CHEMICAL PROFILING, ADME PREDICTION OF BOERHAVIA DIFFUSA LINN. AND CRATEVA NURVALA BUCH. HAM IN THE MANAGEMENT OF UROLITHIASIS

CHEMICAL PROFILING, ADME PREDICTION OF BOERHAVIA DIFFUSA LINN. AND CRATEVA NURVALA BUCH. HAM IN THE MANAGEMENT OF UROLITHIASIS

S. R. Gophane ^* and C. N. Khobragade

School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded - 431606, Maharashtra, India.

ABSTRACT: Background: This study was carried out to investigate qualitative and quantitative phytochemical analysis and free radical scavenging activity of Boerhavia diffusa (root) and Crateva nurvala (bark) along with certain computational ADME (Absorption, Distribution, Metabolism, Excretion) parameters of compounds analyzed from Boerhavia diffusa (root) and Crateva nurvala (bark). Methods: The dried powder of plants were extracted with increasing polarity of solvent namely ethyl acetate, acetone, ethanol and distilled water by a three-step sequential extraction procedure. The chemical compositions of extracts were investigated using UV-Vis, FTIR and PerkinElmer Gas Chromatography-Mass Spectrometry. Ethanolic extract of Boerhavia diffusa and Crateva nurvala was evaluated in vitro for its ability to inhibit the major enzyme activities of GOX and LDH through spectrophotometrically and mode of inhibition were evaluated using Lineweaver-Burk plots while ADME parameters were derived from ADMET server admet SAR. Results: Among all extracts, ethanolic extracts showed the highest phenol, flavonoid, proanthocyanidins, and coumarin content. High DPPH free radical quenching activity was observed in ethanolic, ethyl acetate and acetone extracts of the plant which was significantly comparable with ascorbic acid. Also, plant extracts, analyzed by UV-FTIR and GC-MS which showed promising Glycolate oxidase and Lactate dehydrogenase inhibition and found to contain phenolic, glycosides and flavonoids compounds. Conclusion: The FTIR and GC-MS spectrum profile of the medicinally important plant extract having various bioactive compounds and interestingly from the absorption, distribution, metabolism, and excretion (ADME)-Toxi-city analysis the compound have enhanced pharmacological properties and can be used as an herbal alternative for the synthesis of antiurolithiac agents.

DPPH Assay, ADME, UV-VIS, FTIR, GC-MS analysis,  Boerhavia diffusa, Crateva nurvala

INTRODUCTION: Urolithiasis is a complex process that results from several physicochemical events including crystal nucleation, aggregation, and growth of insoluble particles in the kidney ¹.

Calculogenesis is influenced by the interplay of critical factors, viz. stone inhibitors, complexing agents and stone promoters. The sequence of events in the formation of any urinary stone can be:

Urinary saturation     super saturation      nucleation

crystal growth crystal aggregation crystal retention    stone formation.

Urolithiasis affects 10-12% of the world population, especially in the industrialized countries. It has a recurrence rate of 50% ².

In spite of substantial progress in the study of the biological and physical manifestations of kidney stones, there is no satisfactory drug to use in clinical therapy ³. Therefore, it is worthwhile to look for an alternative for the management of urolithiasis. Herbal drugs are essential components of traditional medicine in several countries including India. Boerhavia diffusa and Crateva nurvala are important medicinal plants much used in Ayurveda and Unani medicines and other traditional medicines in many parts of the world. Crataeva nurvala Buch.-Ham. belongs to the family Capparaceae. The stem bark is being widely used as a single drug or in compound formulations for the treatment of urinary disorders including urolithiasis, prostatic hypertrophy, blood purifier, skin infections, neurogenic bladder and chronic urinary infections ⁴. Boerhaavia diffusa Linn. belongs to the family Nyctaginaceae and is commonly known as, punarnava. Traditionally, this plant has been extensively used in the treatment of dyspepsia, jaundice enlargement of liver, abdominal pain and as an antistress agent. It also has diuretic, anti-inflammatory, antidiabetic, antibacterial and anticancer properties ^{5, 6}.

According to the World Health Organization (WHO) in 2008, more than 80% of the world's population relies on traditional medicine for their primary healthcare needs ⁷. Phenolics and flavonoids are polyphenols, an important class of secondary plant metabolites possessing an impressive array of pharmacological activity which includes free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action, etc. ⁸ Oxidation reaction can produce free radicals, which can damage cells and tissues. Recently, there has been an upsurge of interest in the therapeutic potential of medicinal plants as antioxidants in reducing such free radical-induced tissue injury.

As a result, antioxidants are often reducing agents such as phenolic compounds (flavonoids, phenolic acids), sugars, vitamins, saponins, ethereal oils, polyunsaturated fatty acids, phospholipids, enzymes, amino acids, thiols, etc. As every plant has its metabolite fingerprinting, profiling by some platform technology such as gas chromatography-mass spectrometer (GC-MS), liquid chromatography-mass spectrometer and nuclear magnetic resonance, it is crucial to have a holistic overview of all metabolites present in the extracts ⁹. Spectroscopic (UV-Vis, FTIR) methods are a simple, cost-effective and rapid test for detecting phytocomponents. Hence, the present study is designed to evaluate the phytochemical profile of Boerhavia diffusa and Crateva nurvala with the aid of GC-MS, UV-VIS, and FTIR Techniques and computer analysis using ADME to ascertain the rationale for its use in traditional medicine.

MATERIALS AND METHODS:

Drugs: Boerhavia diffusa (root) and Crateva nurvala (bark) were procured from Yogesh pharma Pvt. Ltd., Nanded (MS), India. The barks and roots were washed thoroughly with water to remove dust and dried under the shade at room temperature for 5 days. The dried parts were ground using a blender to obtain the coarse powder and kept in an airtight container until further use. Powdered plant material (150 g) were successively extracted with Soxhlet apparatus using the solvents in order of increasing polarity viz., C), ethyl acetate, acetone, ethanol, and water. Each time the marc was dried and later extracted with other solvents. All the extracts were concentrated by distilling the solvent in a rotary vacuum evaporator.

Phytochemical Screening of Bark Extract: The extracts were subjected to phytochemical tests to investigate the presence or absence of active secondary metabolites using standard procedures ¹⁰.

Quantitative Estimation of Phytoconstituents:

Quantitative Estimation of Total Phenols: Folin-Ciocalteu reagent was used to determine the total phenolic content (TPC) of the various organic crude extracts with slight modification ¹¹. 1.5 ml Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) was added to the 0.5 mL of the plant extract sample, and was vortexed for 5 min, followed by addition of 3 mL of sodium carbonate solution (7.5%). This reaction mixture was incubated for 30 min at room temperature in the dark. The absorbance of the resulting blue color was measured by using double beam UV-Vis spectrophotometer (UV Shimadzu- 1800) at a fixed wavelength of 760 nm. The calibration curve was prepared by employing gallic acid at concentrations of 10 to 100 μg/ml Fig. 1.

The TPCs were determined using a linear regression equation obtained from the standard plot of gallic acid. The content of total phenolic compounds was calculated as mean ± SD (n=3) and expressed as mg/g gallic acid equivalent (GAE) of dry extract.

Quantitative Estimation of Total Flavonoid: Aluminum chloride colorimetric method was used for flavonoids determination as described by ¹¹. 0.5 mL of each plant extract was mixed with 1.5 mL of ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. The reaction mixture was allowed to stand at room temperature for 30 min, and the absorbance of the reaction mixture was measured at 415 nm.

The calibration curve was prepared by using rutin at concentrations of 10 to100 μg/ml in ethanol Fig. 1. The amount of flavonoid was calculated from the linear regression equation obtained from the rutin calibration curve. The flavonoid content was calculated as mean ± SD (n=3) and expressed as mg/g of rutin equivalent (RE) of dry extract.

Quantitative Estimation of total Proanthocyanidins: The total proanthocyanidin was determined using the procedure reported by Sun et al. ¹² A volume of 0.5 mL of 0.1 mg/mL of extract solution was mixed with 3.0 mL of 4% vanillin-methanol solution and 1.5 mL hydrochloric acid, the mixture was allowed to stand for 15 min at room temperature, the absorbance was measured at 500 nm. Total proanthocyanidin contents were expressed as catechin (mg/g) using the following equation of the curve: Y= 0.69x + 0.076, R2 = 0.970, where x is the absorbance and Y is the catechin equivalent Fig. 1.

Quantitative Estimation of Total Coumarin: Coumarin content was determined with slight modification in procedure reported by Bruna Medeiros-Neves et al. ¹³ A volume of 0.5 mL of the extract was mixed with 2 ml of distilled water and 0.5 mL of lead acetate solution. The sample was shaken, and then 7 mL of distilled water was added before transferring 2 mL of this solution to a new test tube and added 8 mL of hydrochloric acid solution. The sample was incubated at room temperature for 30 min, and the absorbance was measured at 500 nm. Total coumarin contents were expressed as coumarin (mg/g) using the following equation of the curve: Y= 0.050x + 0.022, R2 = 0.992, where x is the absorbance and Y is the coumarin equivalent Fig. 1.

Free Radical Scavenging Activity by DPPH Assay Method: The capacity of the extracts and ascorbic acid to scavenge the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical was measured as per reported method ¹⁴. 0.1 mL of extracts and ascorbic acid of different concentrations (10 to 200 μg/ml) was mixed with 2.9 mL of 0.1mM DPPH solution. The solution was rapidly mixed and allowed to stand in the dark at room temperature for 30 min. The blank was prepared similarly without extract or ascorbic acid. The decrease in absorbance of each solution was measured at 517 nm using UV-Vis spectrophotometer.

The negative control was prepared by mixing 0.1 mL of ethanol with 2.9 mL of DPPH solution. The percentage of radical scavenging activity of tested extracts and positive control ascorbic acid was calculated by using the following formula:

Free radical scavenging activity (%) = [Ac- As] × 100 / Ac

Where Ac=Absorbance of control at 517 nm and As=Absorbance of the sample.

The concentration of the sample required to scavenge 50% of DPPH free radical (IC₅₀) was determined from the curve of percent inhibitions plotted against the respective concentration.

UV-Vis and FTIR Spectroscopic Analysis: The ethanolic extracts were examined under visible and UV light for proximate analysis. For UV-VIS and FTIR spectrophotometer analysis, the extracts were centrifuged at 5000 rpm for 15 min and filtered through Whatman no. 1 filter paper. The sample is diluted to 1:10 with the same solvent. The extracts were scanned in the wavelength ranging from 200-1100 nm using UV-1800 Shimadzu UV Spectrophotometer, and the characteristic peaks were detected.

FTIR analysis was performed using Thermofisher Scientific, model- Nicolet 6700 FTIR system in a scan range of 400-4000 cm^-1 and characteristic peaks and their functional groups were detected.

Gas Chromatography-Mass Spectrometry Analysis: The GC-MS analysis of the ethanolic extracts were carried out using a Agilent 7890 A gas  chromatogram equipped  and coupled to a mass detector 5975 MSD spectrometer with DB 5 MS and 30 m × 0.25 µm DF of the capillary column. Ultra-high purity helium (99.99%) was used as carrier gas at a constant flow rate of 1.0 mL/min. The injection, transfer line and ion source temperatures were at all 290 °C. The ionizing energy was 70 eV. Electron multiplier voltage was obtained from autotune. The oven temperature was programmed from 60 °C (hold for 2 min) to 320 °C at a rate of 3 °C/min. The crude sample was diluted with an appropriate solvent (1/10, v/v) and filtered.

The particle-free diluted crude extracts (1 μL) were taken in a syringe and injected into injector with a split ratio of 30:1. All data were obtained by collecting the full-scan mass spectra within the scan range 30-600 amu. The percentage composition of the crude extract constituents was expressed as a percentage by peak area. The identification and characterization of chemical compounds in ethanolic crude extract were based on GC retention time. AMDIS and NIST Version-Year 2011 was used MS data library and comparing the spectrum obtained through GC-MS compounds present in the plant’s sample was identified.

Lactate Dehydrogenase Enzyme Inhibition Assay: ¹⁵ Assay mixture contained 0.3mM NADH, 2.0 mM pyruvate and 100 µL enzyme in a volume of 3.0 ml. The reaction was started by addition of enzyme. Lactate dehydrogenase inhibitory activity of test plant extracts was monitored spectrophotometrically following the absorbance at 340 nm under aerobic condition. The reaction mixture containing 0.3 mM NADH, 100 µL enzymes in the volume of 3.0 mL and a solution of test plant extracts in DMSO was incubated at room temperature for 15 min. The reaction was started by addition of 2 mM pyruvate, and l-lactate formation was then followed by a measure of decreasing absorbance at 340 nm. The inhibitory activity of each test compound was indicated by their IC₅₀ values calculated using linear regression curve.

The percent inhibition of enzyme activity was calculated using standard formula ¹⁶.

Glycolate Oxidase Enzyme Inhibition Assay: ¹⁵ Each assay contained 200 µM potassium phosphate (pH 7.0), 1 mg of bovine serum albumin, 3 µM EDTA, 0.1 µM DCIP, enzyme and water to a volume of 3 ml and a solution of test plant extracts in DMSO was incubated at room temperature for 15 min.

The reaction was started by addition of 2 µM Sodium glycolate was then followed by a measure of the decrease in absorbance at 600 nm. The inhibitory activity of each test compound was indicated by their IC₅₀ values calculated using linear regression curve. The percent inhibition of enzyme activity was calculated using standard formula ¹⁶.

Lineweaver-Burk Plots: This kinetics study was carried out in the absence and presence of active compounds with varying concentrations of substrate. The initial velocity was expressed as the absorbance decrease at 340 nm for lactate dehydrogenase and 600 nm for glycolate oxidase per 10 s in the assay and Lineweaver–Burk plot analysis was performed ¹⁷.

ADMET Predictions: ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analyses constitutes the pharmacokinetics of a drug molecule ¹⁸. In this study, prediction and significant descriptors of drug-likeness such as mutagenicity, toxicological dosage level and pharmacologically relevant properties of the compounds were predicted using Swissadme (http://www. swissadme.ch) and admetSAR (lmmd.ecust.edu. cn:8000) servers.

Data Analysis: The results are expressed as mean ± SD. Student’s t-test and one way ANOVA were applicable and used to analyze the level of statistical significance between groups. P<0.05 were considered statistically significant. Linear regression analysis was used to calculate the IC₅₀ values.

RESULTS:

Preliminary Phytochemical Screening: Preliminary phytochemical analysis showed the presence of major classes of secondary metabolites such as Alkaloids, tannins, saponins, flavonoids, cardiac glycosides, etc. in extracts Table 1a and 1b.

TABLE 1A: PRELIMINARY PHYTOCHEMICAL SCREENINGS OF BOERHAVIA DIFFUSA (PUNARNAVA) ROOT EXTRACT

TABLE 1B: PRELIMINARY PHYTOCHEMICAL SCREENINGS OF CRATAEVA NURVALA (VARUNA) BARK EXTRACT

Quantitative Estimation of phytoconstituents and Free Radical Scavenging Activity: The present study revealed a relatively good amount of phenols, flavonoids, coumarin and proantho-cyanidins contents of ethanolic extract of Boerhaavia diffusa and Crataeva nurvala as shown in Table 2. The correlation coefficient of total phenols, total flavonoids, coumarin, and total proanthocyanidins were (r =0.993, 0.984, 0.992 and 0.970) respectively Fig. 1.

TABLE 2: QUANTITATIVE ESTIMATION OF TOTAL PHENOLICS, FLAVONOIDS, FLAVONOLS, PROANTHOCYANIDINS AND COUMARIN (n=3)

FIG. 1: STANDARD CALIBRATION CURVE FOR TOTAL STANDARD CALIBRATION CURVE FOR TOTAL PHENOLIC CONTENT (A), TOTAL FLAVONOIDS CONTENT FOR STANDARD RUTIN (B), TOTAL PROANTHOCYANIDINS CONTENT FOR STANDARD CATECHIN (C) AND TOTAL COUMARIN FOR STANDARD COUMARIN (D).

The antioxidant activity of Boerhavia diffusa and Crataeva nurvala extracts in solvents of varying polarity were measured regarding hydrogen donating or radical scavenging ability. The method is based on the reduction of alcoholic DPPH solutions in the presence of a hydrogen donating antioxidant. DPPH solutions show a strong absorption band at 517 nm appearing as a deep violet color. The absorption vanishes and the resulting decolorization is stoichiometric concerning the degree of reduction. The results of the free radical scavenging activity (%) of the Boerhavia diffusa and Crataeva nurvala extracts assessed by DPPH assay and amount of the sample needed for 50% inhibition of free radical activity, IC₅₀ values were summarized in Fig. 2 and Table 3.

FIG. 2: FREE RADICAL SCAVENGING ACTIVITY (%) OF BOERHAVIA DIFFUSA AND CRATAEVA NURVALA

TABLE3. IC₅₀ VALUE OF FREE RADICAL SCAVENGING ACTIVITY BY DPPH METHOD

Values are expressed in mean ± SD; *: P<0.05

Boerhaavia diffusa ethonolic extract was having phenolic (23.9 ± 0.25 mg/g of gallic acid equivalent), flavonoids (13.8 ± 0.13 mg/g of rutin equivalents), coumarin (1.48 ± 0.1 mg/g coumarin equivalent) and proanthocyanidins contents (1.6 ± 0.11 mg/g equivalent of catechin). The Crataeva nurvala extract showed the least concentration of phenol (18.2 ± 0.50 mg/g of gallic acid equivalent), flavonoids (11.9 ± 0.19 mg/g of rutin equivalents), coumarin (1.527 ± 0.2 mg/g coumarin equivalent) and proanthocyanidins (1.7 ± 0.25 mg/g equivalent of catechin) contents.

Spectrophotometric Analysis: The qualitative UV-Vis and FTIR spectrum profile of Boerhavia diffusa and Crataeva nurvala ethanolic extracts were selected at a wavelength from 200 to 1000 nm due to the sharpness of the peaks and proper baseline. The profile showed the peaks with the absorption mentioned in Fig. 3a and 3b. The results of UV-Vis and FTIR peak values and functional groups were represented in Table 4 and 5. The FTIR spectrum confirmed the presence of alcohols, phenols, alkanes, alkynes, alkyl halides, aldehydes, carboxylic acids, aromatics, nitro compounds and amines in different plant extracts.

Chemical Composition of Extracts by GC-MS Analysis: GC-MS chromatogram of the ethanolic extract of Boerhaavia diffusa and Crataeva nurvala Fig. 4 showed peaks indicating the presence of phytochemical constituents. All the constituents were characterized and identified by comparison of the mass spectra of the constituents with the AMDIS and NIST Version-Year 2011 library. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) are presented in Table 6.

TABLE 4: UV-VIS PEAK VALUES OF ETHANOLIC EXTRACTS OF BOERHAVIA DIFFUSA AND CRATAEVA NURVALA

TABLE 5: FTIR PEAK VALUES AND FUNCTIONAL GROUPS OF BOERHAVIA DIFFUSA AND CRATAEVA NURVALA

FIG. 4: TOTAL ION CHROMATOGRAM (GC-MS) OF PLANTS (ETHANOLIC EXTRACTS)

TABLE 6: BIOLOGICALLY ACTIVE CHEMICAL COMPOUNDS OF ETHANOLIC EXTRACT OF BOERHAVIA DIFUSA L. AND CRATEVA NURVALA. A: BOERHAVIA DIFUSA L. (ETHANOLIC EXTRACT)

B: CRATEVA NURVALA BUCH-HAM (ETHANOLIC EXTRACT)

TABLE 7: BIOACTIVITY OF PHYTOCOMPONENTS IDENTIFIED IN THE ETHANOLIC EXTRACT OF BOERHAVIA DIFUSA L. AND CRATEVA NURVALA

(**Activity source: Dr. Duke's Phytochemical and Ethnobotanical Database)

Glycolate Oxidase and Lactate Dehydrogenase Inhibition and Mode of Inhibition: The experimental evidence indicates that all ethanolic extracts of Boerhavia diffusa and Crateva nurvala showed a good activity profile for inhibition of glycolate oxidase and lactate dehydrogenase as indicated by IC₅₀ (μM) Fig. 5 and 6; Table 8. V_max and Km of B. diffusa and C. nurvala (ethanolic extracts) for lactate dehydrogenase (LDH) Inhibition and glycolate oxidase inhibition are mentioned in Fig. 7 and 8; Table 9 and 10.

FIG. 5: LACTATE DEHYDROGENASE ENZYME INHIBITION ACTIVITY (%)

FIG. 6: GLYCOLATE OXIDASE ENZYME INHIBITION ACTIVITY (%)

FIG. 7: LINEWEAVER-BURK PLOT FOR ENZYME GLYCOLATE OXIDASE

FIG. 8: LINEWEAVER-BURK PLOT FOR ENZYME LACTATE DEHYDROGENASE

TABLE 8: IC₅₀ VALUE OF ENZYME GLYCOLATE OXIDASE AND LACTATE DEHYDROGENASE

I: GLYCOLATE OXIDASE

II: LACTATE DEHYDROGENASE

Values are expressed in mean ± SD; *: P<0.05

TABLE 9: V_max AND Km OF B. DIFFUSA AND C. NURVALA (ETHANOLIC EXTRACTS) FOR LACTATE DEHYDROGENASE (LDH) INHIBITION

TABLE 10: V_max AND Km OF B. DIFFUSA AND C. NURVALA (ETHANOLIC EXTRACTS) FOR GLYCOLATE OXIDASE INHIBITION

ADMET Predictions: The potential ADME profiles of the compounds as predicted using an admetSAR server, while the distribution profile of the compounds as obtained from the admetSAR server is shown in Table 11. Computational study for the prediction of the relevant properties influencing bioactivity of the lead compounds was performed. The ADME properties of the compounds were evaluated, and the selected properties are linked to metabolism, cell permeation.

TABLE 11: ADMET PREDICTIONS

Compound 1: à-D-(+)-Talopyranose, pentakis (trimethylsilyl) ether

ADMET PREDICTED PROFILE - CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

Compound 2: Arabinopyranose, tetrakis-O-(trimethylsilyl)-, à-D-

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

Compound 3: Glucofuranoside, methyl 2,3,5,6-tetrakis-O-(trimethylsilyl)-, à-D-

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

Compound 4: Glucopyranose, 1,2,3,4,6-pentakis-O-(trimethylsilyl)-, D-

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

Compound 5: Hexadecanoic acid, methyl ester

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 6: Lup-20(29)-en-3-one

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 7: Lupeol

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 8: Mannose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, D-

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 9: Mannose, 6-deoxy-2,3,4,5-tetrakis-O-(trimethylsilyl)

ADMET PREDICTED PROFILE - CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 10: Myo-Inositol, 1,2,3,4,5,6-hexakis-O-(trimethylsilyl)-

ADMET PREDICTED PROFILE – CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 11: Pinotol, pentakis(trimethylsilyl) ether

ADMET PREDICTED PROFILE - CLASSIFICATION

ADMET PREDICTED PROFILE – REGRESSION

Compound 12: β-Sitosterol trimethylsilyl ether

ADMET PREDICTED PROFILE - CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

Compound 13: Sorbopyranose, 1,2,3,4,5-pentakis-O-(trimethylsilyl)-, L-

ADMET PREDICTED PROFILE - CLASSIFICATION

ADMET PREDICTED PROFILE - REGRESSION

DISCUSSION: The concentrations of phenolic, flavonoids compounds were high in all extracts while proanthocyanidin and coumarin contents were low when compared with other compounds. The differences in polarity of the antioxidant components are noticeably the reason why phenolic compounds and antioxidant activity of the extract correlate. Polyphenolic compounds which influence the radical scavenging, inhibition of hydrolytic and oxidative enzymes and also act as anti-inflammatory agent ¹⁹. The mechanisms of action of flavonoids are through scavenging or chelating process ^{20, 21.}  Flavonoids are free radical scavengers, super antioxidants, and potent water soluble which prevent oxidative cell damage and have strong anti-cancer activity ²² and provide anti-inflammatory actions ²³. Cardiac glycosides are drugs and can be used in the treatment of congestive heart failure and cardiac arrhythmia. Tannins possess astringent, anti-inflammatory, antidiarrhoeal, antioxidant and antimicrobial activities ²⁴.

Among all extracts, the phenolic content decreased in the order of ethanol>acetone>ethyl acetate> distilled water. Hence it was observed that ethanol is a good solvent for the extraction of phenols and these results are in agreement with the previously reported study ^{25, 26}. The interest in the phenols has increased outstandingly due to their prominent free radical scavenging activity. Polyphenols possess ideal structural chemistry for free radical scavenging activities which have shown to be more effective antioxidant in-vitro than vitamins E and C on a molar basis ²⁷. Phenol compounds could be classified as simple phenols, a single aromatic ring bearing at least one hydroxyl group and polyphenols with at least two phenol subunits like flavonoids or three and more phenol subunits called tannins ²⁸.

The phenolic compounds of plant origin showed their anti-oxidative effect by various mechanisms, including their ability to scavenge free radicals or activate various antioxidant enzymes and inhibit oxidase ²⁹. Flavonoids are reported to be responsible for antimicrobial activity associated with some ethnomedicinal plants ³⁰. Flavonoids play an important role in effective antioxidant and show strong anticancer activities ^{31, 32}. Flavonoids are water-soluble antioxidants and possess pharmacological roles including anti-microbial, anti-inflammatory, cardioprotective, anti-allergic and anti-cancer activities ³³. The concentration of the flavonoids in plant extracts depends on the polarity of solvents used for the extracts which support the present results. The result showed a higher flavonoid content of ethyl acetate extract than of others extracts. It was expected that ethanol extract would have the higher antioxidant activity than that of other extracts, but surprisingly the acetone and ethyl acetate extracts have the good antioxidant activity in spite with less phenolic, flavonoids content Table 3. Since the natural antioxidant include many different compounds such as phenol, nitrous compounds, carotenoids and many unidentified compounds ³⁴, compounds other than the phenolic compounds may be responsible for antioxidant activity in the water, ethanolic or acetone extract or sometimes the structure of flavonoids in ethyl acetate extract may decrease its antioxidant activity. Rice-Evans et al. showed that hydroxyl groups, the amount of conjugation and its kind, are two important factors in the antioxidant potential of phenolic compounds.

Stronger antioxidants usually are more conjugated and have more hydroxyl groups that make the antioxidant strong enough to scavenge the free radicals. The occurrence of peaks at 234-676 nm reveals the presence of phenolic and alkaloids.

The FTIR spectrum was used to identify the functional group of the active components based on the peak value in the region of infrared radiation. The biological activities listed Table 7 are based on Dr.Duke’s Phytochemical and Ethnobotanical Databases by Dr. Duke of the Agricultural Research Service/USDA ³⁵. Phytol was proven to exhibit antioxidant and antinociceptive effects ^{36, 37}. These sterols even blocked inflammatory cytokines and induced apoptosis ^38-40. Lupeol exhibited marked anti-inflammatory and anticancer properties ⁴¹. Several investigations revealed that lupeol blocks tumorigenesis by affecting molecular growth pathways which are involved in cell proliferation and cell death ⁴¹.

In-vivo and in-vitro tests revealed the potent anti-mutagenic property of lupeol ^42-44. Identification of these compounds in the plant serves as the basis in determining the possible health benefits of the plant leading to further biologic and pharmacologic studies concerning urolithiasis and related renal disorders. In the case of glycolate oxidase inhibition,