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

This study explored the antibacterial ecacy 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.


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 signi cance 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 signi cant 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 con rm 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 rst 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  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-2thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) based cell metabolic assay 19,20 . After subculturing, grown cell colonies were harvested using proteolytic trypsin enzyme. The cellular content along with su cient 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 nal concentration and 10 μL of media was used in control well. Carboplatin at a nal 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.

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 ve 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 signi cance 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.

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 nding was observed by Ahmad et al., (2006) 12 . While in contrast with Uddin et al., 2013 who also conducted study to determine the anticancer e cacy 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 owers 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 signi cant 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.     Total ion chromatogram of hexane extract of P. integerrima gall Figure 5 Total ion chromatogram of chloroform extract of P. integerrima leaf Figure 6 Total ion chromatogram of chloroform extract of P. integerrima bark