Chemical profiling, in vitro antioxidant, membrane stabilizing and antimicrobial properties of wild growing Murraya paniculata from Amarkantak (M.P.)

The excessive usage of antibiotics in humans and veterinary medicine has lead to the emergence of antibiotic resistance and now requires the use 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-inflammatory activities of the leaf extracts of Murraya paniculata, a plant traditionally used in Indian medicinal system. Evaluations were made for phytochemical analysis, antioxidant, membrane stabilizing, and antimicrobial activities. The methanol extract displayed the highest flavonoid and phenolic content, the acetone extract demonstrated considerable ABTS inhibitory activity (IC50value:555.18 ± 1.68 µg/mL) and the hexane extract exhibited highest H2O2 radical scavenging activity (IC50value: 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 the 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%) as analyzed by GC–MS/MS technique. The current results validate the traditional use of the M. paniculata and warrant its potential in drug development programs in further investigations.

www.nature.com/scientificreports/ Phytoconstituents analysis. Alkaloids, flavonoids, 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).
Total phenolic and total flavonoid content. The total phenolic and total flavonoid content in the different extracts of M. paniculata is represented in Table 1. Among the five extracts, methanol extract possesses highest phenolic content (1060 ± 52.83 mg gallic acid equivalent/g dry material) followed by acetone (849.8 ± 49.2 mg/g), water (114.4 ± 10.49 mg/g), chloroform (21.32 ± 3.6 mg/g), and hexane (22.17 ± 5.5 mg/g). The total flavonoid concentration in different solvent extracts was estimated using quercetin as a standard and values expressed in mg quercetin equivalent /g dry wt. Out of five extracts the methanol (318.4 ± 9.16 mg) contains highest flavonoid content followed by water (244.8 ± 7.98 mg), acetone (121.8 ± 4.42 mg), hexane (43.75 ± 1.5 mg), and chloroform (42.83 ± 6.66 mg).
Antioxidant activity. Supplementary figure (Fig. S1) shows the concentrations of plant extract with percentage inhibition of ABTS radical. The acetone extract demonstrated elevated ABTS + inhibition with increased concentration. For ABTS + inhibition the IC 50 valuesranged between 500-800 µg/mL ( Table 2). The IC 50 value of acetone (555.18 ± 1.68 µg/mL) were less than the standard (ascorbic acid) used. The percentage of 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 ± 3.03 µg/mL) was lowest as compared to the standard (ascorbic acid) or other extracts that were analyzed. These result demonstrate 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 ± 0.002 Fe2+/gram sample) showed highest value, followed by acetone, water, chloroform, and hexane (Table 2) The reducing power 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 membrane stabilization assays are presented in Table 3. It was observed that all the extracts of M. paniculata exhibited concentration-dependent anti-inflammatory properties as compared to the standard drug, indomethacin. The maximum concentration analyzed was 5000 µg/mL and extracts were found to possess anti-inflammatory properties 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. 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 1. Quantification of total Phenolic and Flavonoid contents in M. paniculata leaves extracts. 1 Total phenolic content and 2 Total flavonoids content expressed as Mean ± SE (n = 3). The superscripted alphabets represent the number of extracts over which the values of the given extract are very highly significant (***). a : four extracts, b : three extracts, c : two extracts.  (Fig. 2). The growth of P. aeruginosa was inhibited by all extracts of M. paniculata. Growth inhibition was observed in standard ciprofloxacin (MIC of 25 µg/mL) against all tested bacterial strains. The MIC for different extracts was as 25 µg/mL for aqueous extract while 250 µg/mL for hexane, methanol and chloroform extracts and 500 µg/mL for acetone extract. The hexane extract displayed good inhibition activity against all the bacterial strains as compared to other tested extracts. All extracts were found inactive against fungi as we did not observe any zone of inhibition even after repeated experiments.
Fractionization of hexane extract. 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 spots 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, and PC22, and were used for further antibacterial activities.  www.nature.com/scientificreports/ Antibacterial activity. Table 5 presents antimicrobial activity against selected multi resistance bacteria (E.

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 for snakebite. Therefore, M. paniculata leaves were selected for investigation of antioxidant, membrane stabilizing, and antimicrobial properties stemming from phytochemical features. Phytochemical screening determines the presence of alkaloids, flavonoids, steroids, tannins, saponins and glycosides that are known to have protective or disease preventive properties. Several phytochemicals play vital roles in alleviating the several health problems like malaria, asthma, cancer, arthritis, jaundice, diabetes, dengue, diarrhea, dysentery, and microbial infections 45 . In the current study, secondary metabolites such as alkaloids, flavonoids, steroids andtannins were present in majority of extracts, corroborated by earlier studies 46 Alkaloids, tannins flavonoids and other metabolites are reported to have antioxidant, antifungal, anticancer, antiviral, anti-inflammatory and antiophidic activities 47 . Alkaloids have been the basis of several antibacterial drugs and serve as scaffolds for important antibacterial drugs such as metronidazole, quinolones, linezolid, and trimethoprim 48,49 . More than 8000 flavonoids have been reported from different plants species 50 . That play vital role in stimulation, protection, flavoring, communication, and pigmentation 51 . Flavonoids are rich source of antioxidants mainly due to their free radical scavenging activity by the transfer of an H atom or of a single electron to the radical stabilizing it or due to their metal chelating activity 52,53 . The flavonoids are known to possess anti-allergic, hepatoprotective anticancer, antibacterial, anti-inflammatory, anti-diabetic, and anti-viral properties [54][55][56][57][58] . Saponins, a vast group of compounds are characterized by their foam forming and detergent properties [59][60][61] . They are known to have several biological properties like antimicrobial, immunomodulatory, anti-malarial, anti-allergic, anti-diabetic, insecticidal, and anti-inflammatory [62][63][64][65][66] . The tannins are water soluble polyphenolic compounds synthesized by plants 62 and are responsible to protect plants against herbivores and insects 67,68 . Tannins have also been reported to have anti-cancer, anti mutagenic, anti-oxidant, antibacterial, www.nature.com/scientificreports/ anti-viral, anti-tumor, anti-inflammatory effects and wound healing capabilities [68][69][70][71][72][73] . The presence of all these phytochemicals makes it understandable why M. paniculuta leaves are a prominent medicine for treatment of various diseases within tribal communities.
Here, the total phenolic content was present in high concentrations in methanolic extract followed by acetone, water, chloroform, and hexane while the total flavonoid content was highest in methanol extract followed by water, acetone, hexane and chloroform. Antioxidant activity of flavonoids is due to their ability to reduce free radical formation and to scavenge free radicals. Previously  Significant concentration-dependent inhibition of ABTS free radicals was observed in all the tested extracts. The IC 50 value observed for acetone extract was less than the standard ascorbic acid, indicating the strong ability of the extracts to act as ABTS and H 2 O 2 scavenger. Significant 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 efficient. The reducing capacity of antioxidant is mainly because of their electron transfer property for instance polyphenols and flavonoids 81 . 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 82 . The higher values shows, higher antioxidant property of extracts. Our study clearly indicated that the methanolic extract of the leaves exhibits high scavenging capacity, demonstrating that the high antioxidant capacity of the methanol extract correlates with the total content of phenolic and flavonoid content (i.e., higher the absorbance shows higher antioxidant properties). The present study also demonstrated the results that total antioxidant reducing power of compound/extract is depending on the amount of phenolic and flavonoid content. The comparison between the results of total phenolic content and reducing power assay is found to be in similar order: MPM > MPW > MPA > MPH > MPC. Whereas the results of total flavonoid content and FRAP values found to be in order of: MPM > MPA > MPW > MPC > MPH. Many studies have demonstrated a 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 83,84 .
Low-grade inflammatory state is correlated with various disorders and chronic health conditions, such as obesity, diabetes, cancer, and cardiovascular diseases. Living tissues respond to injury, infection or irritation by releasing inflammatory lysosomal enzymes that may damage macromolecules and cause lipid peroxidation of membranes. Plant extracts can be utilized for stabilization of lysosomal membranes or control of major pro-inflammatory mediators by inhibiting the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases. Human red blood cell (HRBC) or erythrocyte membranes are analogous to lysosomal membranes and have been used in stabilization assays. The antioxidant activity of the M. paniculata extracts correlated well with the RBC membrane stabilization properties. At the concentrations of 4000-5000 µg/ mL, 60-80% protection was observed, which was comparable to the standard drug indomethacin in RBC hemolysis with water and methanol extract. Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, acts by inhibiting the enzyme cyclooxygenase, but the use of NSAIDs is questionable due to the emerging evidence suggesting a high risk of acute myocardial infarction, stroke, heart failure, renal failure, and arterial hypertension 85 . Several medicinal plants have been reported to have phytoconstituents such steroids, flavonoids, alkaloids, polyphenols, glycosides, terpenoids, curcumins, GLA, linear aliphatic alcohols, harpagoside, phenolic diterpenes, which have anti-inflammatory properties with minimal side effects 14 . The results of our study clearly suggest that M. paniculata extracts may provide us with some potent compounds for the management of inflammatory conditions. The presence of saponins and flavonoids in the methanolic and water extracts also support the current findings as they have been known to have profound stabilizing effect on lysosomal membrane 86 . It has been reported that flavonoids isolated from M. paniculata displayed anti-inflammatory activity by inhibition of NO and IL-6 production 87 . The development of flavonoid based phytotherapeutic anti-inflammatory agents, Acheflan and Daflon, gives us hope for the development of drugs without adverse effects 85 . Novel safe and promising antimicrobial agents are urgently required to fight against drug-resistant bacterial and fungal Table 5. Anti bacterial activity of fractions of M. paniculata hexane extracts. Ec: Escherichia coli, Bs: Bacillus subtilis, Pa: Pseudomonas aeruginosa, Sa: Staphylococcus aureus. NF: Not found any zone of inhibition. 1 n = 3 Standard error was < 15% of mean in all cases. P value (***) represents very highly significant difference over the standard group. www.nature.com/scientificreports/ www.nature.com/scientificreports/ strains. Flavonoids are known to inhibit microbial growth by inhibition of nucleic acid synthesis, cytoplasmic membrane function and energy metabolism pathways 88 . 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 89 . Tannins have been shown to possess inhibitory activities against Methicillin-resistant Staphylococcus aureus (MRSA) 90 or bacteria, yeast and fungi 91 . In the current study all the extracts of M. paniculata displayed some level of inhibition of bacterial growth, but no antifungal activity was observed displaying its potency for the development novel antibiotics. Here, 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. aeruginosa. Fractionization of hexane extract yielded six fractions that revealed promising antibacterial potency. 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. P. aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals or cystic fibrosis patients 92 and displays remarkable antibiotic resistance capacities. It has been listed by WHO as one of the three bacterial species against which there is critical need for the development of new antibiotics 93 . Similarly, methicillin-resistant S. aureus have been associated with hospitals settings and has emerged as a widespread cause of community infections. The inhibition of both these strains by PC4 predicts it to be a source of novel antibacterial compound/s specifically against P. aeruginosa and S. aureus. The GC-MS/MS analysis of PC4 fraction revealed a presence of cyclohexane and 3-(6-Methoxy-3-methyl-2benzofuranyl) cyclohexanone that might be a reason for the observed 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 94 . The heterocyclic ring compounds, such as benzofuran, is a significant class of compounds obtained. This Table 6. The major components found in the PC4 fraction (Acetone : Methanol) of Hexane extract.  www.nature.com/scientificreports/ class of compounds is vital in numerous pharmacological areas and could be a one such reasons for the active biological properties of natural products 95 . The derivatives of benzofuran exhibited many noteworthy activities against viruses, fungi 96,97 protozoan, tuberculosis 98 and so on. Therefore, the wide range of biological importance coupled with this scaffold has resulted in the cyclohexane and benzofuran ring systems being considered as a privileged structure. Much attention is warranted for cyclohexane derivatives and benzofuran-based medicinal agents, hence it is important that research and development on these compounds be increased across a wide range of medicinal areas. Efforts have been invested in the past several decades to develop more effective and less toxic agents to treat many infectious and resistant strains of microbes. Cyclohexane and benzofuran based compounds as microbial agents could be a promising, as they possess structural diversity and excellent therapeutic potency. Our antibacterial activity results coupled with previous studies emphasizes the importance of using plants as critical therapeutic agents.

Conclusion
The current study illustrates that the M. paniculata leaves extracts possess potent antioxidant activity and could stabilize the human RBC membranes in a dose-dependent manner, thus providing novel anti-inflammatory compounds. A preliminary chemical examination of different extracts reveals the presence of flavonoids, alkaloids, tannins, and polyphenols that might be responsible for the antimicrobial, antioxidant, RBC membrane stabilization, and anti inflammatory properties. The PC4 is the most potent hexane fraction that was bactericidal against E. coli, B. subtilis, P. aeruginosa, S. aureus, and further characterization revealed the presence of cyclohexane and banzofuran derivatives that might be responsible for the observed antimicrobial activity. The overall results of the present study demonstrate that traditionally used plants provide extremely promising prospects compounds for the development of novel anti-microbial drugs ; especially as treatments against multi-drug resistant bacterial strains.

Materials and method
All the methods were carried out in compliance with relevant guidelines. Total phenolic content. The total phenolic content was determined for different M. paniculata extracts using spectrophotometric methods 102 . 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 = 0.00024x + 0.03510, R2 = 0.9963) and expressed in terms of Gallic acid equivalent (mg of GA/g of extract).

Preparation of extracts.
Total flavonoids content. The total flavonoid content was performed as per Dowd method with some modifications for the different extracts [103][104][105] . 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 (0.1 mL), 1 M potassium acetate (0.1 mL) and distilled water (4.3 mL). Reaction mixture was incubated at room temperature for 30 min and absorbance was measured at 510 nm .Quercetin (1 mg/mL) was used for preparing the standard calibration curve. The total flavonoid content in the test samples was calculated using the calibration plot (Y = 0.002x + 0.158, R 2 = 0.997) and expressed as mg quercetin equivalent (QE)/g of dried plant material.
Anti oxidant assays. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. ABTS is a synthetic radical widely used for both the polar and non-polar samples 106  A 0 --Absorbance of blank, A 1 --Absorbance of Sample. Reducing power assay (RPA). The total reducing power of extracts was estimated by the method of Oyaizu et al. (1986) 109 . The different concentrations of plant extracts (1 mL of 1000, 2000, 3000, 4000, and 5000 μg/mL) were mixed with 5 mL of 0.2 M phosphate buffer (pH-6.6), and 5 mL of 1% ferricyanide was added and incubated for 20 min at 50 0 C. After incubation the 10% trichloro acetic acid (TCA) added and centrifuged at 3000 rpm for 10 min. Equal amounts of water and 1 mL of 1% ferric chloride were added to the supernatant, absorbance read at 700 nm. Higher the absorbance value of the reaction mixture indicated better reducing power.
Membrane stabilizing property. The HRBC membrane stabilization method is mainly used to estimate the anti-inflammatory activity of plant extracts 110 . 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). The administration of NSAIDS for 2 weeks before collecting the blood was avoided in sampled participants. The collected blood was further centrifuged at 3000 rpm for 10 min, the pellet (packed cells) was washed three times with isosaline (0.85%, pH 7.2), and finally 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 0.5 mL 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 111 . The percentage hemolysis was calculated using the following equation: The percentage of HRBC membrane stabilization was calculated using the following equation: Antimicrobial assay. Crude extracts were evaluated for their antimicrobial properties against selected bac- 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 500 g 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 5 g of extracting 25 ml of hexane and 20 g 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 spot were concentrated and allowed for further purification 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. ried out on Agilent technologies model 7890A GC coupled with a mass detector 5975C MS system. The Analytic column was Agilent J&W non polar column DB-5MS ((5%-Phenyl)-methylpolysiloxane, 30 m × 0.25 mm, ID 1.8 micron thickness). Flow rate of helium gas is 1.3 ml/min, used to separate components. The different GC conditions were standardized as follows: injector parameters were injection volume 1 μL under split of 3:1, while injector temperature was set at 280 °C (mass analyzer). During GC extraction the program of oven temperature was 1 min at 75 °C, increased to a temperature of 300 °C at a rate of 30 °C/min for 2 min; inlet and transfer line temperature was 250 °C and 290° C respectively. Mass spectra were taken at an ionization mode with an electron impact at 70 eV. Interpretation of mass spectrum GC-MS/MS analysis was done by matching list of known compound's spectrum with Agilent's GC/MS Chemstation, NIST MS Library and NIST's Automated Mass Spectral Deconvolution and Identification software 114 .

Statistical analysis
All data were expressed as mean ± SE (n = 3) . Comparison of mean values between various extracts of M. paniculata was performed by one way-ANOVA, correlation coefficient (r), and coefficient of determination (r2) calculated using prism 8.0.1(244). The criterion of evaluating statistical significance was as follows: P value < 0.033 was considered significant and marked as *, P < 0.002 as highly significant and marked as **, P < 0.001 was very highly significant and marked as ***.
Ethics approval and consent to participate. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Ethics Committee of IGNTU (Approval no. IGNTU/IEC/03/2019) and with the 1964 Helsinki declaration and its later amendments. Researcher's blood was used for the membrane stabilizing assay and no external participant was enrolled for the study.

Data availability
The data can be accessed/shared to the public. www.nature.com/scientificreports/