In Vitro Antioxidant, Membrane Stabilizing and Antimicrobial Properties of Murayya paniculata Wild Plant of Amarkantak (M.P)

The excessive usage of antibiotics in humans and veterinary medicine has lead to the emergence of antibiotic resistance and requirement of novel antibiotics. There has been increased interest towards plants as source of drugs because of their pharmacological potency and long traditional usage. The aim of the current study was to evaluate bioactive components, antioxidant and anti-inammatory activity from the leaf extracts of Murayya paniculata, a traditionally used Indian medicinal plant. Evaluations were made for phytochemicals, antioxidant, membrane stabilizing and antimicrobial activities. The methanol extract displayed highest avonoid and phenolic content while the acetone extract demonstrated considerable ABTS inhibitory activity (IC 50 value:555.18 ± 1.68 µg/mL) and hexane extract exhibited highest H 2 O 2 radical scavenging activity (IC 50 value: 509.84 ± 3.03 µg/mL). The aqueous extract displayed 19.4 ± 0.66% RBC hemolysis and 80.5 ± 0.66% protection caused by hypotonic solution at high concentration of extract. The fractions of hexane extract revealed a higher zone of inhibition than crude extract. The major components found in the fractions were cyclohexane (40.11%) and 3-(6-Methoxy-3-methyl-2-benzofuranyl) Cyclohexanone (13.68%) analyzed by GC-MS technique. The current results validate the traditional use of the plant and warrant its potential in drug development programs in further investigations. µg/mL) was observed for the hexane extract that was lower than that of ascorbic acid (528.01 µg/mL) although it was observed that at low concentrations ascorbic acid was found to be more ecient. The reducing capacity of antioxidant is mainly because of their electron transfer property for instance polyphenols and avonoids The FRAP assay is a relatively simple, quick, and inexpensive direct method of measuring the combined antioxidant activity of reductive antioxidants in a test sample [42]. The higher values shows, higher antioxidant property of extracts. Our study clearly indicated that the methanolic extract of the leaves exhibited high scavenging capacity demonstrating that the high antioxidant capacity of the methanol extract that correlated well with the total content of phenolic and avonoid content. Higher the absorbance shows higher antioxidant property. The present study also demonstrated the results that total antioxidant reducing power of compound/extract is depending on the amount of phenolic and avonoid content. The comparison between the results of total phenolic content and reducing power assay found to be in similar order of MPM > MPW > MPA > MPH > MPC. Whereas the results of total avonoid content and FRAP values found to be in similar order of MPM > MPA > MPW > MPC > MPH. Many studies demonstrated the strong correlation between the phenolic content and reducing property of compounds. The redox properties of phenolic are major reason for the antioxidant property of an extract Living tissues respond to injury, infection or irritation in the form of inammation. Lysosomal enzymes are released leading to the tissue injury by damaging the macromolecules and lipid peroxidation of membranes. Plant extracts can be utilized for stabilization of lysosomal membrane and thus limiting the inammatory response by inhibiting the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases. Human red blood cell or erythrocyte membrane is analogous to the lysosomal membrane and has been used in stabilization assays. The antioxidant activity of the M. paniculata extracts correlated well with the RBC membrane stabilization properties. the of µg/mL, we observed 60–80% that was comparable to the standard drug indomethacin in RBC hemolysis with water and methanol extract. The results clearly suggest that M. paniculata extracts may provide us with some potent compounds for the management of inammatory conditions. Presence of saponins and avonoids in the methanolic and water extracts also support the current ndings as they have been known to have profound stabilizing effect on lysosomal membrane [45]. to strains MIC µg/mL B. subtilis MIC µg/mL P. aerugonesa. Fractionisation six fractions M. but PC2


Introduction
The discovery of compounds with antimicrobial properties has revolutionized modern medicine system. Use of antibiotics has provided us with successful interventions in surgical procedures, organ transplants and patient care. However, the evident reports of antimicrobial resistance amongst common bacterial pathogens are now threatening this therapeutic accomplishment [1]. A large number of diseases such as pneumonia, tuberculosis, gonorrhoea, and salmonellosis are becoming harder to treat with the currently available antibiotics. It has been estimated that around 300 million premature deaths will occur by 2050 with a global economic loss of up to $100 trillion [2]. The resistance against antibiotics is an immediate threat to the treatment of infectious diseases as the development of new antimicrobial agents is declining and very few drugs have been approved in the recent years [3]. Phytochemicals have displayed potential antibacterial properties against sensitive and resistant pathogens and thus priority research for screening plant extracts for novel antimicrobial properties can be a possible source for a new drug development [4] [5]. Plants possess variety of secondary metabolites that can be utilized for the development of biocompatible therapeutics. Traditional medicinal systems, being practiced in major parts of the world are based on therapeutic use of such medicinal plants. WHO have also emphasized that several countries are gradually accepting the contribution of traditional medicine to the health and wellbeing of individuals and the need to integrate it to their healthcare systems [6]. Thus, it is quite rational that plants be selected based on traditional medicinal knowledge and screened for characterization of natural products as novel antimicrobials.
Murayya paniculata (Synonyms: Cholcas paniculata L, Chalcas exotica L and Murayya exotica L.,) commonly known as Orange Jasmine, mock orange, chinese box, Kamini, is a tropical, evergreen plant with tiny, white, scented owers, and is cultivated as an ornamental tree or hedge [7]. The leaves have been used as a food additive in many Indian and Malay dishes due to their strong fragrance. The plant is known for its therapeutic usage and has been traditionally used for the treatment of tuberculosis, diarrhea, abdominal pain, dysentery, headache, edema, thrombosis, and stasis of blood. Traditional medicinal healers of Amarkantak region utilize medicinal plants for the treatment of various diseases. In our initial studies we found M. paniculata to be one such promising plant used by the healers for the successful treatment of wounds, tuberculosis, malaria and as an antidote. The different parts of the plant such as leaves, roots, and stem are used in the preparation. As to the best of our knowledge, there is no reference about the safe prescribed amount of M. paniculata in traditional medicine so it was considered worthwhile to do the pharmacological activities such as antioxidant, antimicrobial, anti-in ammatory etc. The present study was undertaken to compare e ciency of different leaf extracts to nd out the phyto-constituents, total phenolic, total avonoid content, total reducing power and antimicrobial property against selected microbial strains. As per our knowledge this is the rst reporting of the comparative account of the pharmacological e ciency of different leaf extracts of M. paniculata.

Extract Preparation
The extracts were prepared using dried leaves of the plant in different solvents such as hexane, acetone, chloroform, methanol and water. The results display that maximum yield per gram is obtained from methanol (4.8% w/w) followed by acetone (3.7% w/w), water (3.47% w/w), hexane (1.78% w/w) and chloroform (1.07% w/w). This highlights that methanol is e cient solvent for extraction of phytochemicals from leaves of M. paniculata.

Phytoconstituents analysis
Alkaloids, avonoids, steroids and terpenoids were detected as major secondary metabolites in the plant extracts. Tannins, saponins and glycosides were present in minor quantities while phlobatanins and terpenoids were not present in any of the extracts (Table S1).

Antioxidant activity
The total phenolic and total avonoid content in the different extracts of M. paniculata is represented in Table 1. Among the ve extracts methanol extract possesses highest phenolic content (14.45±0.63 mg gallic acid equivalent/g dry material) followed by acetone (12.02±0.59mg/g), water (3.20± 0.12 mg/g) chloroform (1.68± 0.06 mg/g) and hexane (1.01± 0.01 mg/g). The total avonoid concentration in different solvent extracts was estimated using quercetin as a standard and values expressed in mg quercetin equivalent /g dry wt. Out of ve extracts the methanol (1.987± 0.04mg) contains highest avonoid content followed by water (1.619±0.04mg), acetone (1.004±0.02mg), hexane (0.614± 0.08mg) and chloroform (0.609± 0.03mg). Supplementary gure (Fig. S1) shows the different concentration of plant extract with percentage inhibition of ABTS radical, out of which the acetone extract demonstrated elevated ABTS + inhibition with increased concentration. For ABTS + inhibition the IC 50 values ranged between 500-800 µg/mL ( Table 2). The IC 50 value of acetone (555.18± 1.68µg/mL) were less than the standard (Ascorbic acid) used. The percentage H 2 O 2 inhibition by different extracts was found to be concentration dependent (Fig. S2). The hexane extracts exhibited highest H 2 O 2 scavenging ability compared to other extracts. The IC 50 values ranged between 500-800 µg/mL Table 2.
The IC 50 value of hexane (509.84± 3.03µg/mL) was lowest as compared to the standard (Ascorbic acid) or other extracts that were analyzed. The result display that hexane and acetone have better antioxidant property as compared to the standard.
The analysis of total reducing capacity of extracts was carried out using FRAP and reducing power assays. FRAP value of methanolic extract (0.085± 0.002µg/mL) showed highest value, followed by acetone, water, chloroform and hexane (Table 2) The reducing property of compound/ extracts indicates its electron donating capacity. In reducing power assay, the M. paniculata methanol extract displayed maximum absorbance followed by water, acetone, ascorbic acid, hexane and chloroform extracts (Fig. 1).

Membrane stabilizing property
The results of assay are presented in Table 3. It was observed that all the extracts of M. paniculata exhibited concentration dependent anti-in ammatory property as compared to the standard drug indomethacin. The maximum concentration analyzed was 5000µg/mL and extracts were found to possess antiin ammatory property in the order MPW > MPM > MPH > MPA > MPC. The aqueous extract showed the 80% protection whereas the standard drug showed 83% protection at highest concentration tested. The Pearson's correlation was done with concentration against percent protection provided by different extracts (Table S2), where we found strong positive correlation with concentration and percent protection with all extracts. Table 6 presents antimicrobial activity against tested bacterial (E. coli, S. aureus, P. auregenosa, B. subtilius) and fungal (C. albicans, P. chrysogenum, A. niger, A. avus) strains using disk diffusion method (Fig. 2). The growth of P. auregenosa was inhibited by all extracts of M. paniculata. The MIC for different extracts was as aqueous (25µg/mL), hexane, methanol, and chloroform (250µg/mL), acetone (500µg/mL). The hexane extract displayed good inhibition activity against all the bacterial strains as compared to other tested extracts. All the extracts were found not be active against any fungi as we did not observed any zone of inhibition even after repeated experiments.

Fractionization of Hexane Extract
Total 13 fractions were collected initially using column chromatography. The results of TLC showed that F1-F3 contain single spot and were eluted using hexane and dichloromethane (5:5) as the solvent. The fractions were pooled and renamed as PC1. The F9 & 10 showed similar spot and were pooled as PC3. Further F11-13 had similar spots and was renamed as PC4. The F4 to F8 displayed 3 spots on TLC (2 spots in UV and 1 spot in Vis) which were concentrated and further allowed for column chromatography separation using mixture of hexane and acetone as solvent. From the repeated chromatography we obtained fraction PC2 (in visible region), PC11 and PC22 (in UV region). Finally 6 pooled fractions were obtained, named as PC1, PC2, PC3, PC4, PC11, PC22 and used for further antibacterial activities. Table 4 presents antimicrobial activity against selected multi resistance bacteria (E. coli, S. aureus, P. auregenosa, B. subtilius) using fractions obtained from column chromatography. The B. subtilius and P. auregenosa were inhibited by all fractions of hexane extract, out of which PC4 showed MIC of 250 and 25µg/mL respectively (Fig 3).

Discussion
Amarkantak is located in an ecologically diverse region in the states of Chhattisgarh and Madhya Pradesh (India). Several tribal and non-tribal communities reside in this area and utilize the forest produce for their livelihood and other needs. Being biodiversity rich, Amarkantak is a potential source of hundreds of medicinal plants that are being utilized by the tribal communities for the treatment of various health problems. M. paniculata is used by them for treatment of tuberculosis, malaria, wound healing and as an antidote. We therefore selected M. paniculata leaves for investigation of antioxidant, membrane stabilizing and antimicrobial properties. The phytochemical screening divulges the presence of alkaloids, avonoids, steroids, tannins, saponins and glycosides that are known to have protective or disease preventive properties. Several phytochemicals plays vital role in alleviating the several health problems like malaria, asthma, cancer, arthritis, jaundice, diabetes, dengue, diarrhea, dysentery and microbial infections [8]. In the current study, secondary metabolites such alkaloids, avonoids, steroids and terpenoids present in majority while tannins, saponins and glycosides were present in minor quantities. The alkaloids are a class of nitrogenous compounds that have served as scaffolds for important antibacterial drugs such as metronidazole and the quinolones [9] [10]. More than 8000 avonoids have been reported from different plants species [11]. That play vital role in stimulation, protection, avoring, communication, and pigmentation [12]. Flavonoids are rich source of antioxidants and are known to protect plants against various stresses [13]. The avonoids are known to possess anti-allergic, hepatprotective anticancer, antibacterial, anti-in ammatory, anti-diabetic and anti-viral properties [14][15] [16][17] [18]. Saponins are a vast group of compounds distributed widely in the plant kingdom, which are characterized by their foam forming and detergent properties [19][20] [21]. They are known to have several biological properties like antimicrobial, anti-malarial, anti-allergic, anti-diabetic, insecticidal, and anti-in ammatory [22][23] [24][25] [26]. The tannins (commonly referred to as tannic acid) are water soluble polyphenolic compounds synthesized by plants [22] and are responsible to protect plants against herbivores and insects [27] [28]. Tannins have also been reported to have anti cancer, anti mutagenic, anti-oxidant, antibacterial, anti-viral, anti-tumor, and anti-in ammatory activities [28] [29][30] [31][32] [33]. The presence of all these compounds in the leaves highlights the prominent use of M. paniculuta for the treatment of various diseases by the tribal communities. The total phenolic content was present in high concentrations in methanolic extract followed by acetone, water, chloroform and hexane while the total avonoid content was highest in methanol extract followed by water, acetone, hexane and chloroform. The antioxidant activity of avonoids is due to their ability to reduce free radical formation and to scavenge free radicals. Previously Zhang et al. (2011) reported the presence of seventy polymethoxylated avonoids (PMF) in the leaves extract of M. paniculata [34]. These PMFs are a particular group of avonoids that are responsible for a number of biological activities including antioxidant [35] [36]. and antimicrobial activities [37] [38]. Phenolic compounds are good antioxidants as their hydroxyl groups can directly contribute to antioxidant action leading to a wide range of biological effects including antibacterial, anti-in ammatory, antiallergic, hepato-protective, antithrombotic, antiviral, anticarcinogenic and vasodilatory actions [39]. Some phenolic compounds can stimulate the synthesis of endogenous antioxidant molecules in the cell [40]. The results of antioxidant activities correlated well with the presence of phenolic and avonoids in different extracts. Signi cant concentration dependent inhibition of ABTS free radical was observed in all the tested extracts. The IC 50 value observed for acetone extract was even less than the standard ascorbic acid that indicates the strong ability of the extracts to act as ABTS and H 2 O 2 scavenger. Signi cant dose-dependent H 2 O 2 scavenging activities were observed in all the extracts analyzed. The lowest IC 50 (509.84 µg/mL) was observed for the hexane extract that was lower than that of ascorbic acid (528.01 µg/mL) although it was observed that at low concentrations ascorbic acid was found to be more e cient. The reducing capacity of antioxidant is mainly because of their electron transfer property for instance polyphenols and avonoids [41]. The FRAP assay is a relatively simple, quick, and inexpensive direct method of measuring the combined antioxidant activity of reductive antioxidants in a test sample [42]. The higher values shows, higher antioxidant property of extracts. Our study clearly indicated that the methanolic extract of the leaves exhibited high scavenging capacity demonstrating that the high antioxidant capacity of the methanol extract that correlated well with the total content of phenolic and avonoid content. Higher the absorbance shows higher antioxidant property. The present study also demonstrated the results that paniculata extracts correlated well with the RBC membrane stabilization properties. At the concentrations of 4000-5000 µg/mL, we observed 60-80% protection that was comparable to the standard drug indomethacin in RBC hemolysis with water and methanol extract. The results clearly suggest that M. paniculata extracts may provide us with some potent compounds for the management of in ammatory conditions. Presence of saponins and avonoids in the methanolic and water extracts also support the current ndings as they have been known to have profound stabilizing effect on lysosomal membrane [45].
Novel safe and promising antimicrobial agents are urgently required to ght against drug resistant bacterial and fungal strains. Flavonoids are known to inhibit microbial growth by inhibition of nucleic acid synthesis, cytoplasmic membrane function and energy metabolism pathways [46]. The antimicrobial properties of phenolic compounds have been reported due to the alteration of cell membrane permeability leading to the uncoupling of oxidative phosphorylation, inhibition of active transport and cytoplasmic membrane damage that leads to the loss of pool metabolites [47]. Tannins have been shown to possess inhibitory activities against Methicillin-resistant Staphylococcus aureus (MRSA) [48] or bacteria, yeast and fungi [49]. In the current study all the extracts of M. paniculata displayed some level of inhibition of bacterial growth but no antifungal activity was observed. Both the Gram negative and Gram positive bacteria were inhibited by the extracts, however the hexane extract exhibited growth inhibition against all tested bacterial strains with a minimum observed MIC of 50 µg/mL against B. subtilis or MIC 250 µg/mL against P. aerugonesa. Fractionisation of hexane extract yielded six fractions that revealed promising antibacterial potency. This showed that M. paniculata extracts contains potential antibacterial bioactive components but not good antifungal agents. The P. aeruginosa was found to be very sensitive to the fractions of hexane extract displaying an inhibition zone of 9.6 mm against PC2 fraction. Whereas the PC4 fraction exhibited considerable antibacterial activity against all tested strains. Hence result predicts that PC4 fraction would be an active and e cient compound/s against selected bacterial strains.
The GC-MSMS analysis of PC4 fraction revealed a presence of cyclohexane and 3-(6-Methoxy-3-methyl-2-benzofuranyl) Cyclohexanone in highest amount proved to be a reason for antibacterial activity. The cyclohexane and derivates are lipophilic weak acids and possess exceptional nature of mode of action as antibacterial agents. Unlike other cationic antimicrobial compounds, these cyclohexane and derivatives will not interrupt the cytoplasmic membrane instead they block the transport of low molecular weight hydrophobic substances of bacterial cell [50]. Among the heterocyclic ring compounds, such as benzofuran is a signi cant class of compounds obtained its vital place in numerous pharmacological areas and it could be a one such reasons for the active biological properties of natural products [51]. The derivatives of benzofuran exhibited many noteworthy activities against viruses, fungi[52] [53], protozoan, tuberculosis [54] and so on. Therefore, the wide range of biological importance coupled with this scaffold has resulted in the cyclohexane and benzofuran ring system being considered as a privileged structure. There is a strong attention is required on cyclohexane derivatives and benzofuran-based medicinal agents and the extended research and development to increase active domains of research to the whole range of medicinal areas. A great effort have been invested in the past decades to develop more effective and less toxic agents to treat many infectious and resistant strains of microbes. These cyclohexane and benzofuran based compounds as microbial agents could be a promising, as they possess the structural diversity and excellent therapeutic potency. Our antibacterial activity results supported the previous studies and further highlighted the importance of using plants as therapeutic agents.

Conclusion
The current study illustrates that the M. paniculata leaves extracts possess potent antioxidant activity. It was also observed that the M. paniculata leaf extracts could stabilize the human RBC membrane in a dose dependent manner and thus provides us with anti-in ammatory molecules. The preliminary chemical examination of different extracts reveal the presence of avonoids, alkaloids, tannins and polyphenols that might be responsible for the antioxidant and RBC membrane stabilization activities. The PC4 is the most potent hexane fraction that was bactericidal against E. coli, B. subtilius, P. aeruginosa, S. aureus and further characterization revealed the cyclohexane and banzofuran derivatives as novel antimicrobial agents. The overall results of the present study can be considered as very promising in the prospective of new anti microbial drug discovery from traditionally used plant, especially against multi drug resistant bacterial strains.

Materials And Method
5.1 Preparation of extracts:M. paniculata leaves were collected from the Amarkantak region, District Anuppur, Madhya Pradesh (India). The leaves were washed under tap water, shade dried and nally grinded it into ne powder form. The extraction was carried out using different solvents (hexane, acetone, chloroform, methanol and water) for about 7-8 hours using Soxhlet extractor and concentrated using rotary evaporator. The extract was dried and stored in vials at 4 0 C till future use.

Phytochemical screening
The phytochemical screening of all extracts was carried out according to standard methods as mentioned elsewhere with minor modi cations [55][56] [57].

Total phenolic content
The total phenolic content was determined for different M. paniculata extracts using spectrophotometric method [58]. Different extracts were taken at concentration 1 mg/mL for analysis. The reaction mixture was prepared (in triplicate) by mixing 0.5 mL of individual extracts, 2.5 mL of 10% Folin-Ciocalteu's reagent dissolved in water and 2.5 mL 7.5% NaHCO 3 . The samples were incubated for 45mins at 45 0 C and the absorbance was measured at 765 nm using spectrophotometer (Shimadzu UV-Vis spectrophotometer-1800). Based on the measured absorbance, the total phenolic content in the test samples was calculated using the calibration plot (Y= 0.00023x + 0.0380, R2= 0.998) and expressed in terms of Gallic acid equivalent (mg of GA/g of extract).

Total avonoids content:
The total avonoid content was performed as per Dowd method with some modi cations for the different extracts [59][60] [61]. Different extracts were taken at 1 mg/mL concentration for analysis and reaction mixture prepared by mixing 0.5 mL of extracts, 10% aluminium chloride (0.1mL), 1M potassium acetate (0.1mL) and distilled water (4.3 mL). Reaction mixture was incubated at room temperature for 30 mins and absorbance was measured at 510 nm .Quercetin (1mg/mL) was used for preparing the standard calibration curve. The total avonoid content in the test samples was calculated using the calibration plot ( = 0.004 + 0.158, 2 = 0.997) and expressed as mg quercetin equivalent (QE)/g of dried plant material.
ABTS + was diluted with water to obtain equilibration of absorbance 0.70 (±0.02) at 734nm and suitable blank was used without test adding samples.
50mg/mL stock solutions of plant extracts were prepared and different concentrations used for assay. The following formula was used to calculate percentage of inhibition.  [65]. The different concentrations of plant extracts (1mL of 1000, 2000, 3000, 4000, and 5000 μg/mL) were mixed with 5mL of 0.2M phosphate buffer (pH-6.6), and 5mL of 1% ferricyanide was added and incubated for 20 minutes at 50 0 C. After incubation the 10% trichloro acetic acid (TCA) added and centrifuged at 3000 rpm for 10 minutes. To the supernatant equal amount of water and 1mL of 1% ferric chloride were added, absorbance read at 700nm. Higher the absorbance value of the reaction mixture indicated better reducing power

Membrane stabilizing property
The human red blood cell (HRBC) membrane stabilization method is mainly used to estimate the anti-in ammatory activity of plant extracts [66]. The fresh blood was collected and mixed with an equal volume of sterilized Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% sodium chloride in water). (Administration of NSAIDS for 2 weeks before collecting the blood was avoided). The collected blood was further centrifuged at 3000 rpm for 10 min, and the pellet (packed cells) were washed thrice with isosaline (0.85%, pH 7.2) and nally 10% (v/v) suspension was made with isosaline. To the different concentrations of plant extracts, 1 mL phosphate buffer (0.15 M, pH 7.4), 2 mL hyposaline (0.36%), and 0.5mL HRBC suspension was added. Standard and control were prepared without addition of the extracts. Indomethacin at different concentrations (1000, 2000, 3000, 4000, and 5000 μg/mL) was used as the standard drug and compared with respective concentrations of plant extract. The reaction mixtures were incubated at 37 0 C for 30 min and centrifuged at 3000 rpm for 10 min. The hemoglobin content in the supernatant was estimated at 560 nm [67].
The percentage hemolysis was calculated using the following equation: The percentage of HRBC membrane stabilization was calculated using the following equation:  [69]. Minimum inhibitory concentration (MIC) was calculated using the same plate.

Column chromatography of hexane extract
The most active crude extract (hexane extract) was allowed to separate using gravitation column chromatography. The slurry was prepared by mixing 500g of absorbent (silica gel 60-120 mesh size) in n-hexane and stirred well to remove bubbles then poured in to glass column. The sample to be loaded on column was prepared by dissolving 5g of extractin 25ml of hexane and 20g of silica. In Table S3, we review the ratio of solvents and fractions obtained in the column chromatography. Each fraction was spotted on activated TLC plate along with extract spot and mobile phase [Hexane: acetone (3:1)] used. The fractions showing more than one spots were concentrated and allowed for further puri cation using only different solvent mixtures (Hexane and Acetone). The column chromatography was repeated as the eluent system to obtain a single spots on TLC.

Antibacterial activity
The different fractions collected from the hexane extract were further evaluated for antibacterial activity as mentioned earlier and the zones of inhibition were recorded.

GC-MSMS analysis
The