bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 LC/MS-QToF Profiling, Anti-Diabetic and Anti-Adipogenic 2 potential of Divya MadhuKalp: A Novel Herbo-mineral 3 Formulation 4 5 Alpana Joshi1 2, Subrata K. Das1 3*, Laxmi Bisht1, Sachin Sakat1, Vinamra Sharma1 4, Niti 6 Sharma1, Khemraj Joshi1, Sudeep Verma1, Vinay K Sharma1, Acharya Balkrishna1 and CS 7 Joshi1. 8 9 Author’s Affiliation: 10 1Drug Discovery and Development Division, Patanjali Research Institute, NH-58, Haridwar- 11 249405, India. 12 2 Department of Agriculture & AgriInformatics, School of Biological Engineering & Sciences 13 Shobhit Deemed University, Meerut-250110, India. 14 3 School of Medical science and Technology, IIT Kharagpur- 721302, India. 15 4 Amity Institute of Indian System of Medicine, Amity University, Noida- 201313, India. 16 17 * Corresponding author 18 E-mail: [email protected] 19 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 20 Abstract 21 The incidence rate of diabetes mellitus is increasing worldwide. Herbal formulations have 22 recently gained importance as an alternative therapeutic option in controlling diabetes without 23 causing any side effects. In the present study, we have demonstrated maintenance of glycemic 24 homeostasis and anti-adipogenic potential of a herbo-mineral formulation Divya MadhuKalp 25 (DMK). Initially, we evaluated the presence of bioactive compounds in DMK using LC/MS- 26 QToF analysis. In-vitro analysis of DMK in L6 (skeletal muscle) cells showed a significant 27 increase in cellular glucose uptake. Similarly, a human equivalent dose of DMK significantly 28 reduced blood glucose level in normoglycemic and oral glucose tolerance rat model. DMK 29 extract also inhibited formation of advanced glycation end product and showed anti-α- 30 glucosidase activity. Further analysis of DMK in 3T3 L1 pre-adipocytes demonstrated anti- 31 adipogenic activity through reduction in intracellular lipid accumulation and triglyceride contents 32 along with downregulation of major adipogenic transcriptional factors (PPAR-γ and C/EBPα) 33 and, adipocytes marker genes (LPL, AP2 and adiponectin). In conclusion, DMK exhibited anti- 34 diabetic and anti-adipogenic activities by synergistic effect of its bioactive compounds and can 35 be considered as a potent herbo-mineral formulation for treating metabolic diseases. 36 37 Keywords: Anti-diabetic, Adipogenesis, Herbo-mineral formulation, Divya MadhuKalp, Gene 38 expression. 39 40 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 41 Introduction 42 Diabetes mellitus (DM) is a chronic metabolic disorder that has profound effects on 43 patient’s quality of life in terms of health and socio–economic levels. According to International 44 Diabetes Federation's (IDF) projections, approximately 693 million people will be diagnosed 45 with DM by year 2045 1. DM is characterized by abnormally high blood glucose resulting from 46 defective metabolism of carbohydrates, lipids and proteins. Long-term pathophysiological 47 complications associated with DM are cardiovascular diseases, nephropathy, neuropathy and 48 retinopathy. During hyperglycemic condition, there is also development of advanced glycation- 49 end products (AGEs) as a consequence of protein glycation. AGEs are the possible causal factor 50 lead to the development of diabetes related complications. Currently, the core objective of 51 diabetes treatment is to maintain the normal blood glucose levels and, to prevent or delay its 52 metabolic complications. 53 Obesity is the excessive accumulation of fat or adipose tissue in the body 2. It is a major 54 risk factor for the development of type 2 diabetes mellitus (DM). Adipogenesis involves 55 differentiation of pre-adipocytes into mature adipocytes and is regulated by two major 56 adipogenic transcription factors namely, Peroxisome proliferator–activated receptor γ (PPARγ) 57 and CCAAT/enhancer binding protein α (C/EBPα) and downstream induction of adipocyte- 58 related genes including, adipocyte protein 2 (AP2), lipoprotein lipase (LPL), adiponectin etc.3,4,5. 59 Medicinal plants have great importance to human health and communities. The medicinal 60 value of these plants typically results from the presence of many bioactive compounds. In 61 contrast to synthetic drugs based on single molecule, many bioactive compounds of poly herbal 62 formulation exert their beneficial effects through the additive or synergistic action acting at 63 single or multiple target sites associated with a physiological process6,7. Unlike, synthetic drugs, 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 which may induce unwanted side-effects, natural herbal formulations tend to show similar 65 efficacy with minimal to no side-effects and cost effectiveness6, 7, 8. In traditional Indian 66 medicine system, numerous herbal and herbo-mineral formulations have been used for treating 67 various diseases, but their scientific evidence is still lacking. Divya MadhuKalp (DMK) is an 68 Ayurvedic herbo-mineral formulation has been extensively used in clinical practices for 69 managing blood glucose levels during onset of DM. The DMK comprises of eight herbs and one 70 minerals–rich organic component in specific proportion to make complex bioactive formulation 71 (Table 1). The ingredients are; Momordica charantia 9; Picrorhiza kurroa10; Swertia chiraita 11; 72 Azadirachta indica 12; Trigonella foenum-graecum 13; Syzyium cuminii 14; Withania somnifera 15; 73 Aconitum hetrophyllum 16 and Shilajeet (Asphaltum) 17,18. The ingredients in DMK have been 74 used for diabetic therapy in ancient Indian Ayurvedic medicinal system. In order to explore the 75 pharmacological mechanism of poly herbal extracts, accurate characterization of the bioactive 76 compounds is very essential. Moreover, the quality control is very important factor in discovery 77 of a new drug. LC/MS-QToF technology is most appropriate analytical method for the full 78 characterization and quality control of poly herbal extract 19. DMK associated herbal ingredients 79 have been found to be rich in secondary metabolite compositions such as, phenolics, terpenes, 80 and nitrogen containing compounds. These active bio-molecules play an important role in 81 carbohydrate and lipid metabolism leading to efficient management of diabetes progression and 82 associated complications 20, 21. 83 In the present study, we performed the chemical profiling by LC/MS-QToF analysis and 84 determined the anti-diabetic and anti-adipogenic potential of the herbo–mineral extract of DMK 85 using in vitro and in vivo models. 86 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 87 Material and Methods 88 Experimental Material 89 Divya MadhuKalp (DMK) was sourced from Divya Pharmacy, Haridwar, India. 90 Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Insulin and bovine 91 serum albumin (BSA), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], 92 IBMX (3-isobutyl-1-methylxanthine), dexamethasone were obtained from (ThermoFischer, 93 USA). 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) was 94 purchased from Invitrogen (Carlsbad, CA, USA). Trypsin-EDTA solution was purchased from 95 GIBCO BRL (Grand Island, NY, USA). Sterile 96 well plates were obtained from Tarsons India 96 Pvt. Ltd. (Kolkata, India). Oil-Red-O, metformin, α-glucosidase, acarbose, p-nitrophenyl 97 glucopyranoside, aminoguanidine, glibenclamide, D-glucose, and sodium carboxy methyl 98 cellulose were purchased from Sigma-Aldrich (USA). The glucose monitoring system, 99 measurements strips (Life Scan, Switzerland) and sodium chloride injection I.P. 0.9 % w/v 100 (Infutec Healthcare Ltd. India) was procured locally. All other chemicals and reagents used were 101 of the highest commercial grade. HPLC grade reagents of analytical grade toluene, ethyl acetate, 102 formic acid, Gallic acid Vanillic Acid, Caffeic acid, Trigonelline, Adinosine, Rutin Quercetin 103 Picriside I Pcroside II, Trans-Cinnamic acid and Apigenin were obtained from the Sigma- 104 Aldrich, India. 105 106 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.15.950865; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 107 Experimental Animals and Reagents 108 Male Wistar rats (160-180 g) were procured from Liveon Biolabs Pvt. Ltd., Bengaluru, 109 India. Animals were housed