibacterial activity, gall, HeLa cell lines, GC-MS","lastPublishedDoi":"10.21203/rs.3.rs-396639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-396639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explored the antibacterial efficacy of \u003cem\u003ePistacia integerrima\u003c/em\u003e gall, leaf and bark in different solvents. Extracts of these parts prepared in ethanol, methanol and distilled water by using rotary evaporator. To check the antibacterial potential of this plant, minimal inhibitory concentrations of each extract analysed by agar well diffusion method against \u003cem\u003eStaphylococcus aureus, E. coli, Proteus vulgaris, Bacillus subtilis \u003c/em\u003eand\u003cem\u003e Pseudomonas aeruginosa\u003c/em\u003e. For anticancer activity, hexane, chloroform and ethyl acetate fractions of \u003cem\u003eP. integerrima\u003c/em\u003e gall, leaf and bark used against human cervical cancer (HeLa) and baby hamster kidney (BHK–21) cell lines. Results showed that maximum zone of inhibition 25mm formed with ethanolic gall extract in 200µL concentration against \u003cem\u003eB. subtilis.\u003c/em\u003e \u003cem\u003eP. vulgaris\u003c/em\u003e shows resistance to methanol and aqueous extracts but inhibited with ethanolic leaf extract. Among different parts of \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eintegerrima\u003c/em\u003e, n-hexane leaf fraction shown to be most effective against HeLa cell lines with IC\u003csub\u003e50\u003c/sub\u003e of 7.45 µg/mL. In case of BHK-21, highest cell inhibition of 46.8% observed with crude leaf extract than ethyl-acetate bark extract (44.9%) of \u003cem\u003eP. integerrima\u003c/em\u003e. It is concluded that the effective inhibitor was hexane leaf fraction against HeLa cell lines in which Heneicosane was found (39.7%) which might be responsible for anticancer activity.\u003c/p\u003e","manuscriptTitle":"Antibacterial and Anticancer Efficacy of Different Parts of Pistacia Integerrima Plant Extracts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-15 21:06:12","doi":"10.21203/rs.3.rs-396639/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":"b0d92b20-f477-4156-b215-81427c137202","owner":[],"postedDate":"April 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3661107,"name":"Cancer Biology"},{"id":3661108,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-05-25T09:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-15 21:06:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-396639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-396639","identity":"rs-396639","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}