Int J Pharm Pharm Sci, Vol 13, Issue 11, 34-46Original Article

BERGENIA CILIATA: ISOLATION OF ACTIVE FLAVONOIDS, GC-MS ANALYSIS, ADME STUDY AND INHIBITION ACTIVITY OF OXALATE SYNTHESIZING ENZYMES

SHWETA R. GOPHANE*, SAGAR R. JADHAO, PREETI B. JAMDHADE

School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606 India
Email: shweta.gophane@gmail.com

Received: 20 May 2021, Revised and Accepted: 14 Sep 2021

ABSTRACT

Objective: Bergenia ciliata (family-Saxifragaceae) is a well-known herb for kidney stone. The main objective of the study was the identification of flavonoids along with ADME profile. Another supportive objective was to check inhibition of enzymes which perform active role in oxalate synthesis.

Methods: The hydromethanolic extract was fractionated by liquid-liquid extraction to obtain ethyl acetate and ethyl ether fractions. The chemical structures of the purified compounds were identified by gas chromatography-mass spectrometry.

Results: A total of 12 volatile chemical compounds belonging to hydrocarbons, esters, alcohols, fatty acids, ketones, etc. were identified and characterized in ethyl acetate fraction through GC-MS analysis Fractions enriched in flavonoids showed glycolate oxidase and lactate dehydrogenase enzyme inhibition with IC50 value (µg/ml) 65.76 and 69.84 respectively. The kinetic behaviour of the extracts that inhibit the Glycolate oxidase and Lactate dehydrogenase activity was determined by the Lineweaver-Burk plot. The mode of inhibition of the studied plant extract was type of a non-competitive inhibition. ADMET screening of compounds successfully passed all the parameters of screening.

Conclusion: On the basis of the results, it was found that Bergenia ciliata (rhizome) may serve as a novel and rich source of therapeutic compounds and it can be further explored for urolithiasis treatment purposes.

Keywords: Flavonoids, GC-MS, ADME, Glycolate oxidase, Lactate dehydrogenase, Inhibition

© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijpps.2021v13i11.42127. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps.

INTRODUCTION

Flavonols are a class of flavonoids that have the 3-hydroxyflavone backbone (IUPAC name: 3-hydroxy-2-phenylchromen-4-one). Flavonoids help regulate cellular activity and fight off free radicals that cause oxidative stress on your body. In simpler terms, they help your body function more efficiently while protecting it against everyday toxins and stressors. Flavonoids are also powerful antioxidant agents. Flavonoid-rich foods, based on their surprising health effects, are well described as superfoods. These include all plant-origin foods, mainly tea, fruit, vegetables, grains, legumes, nuts, and wine [1]. Flavonoids are important for human health because of their antioxidant, antibacterial, antiviral, antihepatotoxic, antiosteoporotic, antiulcer, immunomodulatory, antiproliferative and apoptotic activity and anti‐inflammatory activities and because they act as free radical scavengers as they are potential reducing agents that protect from oxidative damage [2-10]. Bergenia ciliata Sternb. (family-Saxifragaceae), a high-value plant of the Sikkim Himalaya has been investigated for antioxidant, antiurolithiac activity and bioactive compounds. However, scientific exploration of B. ciliata for phytochemicals and pharmacological properties is in infancy. With this view, the present study was undertaken to investigate B. ciliata rhizome ethanolic extract for antiurolithiac activity and bioactive compounds. Glycolate oxidase (GOX, EC 1.1.3.15) the key enzyme involved in oxalate synthesis. It was first associated with the disease primary hyperoxaluria type1 (PH1). The inhibition of GOX activity is a suitable therapeutic strategy for decreasing endogenous oxalate synthesis. The oxidation of Glycolate to glyoxylate is catalyzed by Glycolate oxidase [11] and the reduction of glyoxylate to Glycolate by lactate dehydrogenase (LDH) (EC 1.1.1.27) [12-14]. In normal pathway, alanine glyoxylate aminotransferase (AGT) converts glyoxylate to glycine and so prevents the conversion of glyoxylate to oxalate which further form complex with calcium to lead into the formation of calcium oxalate stones (fig. 1.) [15]. In the case of AGT deficiency, glyoxylate gets directly converted to oxalate by enzymes GOX and LDH. Hence their inhibition proves potential in the management of urolithiasis [16]. GC-MS analysis can identify pure compounds present at less than 1 gm [17]. Simple, cost-effective spectroscopic (UV-Vis, FTIR, GC-MS) methods together or separate can be used for detecting photo components in this sense as well as conventional methods [18-20]. So far reports on the systematic evaluation and scientific investigation of Bergenia ciliata or their phytoconstituents as glycolate oxidase and lactate dehydrogenase inhibitors are scare. The mode of interaction of extracted flavonoids (inhibitor) with enzyme, including identifying constituent compounds using GCMS, determining IC50 values using inhibition kinetics analysis and determining inhibitory patterns using Lineweaver-Burk plots and computer analysis using ADME were well described using this approach.

Fig. 1: Pathway associated with oxalate synthesis

MATERIALS AND METHODS

Flavonoid extraction from Bergenia ciliata (rhizome)

Bergenia ciliata (rhizome) was procured from Yogesh pharma Pvt. Ltd., Nanded (MS), India. The plant material was washed thoroughly with water to remove dust and dried under the shade at room temperature for 5 d. The dried parts were ground using blender to obtain the course powder and kept in an air-tight container till further use. Extraction of flavonoids was done as per method reported by Subramnian S and Nagarjan S [21]. Hundred grams of finely powdered sample were soxhlet extracted with 80% hot methanol (500 ml) on a water bath for 24 h and filtered. The filtrate obtained was re-extracted successively with petroleum ether (fraction I), ethyl ether (fraction II), and ethyl acetate (fraction III) using separating funnel. Fraction of petroleum ether was discarded due to being rich in fatty substances. The fractions of (ethyl ether-fraction II) and (ethyl acetate-fraction III) were further analysed for free and bound flavonoids, respectively. Ethyl acetate fraction of the sample was refluxed for the hydrolysis using 7% H2SO4 for 2 h (for removal of bounded sugars) and again filtrate was extracted in separating funnel with ethyl acetate. Ethyl acetate extract thus obtained was washed with distilled water to neutrality. Ethyl ether (free flavonoids) and ethyl acetate fractions (bound flavonoids) were dried in rota vapour and weighed.

Gas chromatography-mass spectrometry analysis

The GC-MS analysis of the ethanolic extract was carried out using a Agilent 7890 A gas chromatogram equipped and coupled to a mass detector 5975 MSD spectrometer with DB 5 MS and 30m × 0.25 µm DF of 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 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.

Glycolate oxidase enzyme inhibition assay

Glycolate oxidase enzyme inhibition activity was performed in cuvette with a 1-cm light path using a UV-spectrophotometer. Each assay contained 200μM potassium phosphate (pH 7.0), 1 mg of bovine serum albumin, 3μM EDTA, 0.1μM 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 the addition of 2μM Sodium glycolate was then followed by measure of the decrease in absorbance at 600 nm. The inhibitory activity of each test compound was indicated by their IC50 values calculated using a linear regression curve. The percent inhibition of enzyme activity was calculated using standard formula [22].

Where control represents reaction mixture as described above excluding test compounds instead contain DMSO only whereas sample represents reaction mixture same as described in method.

Lactate dehydrogenase enzyme inhibition assay

Lactate dehydrogenase activity was assayed at pH 7.4 by measuring the decrease in absorbance at 340 nm associated with NADH oxidation. Assay mixture contained 0.3 mmol NADH, 2.0 mmol pyruvate and 100 μL enzyme in volume of 3.0 ml. The reaction was started by the 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 mmol NADH, 100 μL enzymes in 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 mmol pyruvate and l-lactate formation was then followed by measure of decrease in 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 [22].

Where control represents reaction mixture as described above excluding test compounds instead contain DMSO only whereas sample represents reaction mixture same as described in method.

ADMET predictions

ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analyses constitute the pharmacokinetics of a drug molecule [23]. 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.

RESULTS AND DISCUSSION

Flavonoid extraction from Bergenia ciliata (rhizome)

The ethyl acetate fraction of Bergenia ciliata (rhizome) showed many peaks in the chromatogram (fig. 2a), however, only twelve compounds could be identified and characterized (fig. 2b). Plants contain so many phytoconstituents, many of which are biologically active compounds and are responsible for pharmacological activities [24]. The bioactive secondary metabolites have been shown to reduce the risk and progression of diseases such as cancer, renal disorders, etc., through various biological mechanisms. A total of 12 volatile chemical compounds belonging to hydrocarbons, esters, alcohols, fatty acids, ketones, etc. were identified and characterized in ethyl acetate fraction through GC-MS analysis (table 1). 

Glycolate oxidase and lactate dehydrogenase inhibition and mode of inhibition Lineweaver–Burk plots

The experimental evidence indicates that ethyl acetate fraction of Bergenia ciliata showed a good activity profile for inhibition of glycolate oxidase and lactate dehydrogenase as indicated by IC50 (μM) (fig. 3; table 2). To determine the mode of inhibition by active compounds from the plants, Lineweaver–Burk plot analysis was performed [25]. 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. In case of glycolate oxidase and lactate dehydrogenase inhibition, B. ciliata (ethyl acetate fraction) were found to be non-competitive inhibitors as Km values were constant while Vmax consequently decreased with increased inhibitor concentration. These finding suggest that inhibition of glycolate oxidase and lactate dehydrogenase activity, leading to retardation of oxalate synthesis, is one of the mechanism through which the plant tested in this study could be exhibiting their antiurolithiac effect. Modulation of glycolate oxidase and lactate dehydrogenase activity by compounds in these extracts would thus eventually lead to a lowering of oxalate content. Presence of glycolate oxidase and lactate dehydrogenase inhibitor blocks the normal pathway of conversion of glyoxylate to oxalate which further form a complex with calcium to lead into the formation of calcium oxalate stones. Thus, oxalate synthesis can be blocked in hyperoxaluria conditions with glycolate oxidase and lactate dehydrogenase inhibitors. This glycolate oxidase and lactate dehydrogenase inhibition could occur in a concentration-dependent or independent manner depending upon the bioactive compounds. Inhibition of glycolate oxidase and lactate dehydrogenase may occur due to these phytoconstituents. This is the first report as per author’s knowledge.

Fig. 2a: Total ion chromatogram (GC-MS) of Bergenia ciliata (rhizome) (ethyl acetate fraction)

[1]: 2,5-Furandione, dihydro-3-methylene-[2]: α-D-Glucopyranoside,O-α-D-glucopyranosyl-(fwdarw 3)-β-D-fructofuranosyl, [3]: Fumaric acid, [4]: 2-decenoic acid, [5]: 1,2,3-Benzenetriol, [6]: Benzoic acid, 3-hydroxy-

[7]: 1,2,3,5-Cyclohexanetetrol, (1α,2β,3α,5β), [8]: Nonadecane, [9]: Benzoic acid, 4-hydroxy-3,5-dimethoxy-, [10]: 8-pentadecanone, [11]: Docosene, [12]: 2,5- Furandione,3-methyl-

Fig. 2b: Ion chromatograms (GC-MS) of identified compounds from Bergenia ciliata (rhizome) (ethyl acetate fraction)

Table 1: Bioactivity of phytocomponents identified in the ethyl acetate fraction of Bergenia ciliata

Bergenia ciliata (Ethyl acetate fraction)
Name of the compound Biological Activity**
Fumaric acid Acidifier, Acidulant, Arachidonic acid-inhibitor, Increase aromatic amino acid Decarboxylase activity, Inhibit production of uric acid, Urinary-Acidulant, Urine-acidifier.
α-D-Glucopyranoside, O-α-D-glucopyranosyl-(fwdarw 3)-β-D-fructofuranosyl Aldehyde-oxidase-inhibitor, Anticancer, Antidote, Antiretinitic, Antitumor, Catechol-O-Methyl-Transferase-Inhibitor, Increase Osteocalcin, Inhibit Production of Tumor Necrosis Factor, Inhibit production of uric acid, Lower oxalate, Ionic Channel Opener, NADH-Oxidase Inhibitor, Nitric Oxide Synthase Inhibitor, Occuloirritant, Occulotensive, Odontolytic
2-decenoic acid Acidifier, Acidulant, Arachidonic acid-inhibitor, Increased aromatic amino acid Decarboxylase activity, Inhibit production of uric acid, Urinary-Acidulant, Urine-acidifier.
Benzoic acid, 3-hydroxy- 17-beta-hydroxysteroid dehydrogenase-inhibitor, Aryl-Hydrocarbon-Hydroxylase-Inhibitor, Testosterone-Hydroxylase-Inducer, Acidifier, Acidulant, Arachidonic acid-inhibitor, Increase aromatic amino acid Decarboxylase activity, Inhibit production of uric acid, Urinary-Acidulant, Urine-acidifier.
Benzoic acid, 4-hydroxy-3,5-dimethoxy- Acidifier, Acidulant, Arachidonic acid-inhibitor, Increased aromatic amino acid Decarboxylase activity, Inhibit production of uric acid, Urinary-Acidulant, Urine-acidifier, 17-beta-hydroxysteroid dehydrogenase-inhibitor, Aryl-Hydrocarbon-Hydroxylase-Inhibitor, Testosterone-Hydroxylase-Inducer.
2,5-Furandione,3-methyl- Catechol-O-Methyl-Transferase-Inhibitor, Catechol-O-Methyltransferase-Inhibitor, Methyl-Donar, Methyl-Guanidine-inhibitor.

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

Table 2: Vmax and Km of B. ciliata (ethyl acetate extracts) for glycolate oxidase (GOX) and lactate dehydrogenase (LDH) inhibition

Samples Concentrations (µg/ml) Vmax (µg/min) Km (µg/ml) Type of Inhibition IC50 value (µg/ml)
B. ciliata (gox) 10 0.025 0.86 Non-competitive 65.76
25 0.025 0.86
50 0.029 0.8
75 0.0196 0.8
100 0.0181 0.8
B. ciliata (ldh) 10 3.33 1.75 Non-competitive 69.84
25 1.66 1.75
50 1.25 1.63
75 1.25 1.63
100 1.42 1.63

Fig. 3: Lineweaver-Burk plot for enzyme Glycolate oxidase and Lactate dehydrogenase

ADMET predictions

The potential ADME profiles of the compounds as predicted using the 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 and cell permeation.

Compound 1: 1,2,3-Benzenetriol

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB- 0.6478
Human Intestinal Absorption HIA+ 0.9642
Caco-2 Permeability Caco2+ 0.7355
P-glycoprotein Substrate Non-substrate 0.6749
P-glycoprotein Inhibitor Non-inhibitor 0.9691
Non-inhibitor 0.9916
Renal Organic Cation Transporter Non-inhibitor 0.9289
Distribution
Subcellular localization Mitochondria 0.6807
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8234
CYP450 2D6 Substrate Non-substrate 0.9031
CYP450 3A4 Substrate Non-substrate 0.7441
CYP450 1A2 Inhibitor Non-inhibitor 0.7436
CYP450 2C9 Inhibitor Non-inhibitor 0.8271
CYP450 2D6 Inhibitor Non-inhibitor 0.9494
CYP450 2C19 Inhibitor Non-inhibitor 0.9397
CYP450 3A4 Inhibitor Non-inhibitor 0.8682
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.6899
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.9526
Non-inhibitor 0.9299
AMES Toxicity AMES toxic 0.7459
Carcinogens Non-carcinogens 0.8816
Fish Toxicity High FHMT 0.6928
Tetrahymena Pyriformis Toxicity High TPT 0.8900
Honey Bee Toxicity High HBT 0.6927
Biodegradation Ready biodegradable 0.6003
Acute Oral Toxicity III 0.8089
Carcinogenicity (Three-class) Non-required 0.5339

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -0.4107 LogS
Caco-2 Permeability 0.5771 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 2.2344 LD50, mol/kg
Fish Toxicity 1.1417 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 0.5649 pIGC50, ug/l

Compound 2: 2-decenoic acid

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9638
Human Intestinal Absorption HIA+ 0.9908
Caco-2 Permeability Caco2+ 0.8326
P-glycoprotein Substrate Non-substrate 0.6523
P-glycoprotein Inhibitor Non-inhibitor 0.9689
Non-inhibitor 0.7699
Renal Organic Cation Transporter Non-inhibitor 0.9061
Distribution
Subcellular localization Plasma membrane 0.6894
Metabolism
CYP450 2C9 Substrate Non-substrate 0.7322
CYP450 2D6 Substrate Non-substrate 0.9059
CYP450 3A4 Substrate Non-substrate 0.7043
CYP450 1A2 Inhibitor Inhibitor 0.7152
CYP450 2C9 Inhibitor Non-inhibitor 0.8948
CYP450 2D6 Inhibitor Non-inhibitor 0.9474
CYP450 2C19 Inhibitor Non-inhibitor 0.9459
CYP450 3A4 Inhibitor Non-inhibitor 0.9608
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.8882
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.8983
Non-inhibitor 0.9369
AMES Toxicity Non AMES toxic 0.9737
Carcinogens Non-carcinogens 0.5263
Fish Toxicity High FHMT 0.9784
Tetrahymena Pyriformis Toxicity High TPT 0.9996
Honey Bee Toxicity High HBT 0.7359
Biodegradation Ready biodegradable 0.7618
Acute Oral Toxicity III 0.8593
Carcinogenicity (Three-class) Non-required 0.6623

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -3.5855 LogS
Caco-2 Permeability 1.3217 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.9685 LD50, mol/kg
Fish Toxicity 0.8959 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 0.7180 pIGC50, ug/l

Compound 3: 8-pentadecanone

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9882
Human Intestinal Absorption HIA+ 0.9955
Caco-2 Permeability Caco2+ 0.8766
P-glycoprotein Substrate Non-substrate 0.6680
P-glycoprotein Inhibitor Non-inhibitor 0.8320
Non-inhibitor 0.7768
Renal Organic Cation Transporter Non-inhibitor 0.8727
Distribution
Subcellular localization Mitochondria 0.4585
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8589
CYP450 2D6 Substrate Non-substrate 0.8439
CYP450 3A4 Substrate Non-substrate 0.6531
CYP450 1A2 Inhibitor Inhibitor 0.6890
CYP450 2C9 Inhibitor Non-inhibitor 0.9433
CYP450 2D6 Inhibitor Non-inhibitor 0.9502
CYP450 2C19 Inhibitor Non-inhibitor 0.9645
CYP450 3A4 Inhibitor Non-inhibitor 0.9815
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.8752
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.8043
Non-inhibitor 0.7659
AMES Toxicity Non AMES toxic 0.9859
Carcinogens Carcinogens 0.6310
Fish Toxicity High FHMT 0.7423
Tetrahymena Pyriformis Toxicity High TPT 0.8910
Honey Bee Toxicity High HBT 0.7254
Biodegradation Ready biodegradable 0.8731
Acute Oral Toxicity III 0.8455
Carcinogenicity (Three-class) Non-required 0.7622

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -2.2537 LogS
Caco-2 Permeability 1.3709 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.5870 LD50, mol/kg
Fish Toxicity 0.8094 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 0.5873 pIGC50, ug/l

Compound 4: Benzoic acid, 3-hydroxy-

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.5320
Human Intestinal Absorption HIA+ 0.9872
Caco-2 Permeability Caco2+ 0.8937
P-glycoprotein Substrate Non-substrate 0.7493
P-glycoprotein Inhibitor Non-inhibitor 0.9890
Non-inhibitor 0.9927
Renal Organic Cation Transporter Non-inhibitor 0.9078
Distribution
Subcellular localization Mitochondria 0.9063
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8115
CYP450 2D6 Substrate Non-substrate 0.9377
CYP450 3A4 Substrate Non-substrate 0.7652
CYP450 1A2 Inhibitor Non-inhibitor 0.9752
CYP450 2C9 Inhibitor Non-inhibitor 0.9697
CYP450 2D6 Inhibitor Non-inhibitor 0.9827
CYP450 2C19 Inhibitor Non-inhibitor 0.9651
CYP450 3A4 Inhibitor Non-inhibitor 0.9493
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.9554
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.9617
Non-inhibitor 0.9771
AMES Toxicity Non AMES toxic 0.9826
Carcinogens Non-carcinogens 0.8226
Fish Toxicity High FHMT 0.7616
Tetrahymena Pyriformis Toxicity Low TPT 0.8365
Honey Bee Toxicity High HBT 0.7797
Biodegradation Ready biodegradable 0.8413
Acute Oral Toxicity III 0.5472
Carcinogenicity (Three-class) Non-required 0.6300

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -1.3479 LogS
Caco-2 Permeability 1.1511 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.3983 LD50, mol/kg
Fish Toxicity 2.2036 pLC50, mg/l
Tetrahymena Pyriformis Toxicity -0.8949 pIGC50, ug/l

Compound 5: Benzoic acid, 4-hydroxy-3,5-dimethoxy

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.5861
Human Intestinal Absorption HIA+ 0.9165
Caco-2 Permeability Caco2+ 0.7124
P-glycoprotein Substrate Non-substrate 0.6033
P-glycoprotein Inhibitor Non-inhibitor 0.9199
Non-inhibitor 0.8879
Renal Organic Cation Transporter Non-inhibitor 0.9136
Distribution
Subcellular localization Mitochondria 0.8825
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8213
CYP450 2D6 Substrate Non-substrate 0.8899
CYP450 3A4 Substrate Non-substrate 0.6258
CYP450 1A2 Inhibitor Non-inhibitor 0.9052
CYP450 2C9 Inhibitor Non-inhibitor 0.9316
CYP450 2D6 Inhibitor Non-inhibitor 0.9445
CYP450 2C19 Inhibitor Non-inhibitor 0.8579
CYP450 3A4 Inhibitor Non-inhibitor 0.9538
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.8767
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.9858
Non-inhibitor 0.9664
AMES Toxicity Non AMES toxic 0.9342
Carcinogens Non-carcinogens 0.8809
Fish Toxicity High FHMT 0.8272
Tetrahymena Pyriformis Toxicity High TPT 0.9067
Honey Bee Toxicity High HBT 0.7522
Biodegradation Ready biodegradable 0.7199
Acute Oral Toxicity II 0.4765
Carcinogenicity (Three-class) Non-required 0.7159

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -2.1167 LogS
Caco-2 Permeability 0.7627 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 2.5353 LD50, mol/kg
Fish Toxicity 1.6288 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 0.4613 pIGC50, ug/l

Compound 6: Docosene

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9763
Human Intestinal Absorption HIA+ 0.9928
Caco-2 Permeability Caco2+ 0.7989
P-glycoprotein Substrate Non-substrate 0.6522
P-glycoprotein Inhibitor Non-inhibitor 0.7775
Non-inhibitor 0.5881
Renal Organic Cation Transporter Non-inhibitor 0.8619
Distribution
Subcellular localization Lysosome 0.4578
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8261
CYP450 2D6 Substrate Non-substrate 0.8048
CYP450 3A4 Substrate Non-substrate 0.7278
CYP450 1A2 Inhibitor Inhibitor 0.5418
CYP450 2C9 Inhibitor Non-inhibitor 0.9157
CYP450 2D6 Inhibitor Non-inhibitor 0.9426
CYP450 2C19 Inhibitor Non-inhibitor 0.9190
CYP450 3A4 Inhibitor Non-inhibitor 0.9832
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.6838
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.7812
Non-inhibitor 0.8777
AMES Toxicity Non AMES toxic 0.9904
Carcinogens Carcinogens 0.6000
Fish Toxicity High FHMT 0.9954
Tetrahymena Pyriformis Toxicity High TPT 0.9981
Honey Bee Toxicity High HBT 0.7751
Biodegradation Ready biodegradable 0.5000
Acute Oral Toxicity III 0.6572
Carcinogenicity (Three-class) Non-required 0.5494

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -4.9876 LogS
Caco-2 Permeability 1.3776 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.3452 LD50, mol/kg
Fish Toxicity -1.1807 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 1.4804 pIGC50, ug/l

Compound 7: fumaric acid

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9017
Human Intestinal Absorption HIA+ 0.8740
Caco-2 Permeability Caco2- 0.6728
P-glycoprotein Substrate Non-substrate 0.8006
P-glycoprotein Inhibitor Non-inhibitor 0.9850
Non-inhibitor 0.9808
Renal Organic Cation Transporter Non-inhibitor 0.9583
Distribution
Subcellular localization Mitochondria 0.7863
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8262
CYP450 2D6 Substrate Non-substrate 0.9397
CYP450 3A4 Substrate Non-substrate 0.8039
CYP450 1A2 Inhibitor Non-inhibitor 0.9659
CYP450 2C9 Inhibitor Non-inhibitor 0.9490
CYP450 2D6 Inhibitor Non-inhibitor 0.9606
CYP450 2C19 Inhibitor Non-inhibitor 0.9773
CYP450 3A4 Inhibitor Non-inhibitor 0.9554
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.9899
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.9836
Non-inhibitor 0.9891
AMES Toxicity Non AMES toxic 0.9132
Carcinogens Non-carcinogens 0.5130
Fish Toxicity High FHMT 0.8398
Tetrahymena Pyriformis Toxicity Low TPT 0.9808
Honey Bee Toxicity High HBT 0.7308
Biodegradation Ready biodegradable 0.7561
Acute Oral Toxicity III 0.7762
Carcinogenicity (Three-class) Non-required 0.7191

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -0.3321 LogS
Caco-2 Permeability 0.4098 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.6871 LD50, mol/kg
Fish Toxicity 0.9694 pLC50, mg/l
Tetrahymena Pyriformis Toxicity -0.6339 pIGC50, ug/l

Compound 8: Nonadecane

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9821
Human Intestinal Absorption HIA+ 0.9921
Caco-2 Permeability Caco2+ 0.8284
P-glycoprotein Substrate Non-substrate 0.6915
P-glycoprotein Inhibitor Non-inhibitor 0.8985
Non-inhibitor 0.7267
Renal Organic Cation Transporter Non-inhibitor 0.8780
Distribution
Subcellular localization Lysosome 0.5981
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8480
CYP450 2D6 Substrate Non-substrate 0.7762
CYP450 3A4 Substrate Non-substrate 0.7237
CYP450 1A2 Inhibitor Non-inhibitor 0.6175
CYP450 2C9 Inhibitor Non-inhibitor 0.9349
CYP450 2D6 Inhibitor Non-inhibitor 0.9373
CYP450 2C19 Inhibitor Non-inhibitor 0.9540
CYP450 3A4 Inhibitor Non-inhibitor 0.9877
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.8149
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.8620
Non-inhibitor 0.8109
AMES Toxicity Non AMES toxic 0.9965
Carcinogens Carcinogens 0.6420
Fish Toxicity High FHMT 0.9374
Tetrahymena Pyriformis Toxicity High TPT 0.9947
Honey Bee Toxicity High HBT 0.7485
Biodegradation Ready biodegradable 0.7561
Acute Oral Toxicity III 0.6143
Carcinogenicity (Three-class) Non-required 0.6328

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -5.1776 LogS
Caco-2 Permeability 1.3807 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.3444 LD50, mol/kg
Fish Toxicity -0.7109 pLC50, mg/l
Tetrahymena Pyriformis Toxicity 0.3450 pIGC50, ug/l

Compound 9: 2,5-Furandione, dihydro-3-methylene

ADMET predicted profile---Classification

Model Result Probability
Absorption
Blood-Brain Barrier BBB+ 0.9627
Human Intestinal Absorption HIA+ 0.9833
Caco-2 Permeability Caco2+ 0.5452
P-glycoprotein Substrate Non-substrate 0.7919
P-glycoprotein Inhibitor Non-inhibitor 0.6266
Non-inhibitor 0.9951
Renal Organic Cation Transporter Non-inhibitor 0.8772
Distribution
Subcellular localization Mitochondria 0.6548
Metabolism
CYP450 2C9 Substrate Non-substrate 0.8815
CYP450 2D6 Substrate Non-substrate 0.8935
CYP450 3A4 Substrate Non-substrate 0.7178
CYP450 1A2 Inhibitor Non-inhibitor 0.8434
CYP450 2C9 Inhibitor Non-inhibitor 0.9264
CYP450 2D6 Inhibitor Non-inhibitor 0.9524
CYP450 2C19 Inhibitor Non-inhibitor 0.8061
CYP450 3A4 Inhibitor Non-inhibitor 0.9487
CYP Inhibitory Promiscuity Low CYP Inhibitory Promiscuity 0.9099
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition Weak inhibitor 0.9172
Non-inhibitor 0.9875
AMES Toxicity Non AMES toxic 0.8025
Carcinogens Non-carcinogens 0.8480
Fish Toxicity High FHMT 0.9233
Tetrahymena Pyriformis Toxicity Low TPT 0.7182
Honey Bee Toxicity High HBT 0.8293
Biodegradation Ready biodegradable 0.7452
Acute Oral Toxicity III 0.7565
Carcinogenicity (Three-class) Non-required 0.5918

ADMET predicted profile---Regression

Model Value Unit
Absorption
Aqueous solubility -0.6464 LogS
Caco-2 Permeability 0.9549 LogPapp, cm/s
Distribution
Metabolism
Excretion
Toxicity
Rat Acute Toxicity 1.9632 LD50, mol/kg
Fish Toxicity -0.0057 pLC50, mg/l
Tetrahymena Pyriformis Toxicity -0.4252 pIGC50, ug/l

Previous study showed that phytochemical constituents of B. ciliata are gallic acid, bergenin, (+)-afzelechin [26], 11-O-galloyl bergenin [27], paashaanolactone [26], β-Sitosterol [28] and β-Sitosterol-D-glucoside [29]. Pharmacological properties have demonstrated that the root is well known in traditional medicine for protection against diarrhea; cough, in uric acid diathesis and in pulmonary infections [28]; coughs and colds, hemorrhoids, asthma and urinary problems [30]. The juice of B. ciliata leaves is used as drops to relieve earaches [30]. Currently, Various Ayurvedic classical drugs such as Pashanabhedadi kwath, Pashanabhedadi ghrit, Pashanabhedadi Churan etc. are prepared from Pashanbhed rhizome.

CONCLUSION

We have extracted, purified flavonoids by the liquid-liquid extraction method. Finally, ethyl acetate fractions were collected and characterized by GC-MS analysis. These fractions are used to perform glycolate oxidase and lactate dehydrogenase inhibition study. Flavonoids fraction of B. ciliata showed good enzyme inhibitory activity and ADME profile. Lineviewer-Burk plot and mode of inhibition confirmed the potential of it’s as glycolate oxidase and lactate dehydrogenase inhibitors. However, further dose adjustment and molecular mechanism study need to perform for better understanding.

ACKNOWLEDGEMENT

We would like to offer special thanks to Professor C N Khobragade, who although no longer with us, continue to inspire by his example and dedication to students he served over the course of career.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

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