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1,2,3,4,6 Penta-O-galloyl-[beta]-D-glucose, a bioactivity guided isolated compound from Mangifera indica inhibits 11[beta]-HSD-1 and ameliorates high fat diet-induced diabetes in C57BL/6 mice.

ABSTRACT

Methanolic leaf extract of Mangifera indica (MEMI) was subjected to bioactivity guided fractionation in order to identify the active antidiabetic constituent. 32 fractions were evaluated for possible 11[beta-HSD1 inhibition activity under in vitro conditions. The EA-7/8-9/10-4 fraction was evolved as a most potent fraction among all the fractions and it was identified as well known gallotannin compound 1,2,3,4,6 penta-O-galloyl- [beta]-D-glucose (PGG) by spectral analysis. Based on these results the PGG was further evaluated in ex vivo 11[beta]-HSD-1 inhibition assay and high fat diet (HFD)--induced diabetes in male C57BL/6 mice.

Single dose (10, 25, 50 and 100mg/kg) of PGG and carbenoxolone (CBX) have dose dependently inhibited the 11[beta]-HSD-1 activity in liver and adipose tissue. Furthermore, HFD appraisal to male C57BL/6 mice caused severe hyperglycemia, hypertriglyceridemia, elevated levels of plasma corticosterone and insulin, increased liver and white adipose mass with increase in body weight was observed compare to normal control. Also, oral glucose tolerance was significantly impaired compare to normal control. Interestingly, post-treatment with PGG for 21 days had alleviated the HFD-induced biochemical alterations and improved oral glucose tolerance compare to HFD-control. In conclusion, the PGG isolated from MEMI inhibits 11[beta]-HSD-1 activity and ameliorates HFD-induced diabetes in male C57BL/6 mice.

Keywords:

Mangifera indica

Gallotannins

Stress

Diabetes

1,2,3,4,6 Pentagalloyl glucose

11[beta]-Hydroxy steroiddehydrogenase-1

Introduction

11[beta]-Hydroxysteroid dehydrogenases (11[beta]-HSD) are the class of enzymes belongs to family of oxido-reductase, they catalyzes the interconversion of cortisone (inactive) and cortisol (active) and thereby regulates the levels of glucocorticoids in the body (Seckl and Walker, 2001). 11[beta]-HSD-1 converts cortisone to an active glucocorticoid cortisol, while 11[beta]-HSD-2 take part in the backward reaction results in the conversion of cortisol to cortisone (Park et al., 2011). At physiological concentrations cortisol involves in many biological functions such as elevation of blood glucose levels, immunosuppression, detoxification of toxins and plays vital role in the metabolism of carbohydrate, proteins and lipids, However the excess/elevated levels of cortisol results in pathological conditions such as obesity, diabetes, glucose intolerance, hypertension, dyslipidemia, cardiovascular complications and central nervous system related complications such as Alzheimer's disease (Phillips et al., 1998).

Stress activates the HPA (hypothalamo-pituitary-adrenal) axis and thus increases the conversion of cortisone to cortisol in tissues such as liver, brain, skeletal muscle and adipose tissue due to increased expression of 11[beta]-HSD-1 enzyme (Shpilberg et al., 2010). Sequentially, chronic stress also triggers the secretion/release of catecholamines, glucagon and growth hormones, which contributes directly or indirectly in the development of metabolic syndrome and associated complications (Brotman and Girod, 2002). The cortisol-induced metabolic abnormalities includes many mechanisms, mainly impaired insulin sensitivity, altered lipid metabolism, enhanced adipogenesis and gluconeogenesis; gluconeogenesis is increased due to increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase enzymes activities (Radahmadi et al., 2006; Park et al., 2011).

In this context, 11[beta]-HSD-1 is thought to be a better target for treating the metabolic disorder and hence the multinational pharmaceutical companies, such as Merck, Pfizer, Amgen, and Incyt are extensively working in discovering the 11[beta]-HSD-1 inhibitors for treating the diabetes.

Natural products are being identified as an important source of therapeutic agents and demand for herbal drugs has been increasing significantly (Esquijarosa et al., 2009). Even though the actives responsible for biological activity are unknown, they are widely prescribed because of their good therapeutic efficacy, negligible side effects and relatively low cost. In context of treating diabetes and related complications, scientists have reported that phytoconstituents such as polyphenols, coumarins, flavonoids, triterpinoids, alkaloids and others present in the plants will have a potential to show antidiabetic activity (Aiyelaagbe and Mosamudiamen, 2009).

Mangifera indica Linn is commonly grown in many parts of the world and the plant parts have been widely used to treat various ailments in traditional medicines (Sadakane et al., 2005) and various parts of the plant has been scientifically proved to possess medicinal properties such as antidiabetic (Aaderibigbe and Emudianughers, 2001), antioxidant (Martinez et al., 2000), analgesic and anti-inflammatory (Garrido et al., 2001), immunomodulatory (Makare et al., 2001) and so on.

Considering the literature reports of its potentials antidiabetic activity, the present study was under taken to identify the active antidiabetic constituent present in MEM1 by bioactivity guided fractionation technique. The most potent fraction in in vitro 11[beta]-HSD-1 assay was further evaluated in ex vivo and in vivo experimental models.

Materials and methods

Drugs and chemicals

Cortisol assay kit (Cisbio Bioassays, France), Cortisol (Sigma Aldrich, Bangalore), Cortisone (Sigma Aldrich, Bangalore), Corticosterone rat/mouse ELISA Kit (IBL America), Carbonoxolone sodium, Biochemical kits (ERBA diagnostic Mannheim GMBH, Germany) were used and all the solvents used for the extraction and isolation were of analytical grade and purchased from local firms.

Plant materials

M. indica leaves were collected near Madivala Lake, Bangalore. The plant material was authenticated by Dr. K.P. Srinath, Professor of Botany, Department of Botany, Bangalore University, Bangalore. Vouchers Specimen (M. indica) of the plant material is deposited at the Pharmacognosy Department, Government College of Pharmacy, Bangalore.

Extraction and bioactivity guided fractionation of MEMI

The freshly collected leaves of M. indica were dried under shade and powdered. 20 kg of the powdered material was extracted with methanol in soxhlet apparatus (MEM1). The MEM1 extract was concentrated to dryness and made in to four fractions namely ethyl acetate (EA), n-butanol, aqueous and residue fractions and all the fractions were evaluated for 11[beta]-HSD-1 inhibition property under in vitro conditions. In this step, EA was identified as most potent in biological evaluation and therefore it was chromatographed over silica gel column by gradient elution using ethyl acetate and methanol; flow rate was maintained at 5 ml/min throughout the experiment. In this step, 8 subfractions (EA-1 to EA-8) were obtained; in bioevaluation, EA-7 and EA-8 were found to be most potent and TLC pattern of both fractions were identical. Hence EA-7 and EA-8 were combined and subjected to repeated chromatography by gradient elution with petroleum ether, ethyl acetate and methanol. At this stage, 10 subfractions (EA-7/8-1 to EA-7/8-10) were obtained and among all the fractions, EA-7/8-9 and EA-7/8-10 were evolved as most potent fractions and there TLC patterns were found to be identical and hence they were combined rechromatographed by gradient elution with petroleum ether, ethyl acetate and methanol to get 10 sub fractions (EA-7/8-9/10-1 to EA-7/8-9/10-10). Among these ten fractions EA-7/8-9/10-4 fraction showed most potent 11[beta]-HSD-1 inhibition activity and TLC of EA-7/8-9/10-4 showed single spot. The final purification of EA-7/89/1 0-4 was done by preparative HPLC in Kromosil C18 column using 25% acetonitrile in water as mobile phase at 205 nm wavelength. Furthermore, EA-7/8-9/10-4 fraction was subjected to [sup.1]H NMR, [sup.13]C NMR and mass spectral analysis and based on the spectral data it was identified as a well-known gallotannin compound 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose and based on the in vitro findings the PGG was further evaluated forex vivo 11[beta]-HSD-1 inhibition activity and against HFD-induced diabetes in C57BL/6 mice.

Experimental animals

Male C57BL/6 mice (20-25 g) were purchased from Bioneeds, Nelamangala, Tumkur, India and they were maintained in polypropylene cages at a temperature of 25[+ or -]10[degrees]C and relative humidity of 45-55% in clean environment under 12:12 h light-dark cycle. The animals had free access to either normal chow diet (D10001/AIN-76A, Research Diets, US), or high fat diet (D12492, Research Diets, US) and purified water ad libitum. All the experimental protocols were approved by Institutional Animal Ethics Committee (IAEC) and all the animal experiments were conducted according to the principles and guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals), India.

In vitro 11[beta]-HSD-1 inhibition assay

This assay is based on the competition between the sample free cortisol and XL665 labelled cortisol for binding to an anticortisol europium cryptate labelled antibody. It runs in 2 steps, after the dehydrogenase reaction is finished (stimulation step); anti-cortisol cryptate (donor) and d2 labelled cortisol (acceptor) were added to the reaction mixture. The anti-cortisol cryptate and the d2 labelled cortisol will bind to each other and results in a high HTRF signal. Cortisol formed during the enzymatic reaction will compete with the labelled cortisol for binding to the cryptate conjugate, which results in the loss of HTRF signal (detection step). In short, 2 [micro]l of 11[beta]-HSD-1 microsomal preparation (0.1 mg/ml) in 6 [micro]l of substrate buffer (containing 20 mM Tris, 5 mM EDTA buffer with pH 6) containing cortisone 266 nM and NADPH 333 [micro]M were incubated with 2 [micro]l of inhibitor (final concentration of 50 [micro]g/ml) in buffer containing 20 mM Tris and 5 mM EDTA and dispensed into the wells and incubated at 37[degrees]C for 2 h. After 2 h of incubation 5 pi of each HTRF[R] conjugate (anti-cortisol cryptate and cortisol-d2) were added. The plates were incubated for another 2 h at room temperature and fluorescence was read at excitation and emission wavelengths of 620 and 665 nm respectively using PHERAstar (BMG LABTECH's high-end microplate reader). Results were expressed as percentage inhibition with respect to control. The ratio of emission to excitation were calculated for all the samples and results were expressed as percentage inhibition with respect to control (McCormick et al., 2006).

Ex vivo 11[beta]-HSD-1 inhibition assay

Overnight fasted male C57BL/6 mice were divided in to 13 groups (n = 6) and respective groups were treated with vehicle (0.5% carboxy methyl cellulose), carbenoxolone (10, 25, 50 and 100 mg/kg, p.o.), PGG (10, 25, 50 and 100 mg/kg, p.o.) respectively. After 4 h of drug administration, all the animals were sacrificed and about 100 mg of liver and adipose tissue were excised and transferred to 900 [micro]l phosphate buffer saline (PBS) and minced coarsely with scissor. The tissues were thoroughly washed for three times and tissue pellet was collected. About 30 mg of the various tissue pellets were transferred to 85 [micro]l of DMEM (Dulbecco's modified eagles media with 5.5 mM glucose) and 25 [micro]l NADPH (600 [micro]M) and 6 [micro]M of cortisone was added to all the samples to get final concentration of 100 [micro]M NADPH and 1 [micro]M cortisone, and all the sample tubes were incubated for 2 h at 37[degrees]C. At the end of incubation period, all the tubes were centrifuged at 12,000 rpm for 10 min at 4[degrees]C and 100|xl of the supernatant was taken and 1200 |xl of ethyl acetate was added and vortexed for 10 min at 2000 rpm, then the samples were centrifuge at 12,000 rpm for 10 min at 4[degrees]C. The 1000 [micro]l of supernatant was collected, the organic layer was evaporated to dryness, the pellet obtained was subjected to LC-MS/MS analysis. The percentage inhibition of 11[beta]-HSD-1 activity was calculated with respect to vehicle control (Blum et al., 2012; Feng et al., 2010).

Effect of PGG against HFD-induced diabetes in C57BL/6 mice

High fat diet-induced diabetes

Male C57BL/6 mice were randomized in to two groups (G-I and G-II) G-I consists of 10 animals received normal chow diet, while G-II consists of 50 animals fed with high fat diet for 12 weeks. The change in body weight and biochemical parameters was monitored to confirm the induction of diabetes. Animals with fasting blood glucose levels between 180 and 300 mg/dl were selected and observed for consistent hyperglycemia for next 7 days (Feng et al., 2010; Malgorzata and Jonathan, 2007).

Evaluation of antidiabetic activity of PGG

Once the induction of diabetes was confirmed (after 12 weeks feeding), the G-II animals were further divided in to four groups (G1 to G-4) based on body weight and blood glucose. G-1 served as HFD control received only vehicle; G-2, G-3 and G-4 were treated with PGG -10, 25 and 50 mg/kg, p.o. respectively for 21 days. Throughout the treatment period, body weight and blood glucose were weekly monitored and OGTT was carried out on the last day (21st day) of the treatment to see the glucose tolerance. On the last day fasting blood samples were collected for the estimation of plasma corticosterone and insulin levels and all the animals were sacrificed to collect the adrenal glands, liver and adipose tissue. Weight of adrenals, liver and adipose tissue were recorded and expressed with respect to fasting body weight (Feng et al., 2010; Malgorzata and Jonathan, 2007).

Results

In present study, totally 32 fractions were obtained from MEMI and all the fractions were evaluated for in vitro 11[beta]-HSD-1 inhibition activity to identify the most potent fraction and based on the results the most potent fraction was further evaluated in ex vivo and in vivo models.

In vitro 11[beta]-HSD-1 inhibition assay

In in vitro assay, the EA-7/8-9/10-4 fraction was evolved as a most potent among all the fractions obtained from MEMI. Furthermore, EA-7/8-9/10-4 fraction showed 96.7% inhibition at 50 [micro]g/ml concentration. Further, the EA-7/8-9/10-4 fraction was subjected to spectral analysis and shortlisted for further evaluation in ex vivo and in vivo models.

Identification of EA-7/8-9/10-4 fraction

Based on the biological activity and TLC pattern the EA-7/8-9/104 fraction was subjected to physicochemical evaluation and 'H NMR, [sup.13]C NMR and mass spectral studies to elucidate the structure of the compound. The obtained spectral data and physicochemical properties of EA-7/8-9/10-4 fraction are given below.

Physical state and appearance; solid, light white; melting point: 326[degrees]C; molecular formula: [C.sub.41][H.sub.32][O.sub.26]; molecular weight: 940.67; elemental analysis: C (52.35%), H (0.43%), O (44.22%). Purity: 95%.

[sup.1]H NMR spectra: 7.146, 7.085, 7.061, 7.012, 6.985, 6.933, 6.279, 6.258, 5.939, 5.652, 5.622.

[sup.13]C NMR spectra: 167.02,166.38,166.38,166.10,166.00,165.30, 145.60, 145.51, 145.34, 139.84, 139.42, 139.20, 139.08, 12.08, 119.38,119.25,118.75.

By comparing the obtained spectral data with the literature findings, the EA-7/8-9/10-4 fraction was identified as a well known gallotannin class of compound 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose (PGG). Structure of PGG is given in Fig. 1 and scheme of bioactivity guided fractionation is given in Fig. 2.

Ex vivo 11[beta]-HSD-1 inhibition assay

A single dose of PGG was initially evaluated in C57BL/6 mice to explore the possible 11[beta]-HSD-1 inhibition property; CBX was used as reference standard. Single dose of various concentrations of PGG and CBX were administered orally to 4 h fasted C57BL/6 mice and after 4 h of dosing, liver and adipose tissues were excised and looked for conversion of cortisone to cortisol in ex vivo. The findings have revealed that, PGG and CBX could dose dependently inhibit the 11[beta]-HSD-1 activity. Furthermore, at higher dose (100mg/kg, p.o.) PGG and CBX have offered maximum inhibition of 89.1%, 78.21% and 78.7%, 69.8% in liver and adipose tissue respectively (Figs. 3 and 4). Exceptionally PGG was found to be more potent that reference standard CBX (Table 1).

Antidiabetic activity of PGG against HFD-induced diabetes in C57BL/6 mice

In present study, feeding HFD to male C57BL/6 mice for 12 weeks resulted in severe hyperglycemia associated with impaired glucose tolerance, hypercholesterolemia, hypertriglyceridemia and increase in serum LDL, decrease in HDL levels (Table 2). In addition, there was significant increase in body weight, liver and adipose mass (Table 3); our findings in the present study are in line with literature findings. However, post treatment with PGG (10, 25 and 50 mg/kg, p.o.) for 21 days had normalized the elevated levels of blood glucose, total cholesterol, triglycerides and LDL levels also increased the serum levels of HDL and thus alleviated the observed biological perturbances when compare to HFD control, received only vehicle. Furthermore, the effect of EA-7/8-9/10-4 fraction was found to be highly significant and dose dependent. Further, PGG administration has decreased the body weight significantly and dose dependently when compare to HFD control (Fig. 5).

Abbreviations: MEMI, methanolic leaf extract of Mangifera indica; 11[beta]-HSD, 11[beta]-hydroxy steroiddehydrogenase; 11[beta]-HSD-1, 11[beta]-hydroxy steroiddehydrogenase-1; ll[beta]-HSD-2, lip-hydroxysteroiddehydrogenase-2; PGG, 1,2,3,4,6, penta-O-galloyl-[beta]-D-glucose; HFD, high fat diet; CBX, carbenoxolone; HPA, hypothalamo-pituitary-adrenal; PEPCK, phosphoenolpyruvate carboxykinase; ELISA, enzyme linked immunosorbent assay; NMR, nuclear magnetic resonance; TLC, thin layer chromatography; EA, ethyl acetate; IAEC, Institutional Animal Ethics Committee; CPCSEA, Committee for the Purpose of Control and Supervision of Experimentation on Animals; NADPH, nicotinamide diphosphate; EDTA, ethylene diamine tetra acetic acid; OGTT, oral glucose tolerance test; LDL, low density lipoprotein; HDL, high density lipoprotein; HTRF, homogeneous time-resolved fluorescence; DMEM, Dulbecco's modified eagles media.

Effect of PCG on HFD-induced impaired glucose tolerance in C57BL/6 mice

The male C57BL/6 mice upon HFD feeding developed severe hyperglycemia associated with impairment in oral glucose tolerance. However, single dose oral administration of PGG (25 and 50 mg/kg, p.o.) had improved the glucose clearance dose dependently when compare to HFD control received only vehicle (Table 4). Furthermore, upon PGG administration for 21 days showed gradual decrease in serum glucose levels as observed in weekly blood glucose estimation (Table 5). In addition, OGTT conducted on the last day of the treatment (21st day) showed significant improvement in oral glucose tolerance when compare to HFD control and the effect of PGG was found to be dose dependent (Table 6).

Effect of PGG on serum insulin and corticosterone levels in C57BL/6 mice

In present study, feeding HFD to C57BL/6 mice caused an abnormal elevation of serum insulin and corticosteroid levels when compare to normal control animals (p< 0.001). The outcomes of the present study are in line with literature reports in HFD fed C57BL/6 mice. Increase in serum levels of insulin with elevated levels of blood glucose indicates insulin resistance. However, increase in serum corticosterone levels was observed as a result of increase expression of 11[beta]-HSD-1 enzyme activity. In contrast, PGG (10,25 and 50 mg/kg, p.o.) treatment for 21 days has normalized the serum levels of insulin and corticosterone levels. Incidentally, the reduced insulin is associated with normal levels of blood glucose levels and decrease in corticosterone levels was thought to be associated with the inhibition of 11[beta]-HSD-1 activity, which was well established in the other set of experiment (Table 6).

Effect of PGG on adrenal weight in HFD-fed C57BL/6 mice

To explore the possible adverse effects of PGG on the HPA axis activation, adrenal weights and plasma corticosterone levels were measured at the end of the study. The weight of adrenal gland was expressed with respect to 100 g body weight. The findings of the study revealed that, PGG does not have adverse effects on HPA axis activation (Table 7 and Fig. 6).

Discussion

The 11[beta]-HSD-1 enzyme is an endoplasmic reticulum associated oxido-reductase class of enzyme and in recently days it has been identified as novel a therapeutic target for treating various diseases such as obesity, diabetes, cardiovascular disorders, mood disorders and memory impairment (Malgorzata and Jonathan, 2007). 11[beta]HSD-1 is mainly found in the tissues such as liver, adipose, skeletal muscle, brain and immune system, over expression or increased activity of 11[beta]-HSD-1 leads to increase in cortisol levels in the body; especially in liver increased activity of 11[beta]-HSD-1 leads to increase in gluconeogenesis through the activation of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase activity (Radahmadi et al., 2006; Park et al., 2011) and that leads to hyperglycemia. However, enhanced activity of 11[beta]-HSD-1 in adipose tissue leads to increased in adipogenesis which results in obesity and related complications (Alii et al., 2007). It is also been postulated that, prolonged hyperglycemia itself will activate the llp-HSD-1 activity and ultimately leads to obesity, diabetes and other health problems (Wen et al., in press). Apart from this increase in 11[beta]-HSD-1 activity also leads to insulin resistance and impaired glucose tolerance (Wen et al., in press).

The plant based medicines such as carbenoxolone sodium (Andrews et al., 2003; Dhanesha et al., 2012), emodin (Feng et al., 2010), glycyrrhetinic acid (Gomez-Sanchez et al., 1996), Momordica charantia extract (Blum et al., 2012) are already been well proved to possess potent 11[beta]-HSD-1 inhibition activity and hence though to be useful in the treatment of diabetes.

In this context, various parts of M. indica has been scientifically proved to possess excellent antidiabetic activity in experimental animals (Aderibigbe et al., 1999, 2001; Shivanna and Koteshwara, 2010). In present study with an objective of isolating a pure antidiabetic component from methanolic extract (MEM1), the MEMI was made in to total 32 fractions in 4 steps by bioactivity guided fractionation technique. The fractions obtained in the each step were evaluated for 11[beta]-HSD-1 inhibition activity in order to identify the most potent fraction. At the end of the fractionation process EA7/8-9/10-4 was identified as a most potent fraction among all the 32 fractions and it was further evaluated for ex vivo 11[beta]-HSD-1 inhibition activity and also for antidiabetic activity against HFD-induced diabetes in C57BL/6 mice.

Furthermore, EA-7/8-9/10-4 was identified as 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose (PGG) based on spectral studies, PGG is a well known gallotannin compound and proved to be 5 times more potent than reference antioxidant gallic acid (Jinhui et al., 2009) and it is highly enriched in medicinal plants such as M. indica, Rhus chinensis Mill, Paeonia suffruticosa, Paeonia lactiflora, Schinus terebinthifolius, Acer truncatum Bunge and Terminalia chebula (Jinhui et al., 2009).

In the experimental findings, single dose administration of CBX and PGG at 10,25,50 and 100 mg/kg, p.o. doses showed significant and dose dependent inhibition of 11[beta]-HSD-1 activity in liver and adipose tissue in ex vivo conditions. Interestingly, PGG was found to be more potent than reference standard CBX. Furthermore, at higher dose (100 mg/kg, p.o.) PGG and CBX have offered maximum inhibition of 89.1%, 78.21% and 78.7%, 69.8% in liver and adipose tissue respectively.

In line with the literature findings, HFD feeding to male C57BL/6 mice caused severe hyperglycemia with impaired glucose tolerance, hypercholesterolemia, hypertriglyceridemia and increase in serum LDL, decrease in HDL levels. Also, there was significant increase in body weight, liver and adipose mass, serum insulin and corticosterone levels were also increased significantly when compare to normal control. Exceptionally, PGG (10, 25 and 50 mg/kg, p.o.) treatment for 21 days had alleviated the observed biological perturbances when compare to HFD control received only vehicle. Furthermore, the effect of PGG was found to be dose dependent.

Alternatively, to compensate the reduced cortisol levels due to inhibition of lip-HSD-1 gateway adrenal gland will be over activated, it intend activates the HPA axis for producing extra cortisol. The enhanced activity of HPA axis and adrenal gland simultaneously produce excess adrenal androgens along with cortisol. The androgens at circulatory high levels are known to cause poly cystic ovarian syndrome (PCOS), osteoporosis, hypertension, hirsutism and a number of other complications; hence 11[beta]-HSD-1 inhibitors are needs to be screened for their safety profile (Erika and Anne, 2010).

In present study, to explore the possible adverse effects of PGG on HPA axis, plasma corticosterone levels and adrenal weights were recorded and the findings have revealed that PGG was safe and had no adverse effects on HPA activation in all the administered dose levels. These findings suggest that the PGG isolated from methanolic extract of M. indica could dose dependently inhibit the 11[beta]-HSD-1 activity and hence thought to be useful in the treatment of diabetes.

A detailed structure activity relationship (SAR) study conducted by Ren et al. on galloyl esers of glucose like PGG and its analogues relevant to antidiabetic potential showed that, the both D-glucose and galloyl substitution are essential to induce antidiabetic effect. Interestingly, either D-glucose itself or gallic acid and its analogues such as ellagic acid and methyl gallate do not possess glucose clearance activity. These outcomes suggest that D-glucose core must have substituted galloyl groups with unprotected phenyl hydroxyl groups in order to interact with the receptor to induce glucose uptake, the D-glucose core gives a optimal scaffold for the spatial orientation of galloyl groups to show antidiabetic activity. Furthermore, PGG exists in two anomeric forms ([alpha]-PGG and [[beta]-PGG), the a anomeric form was found to be more potent than the [beta] anomeric form in terms of its antidiabetic activity. The galloyl groups at position 1,2,3,4 of glucose are essential, removal of a galloyl group from these positions results in complete loss of activity in both a and [beta] anomeric forms. Essentially, [beta]-PGG require all the five galloyl group at position 1,2,3,4,6 and removal of any one galloyl group results in the formation of corresponding galloyl esters of glucose (mono, di, tri and tetra galloyl glucose derivatives) with lack of bioactivity. However for [alpha]-PGG, galloyl group at C-6 position is not necessary and its replacement with functional groups like chloride (Cl) gives [alpha]-anomeric derivatives of tetra galloyl glucose (a-TGG) with enhanced antidiabetic potential compare to [alpha]-PGG (Ren et al., 2006). Additionally, the PGG has been scientifically proved to possess biological activities such as, anti-oxidant and antimutagenic (Riedl and Hagerman, 2001; Abdelwahed et al., 2007), antiinflammatory (Feldman et al., 2001), cardioprotective (Goto et al., 1996), anticonvulsant (Sugaya et al., 1991), antidiabetic (Li et al., 2005) and also proved to be useful in treating various types of cancers (Pan et al., 1999; Oh et al., 2001; Hua et al., 2006). Furthermore, PGG is been proved to possess glucose uptake enhancing activity and insulin mimetic activity in in vitro (Ren et al., 2006), therefore the possible mechanism behind the antidiabetic activity of PGG is thought to be associated with the combination of its glucose uptake enhancing property and insulin mimetic activity along with anti-adipogenic and 11-[beta]-HSD-1 inhibition property.

Conclusion

The EA-7/8-9/10-4 fraction from the methanolic extract of M. indica was evolved as a potent inhibitor of 11[beta]-HSD-1 enzyme under in vitro, ex vivo and in vivo models and it was identified as 1,2,3,4,6 penta-O-galloyl-P-D-glucose (PGG) based on the 'NMR, 13NMR, and mass spectral data. Thus it can be concluded that, PGG is herbal based novel therapeutic agent can be useful in treating the patients with type 2 diabetes with obesity.

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgement

The authors are thankful to Government College of Pharmacy, Bangalore, India and Management, PES College of Pharmacy, Bangalore, India for providing all the necessary facilities to carry out the research work.

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C.G. Mohan (a), G.L. Viswanatha (b), *, G. Savinay (c), C.E. Rajendra (d), Praveen D. Halemani (c)

(a) Department of Pharmacognosy, Government College of Pharmacy, Bangalore 560 027, India

(b) Department of Pharmacology, PES College of Pharmacy, Bangalore 560 050, India

(c) Deparment of Pharmaceutics, Government College of Pharmacy, Bangalore 560 027, India

(d) Drug Testing Laboratory, Drugs Control Department, Palace Road, Bangalore 560 001, India

* Corresponding author.

E-mail addresses: mohangcp@gmail.com (C.G. Mohan), giv.000@yahoo.com (G.L. Viswanatha).

http://dx.doi.org/10.1016/j.phymed.2012.12.020

Table 1
Effect of fractions of MEMI on 11 [3-HSD-l enzyme activity in in
vitro conditions.

SI. no.   Sample          Concentration   Percentage
                          ([micro]g/ml)   inhibition

                                          Liver   Adipose

1         Ethyl acetate   50              47.4    46.8
2         n-Butanol       50               8.6     6.2
3         Aqueous         50               2.5     3.8
4         Residue         50              12.3    14.6
5         EA-1            50               2.9     2.2
6         EA-2            50               2.7     1.6
7         EA-3            50               2.4     3.4
8         EA-4            50               6.5     7.2
9         EA-5            50              12.5    16.5
10        EA-6            50              19.2    18.6
11        EA-7            50              62.5    67.2
12        EA-8            50              62.9    66.4
13        EA-7/8-1        50               3.3     4.2
14        EA-7/8-2        50               4.5     5.2
15        EA-7/8-3        50               4.6     3.9
16        EA-7/8-4        50               5.2     5.1
17        EA-7/8-5        50              26.6    28.1
18        EA-7/8-6        50              31.2    33.7
19        EA-7/8-7        50              26.1    12.5
20        EA-7/8-8        50              19.5    17.2
21        EA-7/8-9        50              73.9    74.1
22        EA-7/8-10       50              74.5    77.6
23        EA-7/8-9/10-1   50              41.2    35.7
24        EA-7/8-9/10-2   50              32.1    28.5
25        EA-7/8-9/10-3   50              22.4    12.5
26        EA-7/8-9/10-4   50              92.5    96.7
27        EA-7/8-9/10-5   50              19.5    17.4
28        EA-7/8-9/10-6   50              12.5    14.5
39        EA-7/8-9/10-7   50              14.9    14.6
30        EA-7/8-9/10-8   50              19.4    22.5
31        EA-7/8-9/10-9   50              14.5    14.5
32        EA-7/8-9/10-10  50              26.5    28.4

All the samples were analyzed in duplicates at 50 [micro]g/ml
concentration and the values in terms of percentage inhibition is
expressed as mean of duplicate values.

Table 2
Effect of PGG on blood glucose and lipid profile in HFD fed C57BL/6
mice.

Treatment            BG (mg/dl)

Normal control        91.23 [+ or -] 5.2
HFD control          213.21 [+ or -] 4.56 **
PGG-l0mg/kg, p.o.    219.47 [+ or -] 6.72
PGG-25 mg/kg, p.o.   223.14 [+ or -] 6.51
PGG-50 mg/kg, p.o.   219.64 [+ or -] 5.14

Treatment            TC (mg/dl)

Normal control        69.41 [+ or -] 8.3
HFD control          189.21 [+ or -] 5.2 **
PGG-l0mg/kg, p.o.    108.25 [+ or -] 7.4 ([dagger][dagger][dagger])
PGG-25 mg/kg, p.o.    89.24 [+ or -] 6.3 ([dagger][dagger][dagger])
PGG-50 mg/kg, p.o.    71.45 [+ or -] 4.8 ([dagger][dagger][dagger])

Treatment            TG (mg/dl)

Normal control       109.62 [+ or -] 8.4
HFD control          254.32 [+ or -] 12.2 **
PGG-l0mg/kg, p.o.    185.26 [+ or -] 13.6 ([dagger])
PGG-25 mg/kg, p.o.   149.24 [+ or -] 9.3 ([dagger][dagger])
PGG-50 mg/kg, p.o.   126.61 [+ or -] 7.9  ([dagger][dagger])

Treatment            HDL (mg/dl)

Normal control       32.65 [+ or -] 1.4
HFD control          12.32 [+ or -] 0.9 *
PGG-l0mg/kg, p.o.    19.61 [+ or -] 0.8
PGG-25 mg/kg, p.o.   25.62 [+ or -] 1.2
PGG-50 mg/kg, p.o.    29.8 [+ or -] 0.7 ([dagger][dagger])

Treatment            LDL (mg/dl)

Normal control       18.64 [+ or -] 2.0
HFD control          98.23 [+ or -] 8.1 ***
PGG-l0mg/kg, p.o.    61.74 [+ or -] 3.5 ([dagger][dagger])
PGG-25 mg/kg, p.o.   40.98 [+ or -] 3.9 ([dagger][dagger])
PGG-50 mg/kg, p.o.   22.41 [+ or -] 1.7 ([dagger][dagger][dagger])

Notes: PGG = 1,2,3,4,6 penta-0-galloyl-([beta]-D-glucose, TC = total
cholesterol, TG = triglycerides, HDL = high density lipoproteins,
LDL = low density lipoproteins. All the values are expressed as mean
[+ or -] SEM (n = 8). * HFD control vs normal control (* p<0.05,
** p < 0.01, *** p< 0.001). ([dagger]) Treatments vs HFD control
([dagger]) p<0.05, ([dagger][dagger]) p<0.01,
([dagger][dagger][dagger]) p <0.001).

Table 3
Effect of PGG on liver and white adipose tissue (WAT) mass in HFD fed
C57BL/6 mice.

Treatment            Body weight (g)

Normal control       26.385 [+ or -] 0.73
HFD control          32.229 [+ or -] 1.29 **
PGG-10 mg/kg, p.o.   27.242 [+ or -] 0.94 ([dagger])
PGG-25 mg/kg, p.o.   26.611 [+ or -] 0.97 ([dagger][dagger])
PGG-50 mg/kg, p.o.   25.945 [+ or -] 0.96 ([dagger][dagger])

Treatment            Liver weight (g)

Normal control       1.06 [+ or -] 0.01
HFD control          1.68 [+ or -] 0.05 **
PGG-10 mg/kg, p.o.   1.20 [+ or -] 0.04
PGG-25 mg/kg, p.o.   1.12 [+ or -] 0.06 ([dagger])
PGG-50 mg/kg, p.o.   1.06 [+ or -] 0.03 ([dagger])

Treatment            Relative weight of Liver (g/100 g)

Normal control        4.0 [+ or -] 0.05
HFD control           5.2 [+ or -] 0.21 **
PGG-10 mg/kg, p.o.    4.4 [+ or -] 0.26
PGG-25 mg/kg, p.o.   4.22 [+ or -] 0.21 ([dagger])
PGG-50 mg/kg, p.o.    4.1 [+ or -] 0.31 ([dagger])

Treatment            WAT weight (g)

Normal control       0.21 [+ or -] 0.01
HFD control          0.46 [+ or -] 0.03 ***
PGG-10 mg/kg, p.o.   0.33 [+ or -] 0.02 ([dagger])
PGG-25 mg/kg, p.o.   0.25 [+ or -] 0.02 ([dagger][dagger])
PGG-50 mg/kg, p.o.   0.22 [+ or -] 0.03 ([dagger][dagger])

Treatment            Relative weight of WAT (g/100 g)

Normal control       0.81 [+ or -] 0.02
HFD control          1.42 [+ or -] 0.01 ***
PGG-10 mg/kg, p.o.   1.21 [+ or -] 0.03 ([dagger])
PGG-25 mg/kg, p.o.   0.94 [+ or -] 0.01 ([dagger][dagger])
PGG-50 mg/kg, p.o.   0.85 [+ or -] 0.01 ([dagger][dagger])

Note: PGG = 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose. All the
values are expressed as mean [+ or -] SEM (n = 8). * HFD control vs
normal control (* p <0.05, ** p <0.01. *** p <0.001). ([dagger])
Treatments vs HFD control (([dagger]) p <0.05, ([dagger][dagger])
p <0.01, ([dagger][dagger][dagger]) p <0.001).

Table 4 Effect of single dose oral administration of PGG on blood
glucose levels in HFD fed C57BL/6 mice.

Treatment            Blood glucose (mg/dl)

                     0 h

Normal control        91.23 [+ or -] 5.2
HFD control          213.21 [+ or -] 4.56 *
PGG-l0mg/kg, p.o.    219.47 [+ or -] 6.72
PGG-25 mg/kg, p.o.   223.14 [+ or -] 6.51
PGG-50 mg/kg, p.o.   219.64 [+ or -] 5.14

Treatment            Blood glucose (mg/dl)

                     1 h

Normal control        92.45 [+ or -] 2.8
HFD control          210.84 [+ or -] 4.11 *
PGG-l0mg/kg, p.o.    196.23 [+ or -] 7.20 ns
PGG-25 mg/kg, p.o.   174.23 [+ or -] 4.11 ([dagger])
PGG-50 mg/kg, p.o.   149.54 [+ or -] 6.61 ([dagger][dagger][dagger])

Treatment            Blood glucose (mg/dl)

                     2 h

Normal control        92.15 [+ or -] 4.5
HFD control          224.32 [+ or -] 6.12 *
PGG-l0mg/kg, p.o.    186.42 [+ or -] 6.31 ([dagger])
PGG-25 mg/kg, p.o.   169.25 [+ or -] 7.34 ([dagger][dagger])
PGG-50 mg/kg, p.o.   152.34 [+ or -] 7.21 ([dagger][dagger][dagger])

Treatment            Blood glucose (mg/dl)

                     4 h

Normal control        91.54 [+ or -] 3.1
HFD control          218.44 [+ or -] 4.78 *
PGG-l0mg/kg, p.o.    188.25 [+ or -] 5.1 ([dagger])
PGG-25 mg/kg, p.o.   162.44 [+ or -] 5.22 ([dagger][dagger])
PGG-50 mg/kg, p.o.   145.24 [+ or -] 6.87 ([dagger][dagger][dagger])

Notes: PGG = 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose. All the
values are expressed as mean [beta] SEM (n = 8). * HFD control vs
normal control (* p<0.001). ([dagger]) Treatments vs HFD control
(([dagger]) p<0.01, ([dagger][dagger]) p<0.001).

Table 5
Effect of repeated oral dose of PGC on blood glucose levels in HFD
fed C57BL/6 mice.

Treatment            Blood glucose (mg/dl)

                     0th day

Normal control        91.23 [+ or -] 5.2
HFD control          272.84 [+ or -] 3.9 *
PGG-l0mg/kg, p.o.    271.94 [+ or -] 4.8 ([dagger])
PGG-25 mg/kg, p.o.   268.51 [+ or -] 6.2
PGG-50 mg/kg, p.o.   272.84 [+ or -] 5.2

Treatment            Blood glucose (mg/dl)

                     7th day

Normal control        92.45 [+ or -] 2.8
HFD control          296.45 [+ or -] 11.3 *
PGG-l0mg/kg, p.o.    186.24 [+ or -] 7.9 ([dagger])
PGG-25 mg/kg, p.o.   172.35 [+ or -] 6.4 ([dagger][dagger])
PGG-50 mg/kg, p.o.   146.78 [+ or -] 8.6 ([dagger][dagger])

Treatment            Blood glucose (mg/dl)

                     14th day

Normal control        92.15 [+ or -] 4.5
HFD control          311.41 [+ or -] 9.3 *
PGG-l0mg/kg, p.o.    163.42 [+ or -] 7.4 ([dagger])
PGG-25 mg/kg, p.o.   156.11 [+ or -] 9.7 ([dagger][dagger])
PGG-50 mg/kg, p.o.   131.47 [+ or -] 6.8 ([dagger][dagger])

Treatment            Blood glucose (mg/dl)

                     21st day

Normal control        91.54 [+ or -] 3.1
HFD control          284.22 [+ or -] 9.1 *
PGG-l0mg/kg, p.o.    149.65 [+ or -] 8.2 ([dagger])
PGG-25 mg/kg, p.o.   134.85 [+ or -] 5.9 ([dagger][dagger])
PGG-50 mg/kg, p.o.   102.94 [+ or -] 6.7 ([dagger][dagger])

Notes: PGG = 1,23,4,6 penta-O-galloyl-[beta]-D-glucose. All the
values are expressed as mean [+ or -] SEM (n = 8). * HFD control vs
normal control (* p<0.001). ([dagger]) Treatments vs HFD control
(([dagger]) p<0.01, ([dagger][dagger]) p<0.001).

Table 6
Effect of repeated oral dose of PGG on oral glucose tolerance in HFD
fed C57BL/6 mice on 21st day.

Treatment            Blood glucose (mg/dl)

                     0 min

Normal control       93.37 [+ or -] 3.7
HFD control          279.5 [+ or -] 9.8 *
PGG-10 mg/kg, p.o.   147.1 [+ or -] 6.9 ([dagger])
PGG-25 mg/kg, p.o.   132.7 [+ or -] 7.6 ([dagger] [dagger])
PGG-50 mg/kg, p.o.   100.8 [+ or -] 6.7 ([dagger] [dagger])

Treatment            Blood glucose (mg/dl)

                     30 min

Normal control       141.11 [+ or -] 9.2
HFD control          396.45 [+ or -] 13.2 *
PGG-10 mg/kg, p.o.   179.28 [+ or -] 11.3 ([dagger])
PGG-25 mg/kg, p.o.   152.61 [+ or -] 8.9 ([dagger] [dagger])
PGG-50 mg/kg, p.o.   134.85 [+ or -] 10.9 ([dagger] [dagger])

Treatment            Blood glucose (mg/dl)

                     60 min

Normal control       179.42 [+ or -] 6.2
HFD control          349.32 [+ or -] 15.4 *
PGG-10 mg/kg, p.o.   192.22 [+ or -] 9.4 ([dagger])
PGG-25 mg/kg, p.o.   169.41 [+ or -] 9.6 ([dagger] [dagger])
PGG-50 mg/kg, p.o.   115.69 [+ or -] 8.7 ([dagger] [dagger])

Treatment            Blood glucose (mg/dl)

                     120 min

Normal control       133.98 [+ or -] 7.8
HFD control          290.27 [+ or -] 12.3 *
PGG-10 mg/kg, p.o.   142.32 [+ or -] 7.6 ([dagger])
PGG-25 mg/kg, p.o.   122.42 [+ or -] 8.7 ([dagger] [dagger])
PGG-50 mg/kg, p.o.   104.92 [+ or -] 7.6 ([dagger] [dagger])

Treatment            Blood glucose (mg/dl)

                     180 min

Normal control       98.21 [+ or -] 7.4
HFD control          292.44 [+ or -] 16.3 *
PGG-10 mg/kg, p.o.   129.85 [+ or -] 9.3 ([dagger] [dagger])
PGG-25 mg/kg, p.o.   115.69 [+ or -] 9.4 ([dagger] [dagger])
PGG-50 mg/kg, p.o.   104.49 [+ or -] 8.2 ([dagger] [dagger])

Notes: PGG = 1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose. All the
values are expressed as meaniSEM (n = 8). * HFD control vs normal
control (* p< 0.001). ([dagger]) Treatments vs HFD control
(([dagger]) p < 0.01, ([dagger] [dagger]) p < 0.001).

Table 7
Effect of PGC on blood glucose, insulin and cortisterone levels in
HFD fed C57BL/6 mice on 21st day.

Treatment            BG (mg/dl)

Normal control       93.37 [+ or -] 3.7
HFD control          279.5 [+ or -] 9.8 *
PGG-10 mg/kg, p.o.   147.1 [+ or -] 6.9 ([dagger])
PGG-25 mg/kg, p.o.   132.7 [+ or -] 7.6 ([dagger][dagger])
PGG-50 mg/kg, p.o.   100.8 [+ or -] 6.7 ([dagger][dagger])

Treatment            Plasma insulin (ng/dl)

Normal control       69.07 [+ or -] 3.5
HFD control          126.7 [+ or -] 4.4 *
PGG-10 mg/kg, p.o.   96.64 [+ or -] 7.7 ([dagger])
PGG-25 mg/kg, p.o.   82.11 [+ or -] 5.3 ([dagger][dagger])
PGG-50 mg/kg, p.o.   70.02 [+ or -] 6.2 ([dagger][dagger])

Treatment            Plasma corticosterone levels (ng/ml)

Normal control       64.4 [+ or -] 5.4
HFD control          164.5 [+ or -] 7.4 *
PGG-10 mg/kg, p.o.   132.8 [+ or -] 5.8 ([dagger])
PGG-25 mg/kg, p.o.   104.1 [+ or -] 7.7 ([dagger][dagger])
PGG-50 mg/kg, p.o.   60.1 [+ or -] 4.4 ([dagger][dagger])

Notes: PGG-1,2,3,4,6 penta-O-galloyl-[beta]-D-glucose. All the values
are expressed as mean [+ or -] SEM (n = 8). * HFD control vs normal
control (* p<0.001). ([dagger]) Treatments vs HFD control (([dagger])
p<0.01, ([dagger][dagger]) p< 0.001).
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Author:Mohan, C.G.; Viswanatha, G.L.; Savinay, G.; Rajendra, C.E.; Halemani, Praveen D.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Mar 15, 2013
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