In Silico And In Vitro Metabolite Identification Of Swertiamarin From Enicostemma Littorale Blume Hiray Aishwarya¹, Das Priya¹, Thakkar Disha², and Kate Abhijeet¹ ² ¹Department of Natural Products, National Institute of Pharmaceutical Education and Research- Ahmedabad, Gandhinagar, Gujarat- 382355, India ²Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research- Ahmedabad, Gandhinagar, Gujarat- 382355, India
ABSTRACT Enicostemma littorale, a herb, widely used in the traditional system of medicines for the treatment of diabetes. Most of the literature, suggest the hypoglycemic activity of the extract is due to its major component swertiamarin, reported as an active ingredient. Metabolites like gentiandiol are reported from through in vivo studies. However, the report on in silico and in vitro studies on the metabolites of swertiamarin is still out of reach. In this study, we are in the process of exploring the above limitations. The experimental work including in silico prediction on site of metabolism, in vitro incubation and LC-MS analysis. Two metabolites were observed in in vitro study using RLM and HLM having masses 579 m/z and 420 m/z, wherein the structural elucidation of these metabolites is in progress. However, gentianine or gentiandiol were not found in the in vitro metabolites of swertiamarin. This study support the hypothesis of Wang et. al., indicating the microbial enzymes might be responsible for the conversion of swertiamarin to nitrogen containing metabolites like gentianine and gentiandiol.
INTRODUCTION CYP1A2
Enicostemma littorale blume is a perennial herb, distributed in various parts of India as well as found in South America, and Africa. Acts as a potent antidiabetic, laxative, antipyretic. This plant is enriched source of different chemical constituents like alkaloids, flavonoids. Swertiamarin is a seco-iridoid class of glycoside, and a lead active compound reported for wide range of pharmacological activities Certain metabolites of swertiamarin from plant extract like gentianine, gentiandiol, HMIO, erythrocentaurin are reported among which gentianine is one of the popular active metabolite (in vivo) that possesses a pharmacophoric moiety. Our prime intent is to study in vitro and in silico metabolites of swertiamarin.
Gentianine (Wang et al.,2012)
CYP2B6
CYP2C8
CYP2C19
CYP2C9
CYP2E1
Abemaciclib
HPLC Chromatogram of isolated swertiamarin
HLM
CYP2D6
In silico metabolite study by XenoSite This predictive model is built on neural networks of over 680 molecules. Due to neural network, it has merit of shorter run time and site of metabolism (SOM) score derived by neural network directly correlates to likelihood of metabolite generation. White color indicates a zero SOM score means no probability for metabolism at site while red color denotes the highest probability of metabolism with SOM score value one. Swertiamarin (Kumar et al., 2018)
CYP2A6
CYP3A4
XenoSite server’s HLM metabolism predictions on swertiamarin
Gentiandiol (Wang et al.,2012)
In vitro metabolite study of swertiamarin by using RLM & HLM
MATERIALS AND METHODS Chemical and Reagents: Pooled male RLM and HLM were procured from Invitrogen laboratories. All chemicals, solvents and reagents were used of Analytical Grade and purchased from Sigma- Aldrich, Thermo scientific, S D Fine-Chem, Qualigens, Spectrochem chemicals and Hi Media etc. Instruments: Agilent technologies 6545 QTOF MS/MS instrument coupled to Agilent UPLC 1290 infinity II system. The effective separation was obtained on ZORBAX RX- C18 (9.4 ₓ 250 mm) using mobile phase of methanol (solvent A) and 0.1% formic acid in DI water (solvent B). Plant collection and authentication: This plant was collected from Basan forest research center, Gandhinagar and it was authenticated by Botanist, Dr. Nainesh R. Modi, Assistant Professor, Gujarat University.
Took 50 µM of swertiamarin+ 5 mM magnesium chloride + 100 mM of phosphate buffer (pH-7.4). Incubated at 37°C for 5 min. After that reaction was initiated by addition of 1 mM of NADPH solution.
In vitro metabolite study of swertiamarin by using RLM & HLM Swertiamarin was isolated from about 200 gm of powdered plant material. The extraction was carried out in methanol & obtained 25.2 gm of extract. From the extract, 3.197 gm of 84% pure swertiamarin was obtained after column chromatography, which was further purified by preparative HPLC to achieve > 95% purity. According to the extracted ion chromatogram (EIC), the peak for gentianine & gentiandiol was not observed, which indicates the absence of gentianine & gentiandiol in the incubated samples.
37°C in orbital shaker at 150 rpm for 2 h.
A
Swertiamarin:[M+Na] += 397.1087
Quenching by adding of ice-cold acetonitrile.
Centrifugation at 10,000 rpm for 10 min.
B
Took supernatant and inject in to UPLC-ESI-QTOF-MS
Collection
Drying
Powdered by grinding
Extraction of plant materials
Gentianine: [M+H] += 176.0706
RESULT AND DISCUSSION The main aim of the study was to check the in vitro and in silico metabolite of swertiamarin.
Gentiandiol:[M+H] += 210.0761
C
1 1 1 1 1 1 1
Defatting using chloroform
Cold maceration using methanol (5 days)
Filtration using Whatman Filter Paper
Concentratio n (Rotavapor)
Extracted Ion Chromatograms of RLM and HLM treated (in vitro) swertiamarin samples
In silico Prediction
Isolation of Swertiamarin
In vitro incubation
LC-MS Analysis
Data Mining
In vitro and in silico studies have indicated that the enzymes present in RLM and HLM are not responsible for the conversion of swertiamarin to gentiandiol or gentianine. In silico metabolite of Swertiamarin study by XenoSite XenoSite, an in silico metabolite predicting tool analyzes the atomic sites at which xenobiotics undergoes metabolic modification by CYP450 enzyme. This software was used to predict the metabolite site of swertiamarin. Total 10 enzymes - 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 & HLM were used for RLM and HLM
Enicostemma littorale blume
Visual output of swertiamarin shows molecules’ sites of metabolisms labeled by a color gradient. These sites were more prone to get oxidized near the ring in iridoid moiety as shown in the diagram. Column Purification
Fractions
CONCLUSION
Semi-preparative HPLC
ACKNOWLEDGEMENT We would like to thank National Institute of Pharmaceutical Education and Research (NIPER)- Ahmedabad, Palaj, Gandhinagar, Gujarat for their grateful support and provision of all the facilities.
REFERENCES 1] Leong et al., Biomedicine & Pharmacotherapy 1051-1060 (2016) 2] Vaidya et al Cell, 40, 60 (2012) 3] Jun et al., Journal of Molecular Structure, 878, 22-25 (2008) 4] Wang et al., Journal of Asian Natural Products Research 176– 181 (2012)