The Phytochemical analysis, Metabolic profiling, Anti-bacterial and Anti-oxidant activity of Nepeta cataria

Introduction: Nepeta cataria is a naturally growing plant in the regions of Pakistan, in English name is Catnip mint and the local name is Badranj boya. This plant has immense pharmacological and medical importance. It is used in herbal and traditional medicine as an antipathogenic agent. However detailed phytochemical analyses in a wide spectrum of solvents and their antimicrobial analysis against both Gram-positive and negative limited studied. Objectives : This study aimed to identify phytochemicals, metabolome, antimicrobial potential, and antioxidant analysis of Nepeta cataria (Pakistani plant) . Methods: Plant collection, identification, cleaning, and grinding were performed followed by extract preparation in five solvents i.e. methanol, ethanol, water, acetone, and hexane. Qualitative phytochemical analysis was performed by general biochemical tests. . GC/MS was used to identify metabolic compounds in all extracts. Antibacterial analysis for both gram-positive and negative bacteria was performed by Kirby’s Disc method, 96 well test, and Resazurin test. The antioxidant activity was determined by DPPH assay. Anthraquinones, Combined Anthraquinones, Tannins, Alkaloids, and DPPH was positive in the different extracts. The ORAC was 133759.021um/100ml in concentration. GC/MS analysis of methanolic extract (71 identified + 48 unknown),ethanol-based extract ( 80 known + 31unknown), water-based extract ( 28 known + 11 unknown ), acetone-based extract ( 13 known + 9 unknown) , hexane based extract (11 known + 8 unmatched) phytochemicals were detected. Conclusion: Current study concludes the remarkable antibacterial and antioxidant potential of Nepeta cataria extracts.


Introduction
Extremophile plants grow in harsh environments with a high degree of abiotic stress. The plant growth and development of secondary metabolites are determined by environmental conditions besides the plant genome. These plants required drought, heat, cold, and soil salinity, pH, pressure, and water shortage [1], Medicinal plants are known as drug candidates [2]. Different plants used in the medicine range is 4 -20% in various regions of the world, 2500 species are being traded [3].
In a recent study, water-based extracts of Nepeta cataria significantly inhibited the replication of the herpes virus in humans. Moreover, various phenolic compounds and flavonoids were For this test, 100 µL of TSB liquid medium was dispensed in each well, and 100 µL of plant extract concentrations were added to the wells (five wells/dilution). Bacterial inoculum (10 6 CFU/mL) was added to each well. Un-inoculated wells contain TSB medium confirmed the sterility of the medium while negative control contains only the application of TSB, bacterial agents, and Resazurin dye. After overnight at room temperature, 20 µL of Resazurin dye was added to wells and again incubated for 2 -4 h for color change. Standard reading was taken at 550-590 nm absorbances on SPECTRA MAX M2e plate reader. The resazurin-based experiment was carried out in the Food Sciences Department of UMASS, Massachusetts USA [15].

Qualitative Test for Saponin
For this test, plant extract (1 mL ) mixed in 1 ml of distilled water, shaken vigorously, and left for 15 min for the formation of Persistent frothing. Emulsion formation on adding 3 drops of olive oil showed positive results [16].

Qualitative test for Phenolic compounds
The Phenolic compounds in the plant extract sample were tested by adding 2 mL (3 % aqueous Na2CO3 ) solution in 200 µL plant extract. In the reaction mixture, 200 µL of Folin Ciocalteu reagent was added and left for 30 mint. The development of blue/grey color indicated the presence of Phenolic compounds in plant extract samples [17].

Qualitative test for Water Soluble Phenol
To indicate the presence of water-soluble phenol in the plant extract sample, 2 drops of 1 % ferric chloride solution added in plant extract (500 µL). Red color development confirmed the presence of water-soluble phenol in the test sample [18].

Qualitative test for Water Insoluble Phenol
For the analysis of water-insoluble phenol, 500 µL of plant extract added in 500 µL of CH2Cl2, 3 drops of ferric chloride, and 1 drop of Pyridine was added to this solution. The appearance of any color change confirmed the presence of water-insoluble phenols.

Qualitative test for Flavonoids
For this test, 100 µL of aqueous NaOH was added in the plant extract sample (1 mL) and intense yellow color development confirmed the presence of flavonoids in the tested sample [19].

Qualitative test for Poly steroid
Liebermann-Burchard's test was performed to detect the presence of Poly steroid in plant extract sample, 500 µL of plant extract was added with 3 drops of acetic anhydride and concentrated H2SO4 and allowed to stand for 5 min. The development of blue-green color confirmed the positive result [20].

Qualitative test for Terpenoids
Added 1 mL plant extract sample, a mixture of 400 µL Chloroform and 400 µL Concentrated H2SO4 was added. Reddish-brown coloration was noted in positive test results [21].

Qualitative test for Cardiac Glycosides
Cardiac glycosides in the test plant samples were confirmed by treating 500 µL plant extract separately with 500 µL of glacial acetic acid and a few drops of 1 % aqueous FeCl3 and H2SO4.
Green-blue color development confirmed the cardiac glycosides in plant extract [22].

Qualitative test for Free Anthraquinones
For this test 1 ml of the extract was added in 20 ml of chloroform followed by heating for 5 min on steam bath then the filtrate was allowed to cool, 20 ml of 10% Ammonia solution was added and shaken. Rose pink color development in the upper layer indicated a positive result [20].

Qualitative test for Combined Anthraquinones
Combined anthraquinones in plant extracts were confirmed by treating 450 µL of plant extract of each sample with 500 µL of chloroform and 50 µL of concentrated HCl. The appearance of rose-pink color confirmed the positive test results [20].

Qualitative test for Tannins
For this test 2 drops of 1 %, ferric chloride (Fecl3) were added to the 500 µL plant extract sample. Development of blue, green, or black color confirmed positive test results [23].

Qualitative test for Alkaloids
Alkaloids were confirmed in the plant extract sample by treating 200 µL of a plant extract with few drops of aqueous HCl followed by treatment with 500 µL of Mayer's reagent. White precipitation confirmed the alkaloids in plant extract samples [16].

Quantitative Analysis for Phenols (96 well plate method)
In this assay, 75 µL of double distilled water (DDW) was added in well followed by 25 µL sample or standard was added. Folin C (F-C reagent) 25 µL/well (diluted 1: 1 (v/v) with DDW) added , left for 6 min. After that 100 µL of Na2CO3 (75 g/L) was added, mixing, plates were put in dark for 90 mins. Absorbance was measured at 765 nm by using a SPECTRA MAX M2e plate reader. Readings for sample control (sample and DDW) were taken before proceeding further.
Gallic acid was taken as a standard at 12.5-400 µg/ml produces a calibration curve. The standard and plant extract solution was analyzed in three replications and averaged before making standard curves. Phenols were determined as μg of gallic acid equivalents / mL; calculated by the formula, y = 0.6053 x − 0.0567, where y is the absorbance at 765 nm and x is representing the amount of gallic acid equivalent in μg/Ml [24].

Quantitative Analysis for Flavonoids (96 well plate method)
To quantify flavonoids in plant extract sample, 100 µL of double distilled water (DDW) was dispensed in 96 wells, 10 µL of NaNO2 (50 g/L), and 25 µL of standard or plant extract sample added in wells followed by incubation for 5 mint at room temperature. After this, 15 µL of AlCl3 (100 g/L) was added to the mixture and left for 6 mint. After that, 50 µL of NaOH (1 mol/L) and 50 µL of DDW were added to each well and the plate was shaken for the 30s and absorbance was measurement at 510 nm. Catechin was used as a standard at 5-500 µg/mL to draw a calibration curve. All standards and plant extract samples were analyzed by using SPECTRA MAX M2e plate reader in triplicates and obtained readings were averaged before making standard curves. A standard curve of Catechin obtained and flavonoids of plant sample were expressed in µg of Catechin equivalents / mL, and were calculated by the formula, y = 0.5377 x + 0.316, where y is the absorbance at 510 nm and x is representing the amount of Catechin equivalent in μg/mL [24].

Quantitative Analysis for DPPH (96 well plate method)
For this, 200 µL of DPPH (DPPH; Sigma-Aldrich, Germany) solution (150 mmol/L) was added to each well except blank wells. The plant extract, control, or standard solutions were added 25 µL in the wells, mixed and left to react in dark for 6 h, and absorbance measured at 517 nm by using SPECTRA MAX M2e plate reader. Ascorbic acid is used as a standard at 50-500 µmol/L concentrations to draw a calibration curve. All the standards and plant extract samples were analyzed in triplicates and obtained readings were averaged before making standard curves. An ascorbic acid-based standard curve was obtained and DPPH in plant sample was expressed as µmol of Ascorbic acid equivalents / L and were calculated by the formula, y = 0.0319 x + 0.1007, where y is the absorbance at 517 nm and x is taken the amount of Ascorbic acid equivalent in µmol / L [24].

Quantitative Analysis for ORAC
Various dilutions of Trolox (75 mM concentration) and plant extract were made in 10 mM phosphate buffer with pH 7.6. Plant extract sample (20 μL) was added to each well with the pipette. In each working well, 40 µL Fluorescein of 10 nM concentration was added. Phosphate buffer (25 μL) was used as blank. Sealed microplates were incubated in a spectrophotometer at 37 ºC for 30 min. Initial fluorescence was measured after every 90 s at a wavelength of 485 nm and an emission wavelength of 520 nm by using a SPECTRA MAX M2e plate reader. After 3 cycles, 140 μL of 2,2`-Azobis(2-amidopropane) dihydrochloride (AAPH) was added to the sample containing well. After adding AAPH, the test was run again, and readings were taken up to 120 mint. The ORAC value for each plant extract was calculated against standard Trolox concentration by constructing a regression equation and standard curve [25].

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
Gas Chromatography / Mass Spectrometry (GC/MS) analysis was performed to analyze the phytochemical constituents based on the spectral output of all the compounds that get separated from plant extract sample [26]. Helium (He) (99.9 %) used as a carrier gas at a flow rate of 1 ml/min, and a volume of 2 μl was injected (split ratio of 10:1, 15:1 or 20:1) by using the split sampling technique, while injection temperature was kept 250 °C and the oven temperature was set from 60 °C for 5 min, increased at a rate of 20 °C per min to 200°C, then finally to 330 °C at the rate of 10 °C per min to 330 °C for 5 min. The total pressure was maintained up to 66.7 kPa and the total flow was 32.9 mL/min while column flow was 1.12 mL/min with a linear velocity of 38.6 cm/sec. The total GC/MS analysis time was 40 min. The relative percentage of the constituent was found out by comparing its average peak area to the total areas. Mass spectrum results of plant extract fraction were intenerated by using the database of NIST and phytochemicals were characterized and identified.

NMR-based plant metabolomics analysis
Nuclear magnetic resource analysis is a powerful tool used to detect and measure all the metabolites present in test samples both on a qualitative and quantitative basis under certain conditions. NMR is used to detect a variety of secondary metabolites along with primary metabolites. Signals generated in the NMR spectrum are proportional to the molar concentration of the compounds present, and it directly compares the concentrations of all the present compounds without making calibration curves of each compound. This technique is very helpful for structure elucidation. The following procedure is adopted to carry out this analysis.

Harvesting of Plants
Leaves from the plants were carefully harvested and were transferred to the tubes containing liquid nitrogen.

Freeze Dried sample Preparation
Frozen leaves were grind in a pre-cooled pestle and mortar in liquid nitrogen and powdered material was transferred into plastic tubes by using a spatula. Before freeze-drying, samples were kept in the deep freezer. After that, samples were put in the freeze-dryer for 24 to 48 h.

Sample Preparation and NMR Analysis
The freeze-dried sample was weighed in an Eppendorf tube. 0.75 ml of CH3OH-d4 and 0.75 ml of KH2PO4 buffer in D2O (pH 6.0) containing 0.1% (wt/wt) TSP were added to the sample and vortexed for 1 min at room temperature. The prepared sample was ultrasonicated for 10 -20 min at room temperature. The clear supernatant was obtained by centrifugation (17,000 g) of the sample for 5 -10 min at room temperature in a microtube centrifuge. The supernatant was shifted to a 1.5 ml Eppendorf tube and 800 μl of supernatant was shifted to a 5 mm NMR tube. It was then placed into the spectrometer at 25 ºC. Spectrometer frequency was locked to the deuterium resonance arising from the NMR solvents and the most suitable experiment model was used. NMR signals are considered as directly proportional to the molar concentration of the characteristic of a metabolite and the concentration of detected metabolites can be obtained by comparing the peak intensity with an internal standard (TSP).

STATISTICAL ANALYSIS
The results of all the experiments were analyzed under a complete randomized design (CRD) with three replications for each treatment. Results were statistically analyzed using Satistix 8.1 and Microsoft office excel 2010 version. Means were calculated and a One-way analysis of variance (ANOVA) test was performed for multiple comparisons of all the mean values. Mean differences were calculated by least significant difference (LSD) at 0.05 probability.

Kirby-Bauer Disk Diffusion Method for Anti-Microbial Activities
Kirby disk diffusion method used for measuring antimicrobial efficacy of plant extracts under in vitro conditions. Chloramphenicol was used as a standard antibiotic. Results indicated that Nepeta cataria ethanol-based extract showed maximum inhibition of B.
subtilis followed by C. freundii and M. luteus while methanol-based extracts also showed maximum efficacy against S. sonnei, E. coli, M. luteus, and C. freundii. Water, acetone, and Hexane based extracts were equally effective against tested bacterial isolates as shown in

Resazurin based Well Plate Microdilution Method
The Resazurin method is used to check the antimicrobial efficacy of each prepared plant extract against tested bacterial agents. Chloramphenicol was used as a positive control at

Qualitative Test for Saponin
Water-based extracts of N. cataria were positive for saponin.

Qualitative Test for Phenolic Compounds
Water-based, methanol, ethanol, and acetone based extracts showed positive results while hexane showed the negative result.

Qualitative Test for Water Soluble Phenol
Water-based and methanol extracts showed positive results while ethanol, acetone, and hexane showed negative results.

Qualitative Test for Water Insoluble Phenol
Methanol, ethanol, and acetone based extracts showed positive results while hexane and water-based showed a negative result.

Qualitative Test for Flavonoids
Test for flavonoids was carried out and the development of intense yellow color is positive test results indicate. Methanol and acetone-based extracts showed positive results while hexane, ethanol, and water-based showed negative result.

Qualitative Test for Terpenoids
For the triterpenoids testing, reddish-brown coloration development confirms the positive test results. Water-based, methanol, ethanol, acetone, and hexane-based extracts were positive.

Qualitative Test for Cardiac Glycosides
In the cardiac glycosides test, the development of green-blue color is the confirmation of positive results. Acetone -based extracts were positive while water-based, methanol, ethanol, and hexane-based extracts were negative.

Qualitative Test for Free Anthraquinones
The water-based extracts, methanol, ethanol, and acetone based extract were positive while hexane-based extracts were negative.

Qualitative Test for Combined Anthraquinones
This test was performed to confirm the presence of combined anthraquinone in the test plant sample. water-based extracts extract were positive, while methanol, ethanol, acetone, and hexane-based extracts were negative.

Qualitative Test for Tannins
In this test, water-based, methanol, and ethanol extracts were positive, while acetone and hexane-based extracts were negative

Qualitative Test for Alkaloids
In this test, water-based, methanol, ethanol, acetone, and hexane-based extracts were positive.

Quantitative Analysis for Phenols (96 well plate method)
The methanol, ethanol, water, acetone, and hexane extracts of Nepeta cataria, were examined in terms of μg of Gallic Acid Equivalents per mL. Methanol, acetone, and ethanol-based extracts showed the maximum presence of phenols as compared to water and hexane-based extracts. The order of phenol presence in the sample was Methanol extracts > ethanol extracts >acetone extracts > > water extracts > hexane extracts showed in

Quantitative Analysis for Flavonoids (96 well plate method)
The Flavonoids in methanol, ethanol, water, acetone, and hexane extracts of

Quantitative Analysis for DPPH (96 well plate method)
The presence of 2, 2-diphenyl-1-picrylhydrazyl DPPH was determined in Nepeta cataria extracts by using different solvents viz., methanol, ethanol, water, acetone, and hexane which measured by Spectrophotometrically and results are drawn as µmol of ascorbic acid equivalents / L and results are given in Figure 6. Results showed that acetone-based plant extract contained a high concentration of DPPH followed by water-based extracts and results compared with hexane extracts, ethanol, and methanol-based extracts. The presence of DPPH was in order acetone extracts > water extracts> ethanol extracts > methanol extracts > hexane extracts.

Quantitative Analysis for Alkaloids
The alkaloid percentage method was used to quantify the alkaloids in plants. Alkaloids quantity in tested plant extracts was varied from 0.204 / 0.5 g plant.

Oxygen Radical Absorbance Capacity (ORAC)
Oxygen Radical Absorbance Capacity Assay (ORAC) was performed to study the antiradical activity of Nepeta cataria prepared in methanol extract. The results of the ORAC test was 133759.021um/100ml in concentration.  (Table 2). Water-based extracts of Nepeta cataria contain 28 known phytochemicals while 11 un-matched chemicals were also detected ( Table 3). Acetone-based extract confirmed the existence of 13 known compounds extract while 9 chemical constituents were unmatched ( Table 4). Analysis of hexane based extract confirmed the presence of 11 known chemical constituents while 8 unmatched chemicals were detected as given in Table 5. GCMS spectral chromatograms of all the solvent-based extracts are given in Figure; 7-11.          The current study result was remarkable against the antibacterial and antioxidant activity. This plant is a natural source of various pharmacological and organic compounds that can be used against several bacterial infections after processing. It may help out to treat different antibiotic-resistant pathogens. Its chemicals may be used in pharmacology industries as a cheaper and easily available source.

Conclusion
The current study concludes remarkable antibacterial and antioxidant potential in Nepeta cataria extracts. This plant is a natural source of various pharmacological and organic compounds that can be used against several bacterial infections after processing.