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Hypoglycemic and Antiplatelet Effects of Unsaponified Petroleum Ether Fraction and Isolated Compounds of Methanol Extract of Clerodendrum Phlomidis Linn

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Hypoglycemic and Antiplatelet Effects of Unsaponified Petroleum Ether Fraction and Isolated Compounds of Methanol Extract of Clerodendrum Phlomidis Linn International Journal of Pharmacy and Biological Sciences ISSN: 2321-3272 (Print), ISSN: 2230-7605 (Online) IJPBS | Volume 7 | Issue 3 | JUL-SEPT| 2017 | 93-107 Original Research Article – Pharmaceutical Sciences| Open Access| UGC Approved | MCI Approved Journal HYPOGLYCEMIC AND ANTIPLATELET EFFECTS OF UNSAPONIFIED PETROLEUM ETHER FRACTION AND ISOLATED COMPOUNDS OF METHANOL EXTRACT OF CLERODENDRUM PHLOMIDIS LINN. F. LEAVES Muthu K MohanMarugaRaja a, Riyaj S Tamboli b, Shri H Mishrab, Devarajan Agilandeswari c* a Senior Scientist, TanBio R&D Solution, Periyar TBI, Periyar Maniammai University, Thanjavur-613403, Tamil Nadu, India b Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara-390002, Gujarat, India c Department of Pharmaceutics, Ikon Pharmacy College, Bheemanahalli-562109, Karnataka, India *Corresponding Author Email: [email protected] ABSTRACT Methanol extract of the leaves of Clerodendrum phlomidis Linn. f. (Lamiaceae) has been reported for antidiabetic activity. The aim was to explore in detail the various fractions and isolated compounds from methanol extract of the leaves of C. phlomidis for hyopglycemic and antiplatelet effects. Residual fraction of methanol extract (RFME), unsaponified petroleum ether fraction of methanol extract (USPEF) and crude polyamine fraction (CPF) were studied by streptozotocin-nicotinamide (STZ-NAD) induced diabetic rat model (100 and 200 mg/kg BW, p.o. daily for 30 days). Metformin was used as standard drug along with diabetic and normal control. Compounds (CPI–VI) were isolated from bioactive USPEF. CPII and CPVI were studied at 15 and 30 mg/kg BW, p.o. daily for 30 days. All fractions, CPII and CPVI were also studied for protein tyrosine phosphatase 1B (PTP1B) inhibition and adenosine diphosphate (ADP) induced antiplatelet aggregation study. CPI (1-hexadecanol), CPII (clerosteryl palmitate), CPIII (β-carotene), CPIV (β-sitosterol), CPV (lupeol) and CPVI (clerosterol) were isolated from USPEF. CPII and CPVI showed moderate antidiabetic and antiplatelet effects. USPEF 200 mg/kg showed significant changes (p<0.01) in all parameters. USPEF also showed significant IC50 value of 23.50±1.1 and 11.12±0.8 µg/mL in PTP1B inhibition and ADP induced platelet aggregation study respectively. It was concluded that USPEF exhibits synergistic antidiabetic and antiplatelet activities. KEY WORDS Protein tyrosine phosphatase 1B, Platelet aggregation, Histopathological studies, Clerosterol, Clerosteryl palmitate. 1 INTRODUCTION: leptin signalling pathways2. In the insulin signalling Type 2 diabetes is a progressive disease characterized pathway, PTP1B dephosphorylates the insulin receptor 3,4 by insulin resistance in peripheral tissues and/or substrate IRS-1 . Overwhelming evidence suggests impaired insulin secretion by the pancreas. At that inhibiting PTP1B represents a highly promising 5 molecular level, the mechanism of insulin resistance in approach to treat diabetes . type 2 diabetes involves post-receptor defects in insulin Traditional herbal medicines are prescribed widely in signalling1. The knowledge of heterogeneity of type 2 developing countries because of their time-tested diabetes is increasing; hence there is a need to look for effectiveness and relatively low cost. An exhaustive more efficacious agents for its treatment rather than study on medicinal plants may lead to isolation of managing hyperglycemia. Protein tyrosine phosphatase interesting leads of novel chemical entities. 1B (PTP1B) is a negative regulator in both insulin and Additionally, there exists a possibility of deriving new International Journal of Pharmacy and Biological Sciences Devarajan Agilandeswari* et al 93 www.ijpbs.com or www.ijpbsonline.com ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. knowledge on the mechanism of action of therapeutic Association (ADA) recommend antiplatelet therapy as a agents which, in turn, may help in better understanding primary prevention strategy in diabetic patients with of the etiopathogenesis and the course of various increased cardiovascular risk12. Numerous diseases. The World Health Organisation (WHO) has epidemiological studies also support the early recommended evaluation of the medicinal plants used treatment of diabetic cardiovascular complications for the treatment of diabetes. Therefore, it is prudent with antiplatelet agents13. In our current study, to look into medicinal plants as alternative resource for fractions and the compounds isolated from leaf the treatment of diabetes. methanol extract of C. phlomidis were also evaluated Clerodendrum phlomidis Linn. f. (Lamiaceae) is an for platelet aggregation inhibitory activities. important and well known medicinal plant in Ayurveda and Siddha system of medicines. It is commonly known 2 SUBJECTS AND METHODS: as Thalludhalai, Agnimantha or Arani and is a 2.1 Materials constituent of more than 50 indigenous drug All reagents used were of technical or analytical grade formulations. Agnimantha is very essential medicinal and were purchased from Sigma-Aldrich, Spectrochem plant mentioned in texts since vedic period. It is and Merck. All solvents were of analytical grade or HPLC described as one of ten herbs of dashamula. The grade and were distilled prior to use. The 1H NMR and 13 ayurvedic properties of C. phlomidis are: rasa - tikta, C NMR spectra were recorded in CDCl3 or C6D6 using katu, kashaaya and madhura; guna - rooksha and laghu; Bruker (Swiss) Avance II spectrometer (100 or 400 6 veerya – ushna; vipaka – katu . Due to its bitter and MHz). Chemical shifts are reported in δ units downfield pungent nature C. phlomidis is considered to normalize from TMS as an internal standard. The CHNOS analyses 7 the vitiated kapha and vata dosa . C. phlomidis is used were recorded using Thermo Finnigan Flash EA 1112 in different ayurvedic formulations such as (Italy) analyser. Ayushyavardhaak tel, Bhratpanchamula, 2.2 Plant material Chandraprabha vati, Lavanbhasker churna, Leaves of C. phlomidis were collected on October 2009 Abhayarisht, Chavanprasha, Dasamularista, from out-fields of Trichy city, Tamil Nadu, India. The Ashwagandharishta, Mritasanjivani, Dasamula kvatha plant material was identified and authenticated by Dr. churna, Haritakiavleh, Indukanta ghrta, Dhanvantara A. Rajan, Field Botanist, The Survey of Medicinal Plants ghrta, Gorocanadi vati, Narayana taila, Ras pitari, and Collection Unit, Government Arts College, Vrahat panchamuli and Muthu marunthu (a siddha Ootacamund, Tamil Nadu, India. A voucher specimen 8 polyherbal formulation) . Its popular uses include the (Pharmacy/HDT/CP/08-09/MKM/15) has been 9 treatment of inflammation and diabetic conditions . deposited in the Herbarium of Medicinal Plants, Pectolinaringenin, scutellarein, clerodin, clerodendrin, Pharmacy Department, The Maharaja Sayajirao clerosterol, 24β-ethylcholesta-5,22E,25-triene-3β-ol, University of Baroda, Vadodara, Gujarat, India. lup-20(29)-en-3-triacontanoate, 4,2’,4’-trihydroxy-6’- 2.3 Preparation of extract and fractions methoxychalcone-4, 4’α-D-diglucoside, 7- Air-dried leaves (2000 g) of C. phlomidis were grounded hydroxyflavone, 7-hydroxyflavanone-7-O-glucoside and extracted with methanol in Soxhlet apparatus for and α-L-rhamnopyranosyl-(1→2) α-D-glucopyranosyl- 48 h. The extract was evaporated to dryness under 7-O-naringin-4’-O-α-D-glucopyranoside-5-methyl ether reduced pressure on rotary evaporator (Rotavapor, are reported earlier from C. phlomidis in various Buchi) and further dried in desiccator to yield methanol 8 literatures . Preliminary investigation has revealed the extract. The methanol extract was further fractioned by antidiabetic activity of methanol leaf extract of C. centrifuging with 3 x 500 mL of petroleum ether (60-80 10 phlomidis . Thus, it was thought to thoroughly °C) at 1000 g for 15 min. The supernatants were investigate the various fractions of methanol leaf combined, evaporated to dryness under reduced extract for in vivo and in vitro antidiabetic activity. pressure on rotary evaporator and further dried in The American Heart Association (AHA) considers desiccator to yield petroleum ether fraction of platelet hyperaggregability to be one of the important methanol extract. The insoluble residue was designated risk factors for cardiovascular disease in diabetic as residual fraction of methanol extract (RFME). The 11 patients . Hence, AHA and American Diabetes petroleum ether fraction of methanol extract was 94 International Journal of Pharmacy and Biological Sciences Devarajan Agilandeswari* et al www.ijpbs.com or www.ijpbsonline.com ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. saponified as per the reported procedure14 and the administered orally once a day for 30 days. The effects unsaponified matter was designated as unsaponified of all the test samples were determined by measuring petroleum ether fraction of methanol extract (USPEF). plasma glucose17, plasma insulin levels18 and changes in A crude polyamine fraction (CPF) was prepared from body weight. On day 30 the animals were sacrificed, 100 g of air-dried grounded leaves of C. phlomidis livers were isolated for the estimation of hexokinase19, following the earlier described method15. glucose-6-phosphatase20, glycogen21, and pancreas was 2.4 Animals removed for histopathological studies. Liver and Healthy adult Albino rats of Wistar strain weighing 200– pancreas tissues were washed with normal saline and 250g were procured from Zydus-Cadila stored in 10% formalin. The pancreatic
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