Hypoglycemic activity of alkaloidal fraction of Tinospora cordifolia

Phytomedicine 18 (2011) 1045–1052

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Hypoglycemic activity of alkaloidal fraction of Tinospora cordifolia Mayurkumar B. Patel ∗ , Shrihari Mishra Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390 001, Gujarat, India

a r t i c l e

i n f o

Keywords: Hyperglycemia Tinospora cordifolia RINm5F pancreatic ␤-cell line Rat hepatocytes

a b s t r a c t The stem of Tinospora cordifolia (TC) is widely used in the therapy of diabetes in traditional folk medicine of India. In the present study, isoquinoline alkaloid rich fraction (AFTC) derived from stem of TC and three alkaloids viz., palmatine, jatrorrhizine and magnoflorine were evaluated for insulin-mimicking and insulin-releasing effect in vitro and in vivo. Their effect on hepatic gluconeogenesis was examined in rat hepatocytes. Insulin releasing effect was detected in vitro using rat pancreatic ␤-cell line, RINm5F. Furthermore, effects of AFTC and isolated alkaloids on serum glucose and insulin level were studied in fasted and glucose challenged normal rats. AFTC significantly decreased gluconeogenesis in rat hepatocytes as insulin did and it increases insulin secretion in RINm5F cells similar to tolbutamide. In acute 30 min test in vitro, AFTC, palmatine, jatrorrhizine and magnoflorine stimulated insulin secretion from the RINm5F cell line. As in vivo results, administration of AFTC (50, 100, and 200 mg/kg), palmatine, jatrorrhizine and magnoflorine (10, 20 and 40 mg/kg each) orally significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal rats. In vivo study further justified their insulin secreting potential by raising the serum insulin level in glucose fed rats. These results demonstrate the alkaloid present in TC contributed for antihyperglycemic activity. AFTC may have hypoglycemic effects via mechanisms of insulin releasing and insulin-mimicking activity and thus improves postprandial hyperglycemia. © 2011 Elsevier GmbH. All rights reserved.

Introduction Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms is a large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae (Wealth of India 1976; Atal et al. 1986). The stems of Tinospora cordifolia are succulent with long filiform fleshy aerial roots from the branches. It is widely used in folk and ayurvedic system of medicine as general tonic, hepatoprotective, antiperiodic, anti-spasmodic, anti-inflammatory, antiarthritic, anti-allergic and antidiabetic (Atal et al. 1986; Nayampalli et al. 1988; Chintalwar et al. 1999; Prince and Menon 1999; Grover et al. 2000; Bishayi et al. 2002; Prince et al. 2004; Badar et al. 2005). Free radical scavenging and radioprotective actions are reported (Goel et al. 2002, 2004). The plant is used in “Rasayana” therapy of ayurveda to improve the immune system. The root of this plant is known for its antistress, anti-leprotic and anti-malarial properties (Zhao et al. 1991; Nayampalli et al. 1982). T. cordifolia is widely used in Indian traditional medicine for treating diabetes mellitus (Grover et al. 2002). Oral administration of either alcoholic or aqueous extract of TC is reported to have hypoglycemic activity in different animal models (Gupta et al. 1967; Raghunathan and Sharma 1969; Mahajan and

∗ Corresponding author. E-mail address: [email protected] (M.B. Patel). 0944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.05.006

Jolly 1985; Wadood et al. 1992; Dhaliwal 1999; Grover et al. 2000, 2001; Stanely et al. 2000). A variety of constituents are present in TC plant belonging to different classes such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides. Water soluble isoquinoline alkaloids viz., jatrorrhizine, palmatine, berberine, tembetarine, magnoflorine, choline, tinosporine, isocolumbine and hydrastine are present (Qudrat-IKhuda et al. 1964; Pachaly and Schneider 1981; Bisset and Nwaiwu 1983; Padhya 1986; Gangan et al. 1994; Sarma et al. 1995, 1998; Kumar et al. 2000). Since majority of the hypoglycemic activity of TC has been carried out on its aqueous extract, present studies has been undertaken to evaluate a fraction rich in water soluble alkaloid, for its hypoglycemic activity in vitro and in vivo. Fresh plants (stem) hanging on neem tree (Azadirachta indica) was collected and used for preparation of fraction. Materials and methods Plant material and fraction preparation The stems of Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms (Menispermaceae) were collected from the village of Vadodara district, Gujarat, India in September 2008. Plant material was identified by Dr. K.S. Rajput, Botany Department, The M.S. Uni-

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versity of Baroda, Vadodara, India. The herbarium voucher has been deposited in our laboratory for future reference (Accession Number: MS/PHR/TC1/SAMP003-08). Coarse powder (920 g) of TC stem was defatted with petroleum ether (60–80 ◦ C) and dried material was extracted with 70% methanol using soxhlet apparatus at 65–70 ◦ C for 7 days. Solvent was recovered and semisolid hydroalcoholic extract (26.2 g) was made acidified by adding dilute hydrochloric acid (100 ml). It was filtered through Whatman filter paper and residue was washed with dilute hydrochloric acid till filtrate showed positive test for alkaloid. Combined filtrate and washings were made alkaline (pH 8.0) with ammonia (25%) and partitioned with chloroform (3× 150 ml, 2× 100 ml). Chloroform layer was concentrated and labelled as AFTC.

Isolation of compounds An aliquot of AFTC (15.0 g) was fractionated by VLC using silica gel as an adsorbent. The system was sequentially eluted with CHCl3 and MeOH in a polarity gradient fashion, by increasing polarity of MeOH from 0 to 100%, to obtain 36 fractions (200 ml each). Fractions 10–16 were purified by Column chromatography (silica gel) eluting with CHCl3 –MeOH (6:1, 2100 ml) to obtain compound 1 as yellow needles (2.467 g). Fractions 17–31 were subjected to Column chromatography (silica gel), sequentially eluted with CHCl3 –MeOH (4:1, 2000 ml and 3:1, 1200 ml). After crystallization in MeOH, compound 2 (748 mg) was obtained as orange plates. The alkaline aqueous fraction was acidified with HC1 to pH 3.0 and treated with a solution of picric acid for 24 h, forming a ppt. This ppt. was dissolved in MeOH, treated with activated charcoal and filtered over celite. The methanolic solution was passed through an ion-exchange resin (Amberlite IRA400 OH− ) column, evaporated and further purified by preparative chromatography on silica gel (mobile phase: MeOH:water:NH3 (25%), 15:3:1) to obtain compound 3 (Rf 0.3, blue fluorescent band under 365 nm, red after treatment with Dragendorff’s reagent) (yield – 140 mg).

Instrumentation Melting points were determined in open glass capillaries, using a scientific melting point apparatus. IR spectra were recorded on a Shimadzu FT IR 8300 spectrophotometer (Vmax in cm−1 , using KBr pellets). The 1 H NMR spectra were recorded on a 500/125 MHz on a Varian Inova 500 NMR spectrometer. Chemical shifts (ı) are reported in parts per million (ppm) relative to TMS in DMSO-d6 solution. EI-MS data were recorded on a Hewlett-Packard HP 5890 Series II Plus GC-HP 5972 Mass Selective Detector (EI mode with mass range of 20–700 amu) and a Finnigan MAT Incos 50 mass spectrometer.

Fig. 1. HPTLC chromatogram of AFTC. Stationary phase: Aluminium backed precoated silicagel 60F254 HPTLC plates. Mobile phase: Ethylacetate:glacial acetic acid:formic acid:water (100:11:11:32), track scanned at 254 nm. Peak 2: Magnoflorine, Peak 3: Palmatine and Peak 4: Jatrorrhizine.

Material and protocols in vitro Materials in experiments in vitro Rat insulinoma RINm5F cells were obtained from National Centre for Cell Sciences (NCCS, Pune, India). Cell culture media, phosphate saline buffer (PBS), dimethyl sulfoxide (DMSO), lglutamine, sodium bicarbonate, glucose, HEPES, sodium pyruvate, fetal bovine serum, streptomycin, penicillin, and MTT (3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) were procured from Hi Media (Mumbai, India). Dexamethasone (DEX), STZ and 8-(4-chlorophenylthio) adenosine 3 ,5 -cyclic monophosphate sodium salt (pCPT-cAMP) were purchase from Sigma–Aldrich Corporation (Bangalore, India). Tolbutamide was obtained as a gift sample from Zydus Research Centre (Ahmedabad, India). Glucose assay kit purchased from Beackon Diagnostics (Navsari, India) was used. Rat insulin ELISA kit was procured from Linco Research (MO, USA). Cell culture and treatment for insulin secretion assay RINm5F cells were cultured in 75 cm3 culture flasks and incubated at 37 ◦ C in an atmosphere of 5% CO2 and 95% air. Cells were provided with liquid growth media of RPMI-1640 (with lglutamine, 2 mM; sodium bi carbonate, 1.5 g/l; glucose, 4.5 g/l; HEPES, 10 mM; and sodium pyruvate, 1.0 mM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin G sodium and 100 IU/ml streptomycin sulphate. Cell confluency was measured by trypan blue dye exclusion method. Cells were sub-cultured when 80% confluency was reached. The insulin secreting assay was carried out according to the protocol described by Bahekar et al. (2007). Samples for testing were initially dissolved in an amount of DMSO and further diluted with PBS (pH 7.2). The final concentration of DMSO in the test system was not greater than 2% which has no effect on the growth of RINm5F cells.

HPTLC fingerprinting of AFTC HPTLC fingerprinting was performed using aluminium backed precoated silicagel 60F254 HPTLC plates (Merk KGaA, Germany) as stationary phase and ethylacetate:acetic acid:formic acid:water (100:11:11:32, v/v/v/v) as mobile phase. Sample application was done using LINOMATE 5 sample applicator with Hamilton’s glass syringe (100 ␮l). HPTLC plates were developed in Twin trough chamber (10 × 10 cm). Developed plates were scanned at 254 and 366 nm using CAMAG SCANNER 3. Presence of alkaloid was confirmed by dipping the developed plate in dipping chamber containing Dragendorff’s reagent.

Effect on insulin secretion after treatment with AFTC and isolated compounds After trypsinization, RINm5F cells were seeded at a concentration of 2 × 105 cells per well, in 96-well plates. The cells were grown overnight and allowed to reach a 70–80% confluent state. Culture medium was then replaced with 0.5 ml of PBS (pH 7.2) followed by 40 min incubation in fresh Krebs–Ringer Balanced Buffer (NaCl, 115 mM/l; KCl, 4.7 mM/l; CaCl2 , 1.28 mM/l; MgSO4 ·7H2 O, 1.2 mM/l; KH2 PO4 , 1.2 mM/l; NaHCO3 , 10 mM/l; and HEPES, 25 mM/l), supplemented with glucose, 1.1 mM and bovine serum albumin, 0.5% (pH 7.4). The effect of increasing concentrations (5–80 ␮g/ml)

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a

b

c

MeO

HO

1047

MeO

N+

+

N+

MeO

MeO

HO

O

OMe

OMe

CH 3

H

HO

O

CH 3

N

MeO

Fig. 2. Chemical structure of (a) jatrorrhizine (b) palmatine and (c) magnoflorine.

Insulin release (ng/105 cells/30 min)

10

DMSO AFTC

9

Palmatine Jatrorrhizine

8

Magnoflorine Tolbutamide 10 µM

7

#

#

#

#

#

6 5

#

# #

4

#

#

#

*

** *

3

**

** **

**

2 1

80 µg /

40 µg /

m

m

l

l

20 µg /m l

5

µg /m l

10 µg /m l

0

Fig. 3. Influences of alkaloid fraction and isolated alkaloids of Tinospora cordifolia stem on insulin secretion in RINm5F cells in absence of glucose. Each value represents mean ± SEM, n = 3 independent experiments. *p < 0.05, **p < 0.01, # p < 0.001 vs. vehicle control.

Insulin release (ng/105 cells/30 min)

10

DMSO AFTC Palmatine

9 8

Jatrorrhizine Magnoflorine

7

Tolbutamide 10 µM #

#

#

#

6

# #

# # # #

4

# # #

#

#

#

5

#

#

# #

**

#

**

*

3 2 1

/m l

l

µg 80

40

µg

/m

/m l µg 20

µg 10

5

µg

/m

/m l

l

0

Fig. 4. Influences of alkaloid fraction and isolated alkaloids of Tinospora cordifolia stem on insulin secretion in RINm5F cells in presence of glucose (16.7 mM). Each value represents mean ± SEM, n = 3 independent experiments. *p < 0.05, **p < 0.01, # p < 0.001 vs. vehicle control.

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Glucose Production (% of contro)

120 110

Vehicle control

Insulin (10 nM)

AFTC

Palmatine

Jatrorrhizine

Magnoflorine

100 #

#

90

#

#

#

80

# #

#

#

#

70

#

# #

60

# #

#

# #

50

#

40 30 #

20

#

#

#

#

10 µg/ml

20 µg/ml

40 µg/ml

80 µg/ml

10 0

5 µg/ml

Fig. 5. Effects of the AFTC on glucose production in rat hepatocytes. *p < 0.05, **p < 0.001 when compared with control (dexamethasone treated) group.

Vehicle control Tolbutamide

7

AFTC - 400 mg/kg

Jatro. - 40 mg/kg

Palma. - 40 mg/kg

Magno.- 40 mg/kg

Glucose (mmol/L)

6 5

# 4

#

3 2 1 0 0h

1h

2h

Fig. 6. Influence of AFTC and isolated alkaloids from TC on serum glucose levels in normal rats. The rats fasted for 12 h were received AFTC, palmatine, jatrorrhizine and magnoflorine 400, 40, 40 and 40 mg/kg p.o. respectively. The rats of vehicle control group were given 0.5% sodium CMC. Tolbutamide was as the positive control drug. Serum glucose levels were monitored at 0, 1, and 2 h after administration. Each value represents mean ± SEM, n = 6 rats in each group. # p < 0.001 vs. vehicle control. 2.00

1.75

#

Serum Insulin (ng/ml)

1.50

# 1.25

# 1.00

#

# 0.75

#

*

**

0.50

0.25

40

m

m

g/

g/ kg

kg

g/ kg

o. -

20 no .ag

M

M ag n

m o. ag n M

Ja

tro .

-4

0

10

m 20 .ro

Ja t

m

g/ kg

g/ kg

kg g/

kg

m

g/

10

m

-4 a. m

tro .-

0

0 Pa l

Ja

kg

m g/ kg

g/ m 0

-2 a. lm

lm a.

-1

00 -2 TC

Pa

AF

Pa

m

g/

m g/ kg

kg

g g/ k m

00

AF TC

-1

am id e

-5 0

ut

TC AF

To lb

Ve hi

cl e

co nt ro l

0.00

Fig. 7. Effect of AFTC and isolated alkaloids from TC on serum insulin in wistar rats after administration of glucose (2 g/kg, p.o.). Serum insulin levels were measured 1 h after administration of glucose. Each value represents mean ± SEM, n = 6 rats in each group. *p < 0.05, **p < 0.01, # p < 0.001 vs. vehicle control.

5.0 ± 0.04 5.4 ± 0.10# 4.1 ± 0.11# 5.1 ± 0.04 6.1 ± 0.04# 5.1 ± 0.04#

The rats fasted for 12 h were received various doses of AFTC, palmatine, jatrorrhizine and magnoflorine p.o. The rats of vehicle control group were given 0.5% sodium CMC. Tolbutamide was as the positive control drug. One and two hour after administration of glucose (2 g/kg, p.o.), serum glucose levels were measured. Each value represents mean ± SEM, n = 6 rats in each group. ** p < 0.01 vs. vehicle control. # p < 0.001 vs. vehicle control.

40 20

5.1 ± 0.02 5.6 ± 0.08# 5.2 ± 0.04#

10 40

5.2 ± 0.06 5.6 ± 0.06# 4.6 ± 0.06# 5.1 ± 0.07 5.8 ± 0.06# 4.9 ± 0.09# 5.2 ± 0.03 6.3 ± 0.09# 5.1 ± 0.07# 5.0 ± 0.06 5.7 ± 0.06# 4.8 ± 0.06#

20 10 40

5.1 ± 0.04 5.8 ± 0.05# 5.0 ± 0.05# 5.1 ± 0.05 6.3 ± 0.08# 5.1 ± 0.04#

20 10

5.1 ± 0.04 6.1 ± 0.04# 5.0 ± 0.08# 5.2 ± 0.03 6.8 ± 0.06# 6.0 ± 0.05# 5.2 ± 0.07 6.8 ± 0.06# 6.1 ± 0.07**

200 100 50 10

5.0 ± 0.04 4.5 ± 0.10# 3.5 ± 0.10# 5.0 ± 0.04 7.4 ± 0.12 6.5 ± 0.08

Jatrorrhizine (mg/kg) Palmatine (mg/kg) AFTC (mg/kg) Tolbutamide (mg/kg) Vehicle control Time (h)

Table 1 Glucose lowering effect of AFTC, palmatine, jatrorrhizine and magnoflorine in OGTT on wistar rats.

0 1 2

Magnoflorine (mg/kg)

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of AFTC and isolated compounds were studied. The buffer was replaced after 40 min and the cells were incubated (37 ◦ C, 5% CO2 ) with different concentrations of test and the standard (tolbutamide 10 ␮M) compounds for 30 min both in the presence (16.7 mM) and absence (0 mM) of glucose load. The supernatant was collected and the insulin produced was measured by enzyme-linked immunosorbent assay using commercial rat insulin ELISA kit. Hepatic gluconeogenesis in vitro Hepatocytes were isolated by the collagenase perfusion technique (Sarkar et al. 2005). The viability of the hepatocytes was assessed by trypan blue dye exclusion method. The method of Tinstorm and Obrink (1989) was adopted for preparation of primary culture of rat hepatocytes (Tinstorm and Obrink, 1989). The glucose production was measured by incubating the culture in glucose free RPMI-1640 medium. Rat hepatocytes were treated with 500 nM of dexamethasone (DEX) and 0.1 mM of 8-(4chlorophenylthio) adenosine 3 ,5 -cyclic monophosphate sodium salt (pCPT-cAMP) in the presence or absence of insulin (10 nM) or test samples (5–80 ␮g/ml) for 5 h at 37 ◦ C. Cells were incubated for an additional 3 h in glucose production buffer (glucose-free Dulbecco’s modified essential medium, pH 7.4, containing 20 mM sodium lactate and 2 mM sodium pyruvate without phenol red) with DEX/pCPT-cAMP in the presence or absence of insulin or test samples. At the end of this incubation, 0.5 ml of medium was taken to measure the glucose concentration in the culture medium using a glucose assay kit. Materials and animals in experiments in vivo Wistar rats of either sex (180 ± 35 g) obtained from Zydus Research Centre, Ahmedabad, Gujarat, were used. The rats were housed in an air conditioned room (25 ± 5 ◦ C, 60–65% relative humidity) with a lighting schedule of 12 h light and 12 h darkness. Animals had free access to a standard pellet diet and water. The study was conducted after obtaining institutional animal experimentation committee clearance (Approval No. FTE/PHR/HDT/SHM/2008-03). Glucose, tolbutamide, glucose estimation and insulin estimation kit were the same as previously described. Effects on serum glucose levels in normal rats The rats (6 in each group) fasted for 12 h were received various doses of AFTC (50, 100, and 200 mg/kg), compounds 1–3 (10, 20 and 40 mg/kg each) p.o. and the rats of negative control group were given 0.5% sodium carboxy methyl cellulose (Na-CMC). Tolbutamide (10 mg/kg) was administered p.o. as the positive control drug. Serum glucose levels were monitored at 0, 1, and 2 h after administration by retro orbital plexus bleeding and by the glucose oxidase method. Effects on serum glucose and insulin levels in glucose-loaded mice The rats (6 in each group) fasted for 12 h were received various doses of AFTC (50, 100, and 200 mg/kg), compounds 1–3 (10, 20 and 40 mg/kg each) p.o. and the rats of negative control group were given 0.5% Na-CMC. Tolbutamide (10 mg/kg) was administered p.o. as the positive control drug. One hour after administration, glucose (2 g/kg) was given p.o. serum glucose levels were determined soon after glucose administration and 1 and 2 h after glucose loading. Insulin content was estimated after 1 h using rat insulin ELISA kit. Statistics Statistical analysis was performed using GraphPad Prism 3.02 software. One way analysis of variance (ANOVA) was applied to

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assess the statistical significance of mean values of varying concentration of the samples. Groups were considered to be significantly different in case of p < 0.05. When a significant F value was obtained for ANOVA the differences between all pairs were tested using the Tukey multiple comparisons test. Results Air dried TC stem powder yields 1.63% (w/w) of total alkaloids. HPTLC study of the AFTC showed presence of total seven bands when scanned at 254 nm. Out of these, five bands (Peak nos. 2–6, Fig. 1) were identified as alkaloids after treatment with Dragendorff’s reagent. Physical properties and spectral data Compound 1 (palmatine): Yellow needles, the chemical and physical data (m.p., MS and NMR) are in accordance with those of the literature (Keawpradub et al. 2005). Compound 2 (Jatrorrhizine): Orange plates, the chemical and physical data (m.p., MS and NMR) are in accordance with those of the literature (Keawpradub et al. 2005). Compound 3 (Magnoflorine): Yellow powder, the chemical and physical data (m.p., MS and NMR) are in accordance with those of the literature (Barbosa-Filho et al. 1997). Chemical structures of compounds 1–3 are shown in Fig. 2. AFTC and isolated alkaloids increased insulin secretion in RINm5F cells Effects of AFTC and the isolated alkaloids on insulin secretion in RINm5F cells in absence and presence of glucose (16.7 mM) were given in Figs. 3 and 4 respectively. In case of absence of glucose, cells treated with AFTC showed significant increase in insulin secretion only after 40 ␮g/ml concentration where as jatrorrhizine and magnoflorine showed significant increase in insulin secretion after 20 ␮g/ml. In the presence of 16.7 mM glucose, jatrorrhizine and magnoflorine significantly increase insulin secretion in dose dependent manner. Insulin secreting activity of palmatine (5–80 ␮g/ml) and tolbutamide (10 ␮M) was significant (p < 0.001) in both the cases (0 mM and 16.7 mM glucose). AFTC and isolated alkaloids inhibited hepatic gluconeogenesis in rat hepatocytes Influence on gluconeogenesis in response to insulin (at physiological conc. 10 nM) or AFTC, palmatine, jatrorrhizine and magnoflorine (5–80 ␮g/ml) was examined in rat hepatocytes. Hepatocytes produced glucose in response to dexamethasone and pCPT-cAMP. A significant (p < 0.001) inhibition of glucose production was observed in hepatocytes when treated with AFTC alkaloid at 20 ␮g/ml and above. This inhibition of gluconeogenesis was observed in dose dependent manner as shown in Fig. 5. Effects on serum glucose levels in normal rats Effects of AFTC, palmatine, jatrorrhizine and magnoflorine and tolbutamide on serum glucose in normal mice were illustrated in Fig. 6. Tolbutamide decreased serum glucose levels at both 1 and 2 h compared with vehicle control. AFTC (50, 100, and 200 mg/kg), palmatine, jatrorrhizine and magnoflorine (10, 20, and 40 mg/kg) did not cause any significant reduction in serum glucose levels at 1 and 2 h post administration when compared with vehicle control. Suppression of postprandial serum glucose levels by AFTC and alkaloids after glucose loading in normal rats In oral glucose tolerance test (OGTT), effects of AFTC, palmatine, jatrorrhizine and magnoflorine and tolbutamide on serum glucose levels in glucose loaded rat were shown in Table 1. AFTC (50, 100

and 200 mg/kg) and palmatine, jatrorrhizine and magnoflorine (10, 20 and 40 mg/kg) significantly declined blood sugar at 1 h after glucose challenging by oral administration. The effect found to extended up to 2 h post glucose load in all test groups indicating their potent hypoglycemic action. Augmentation of insulin secretion by AFTC and alkaloids in glucose-loaded rats Influence of AFTC, palmatine, jatrorrhizine and magnoflorine at various dose levels was illustrated in Fig. 7. Tolbutamide (10 mg/kg) raised insulin level by three times as compare to vehicle control. AFTC, palmatine, jatrorrhizine and magnoflorine at their higher dose level (200, 40, 40 and 40 mg/kg) significantly (p < 0.001) increased insulin level compare to vehicle control. Magnoflorine showed significant insulin secretogogue activity at 10 and 20 mg/kg dose levels too. Discussion A number of alkaloids from natural sources have been proved efficacious to cure various ailments. Indole alkaloids from Catharanthus roseus (Benjamin et al. 1994; Chattopadhyay, 1999), carbazole alkaloid from murraya koenigii (Dineshkumar et al. 2010), isoquinoline alkaloid berberine (Punitha et al. 2005) are some of these examples which are useful in treatment of diabetes mellitus. As a traditional medicinal herb, TC was studied frequently for the beneficial role in diabetes (Grover et al. 2000; Prince and Menon 1999; Raghunathan and Sharma 1969; Gupta et al. 1967). However mechanisms by which various oral therapeutic agents achieve antidiabetic action could be linked to more than one mechanism, such as insulin sensitizing, insulin releasing, gluconeogenesis inhibiting, and alpha-glucosidase inhibiting (Davis and Granner 2001; Rosak 2002). Many scientific reports are published describing hypoglycemic activity of TC extracts. The plant contains wide range of phytoconstituents like alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, polysaccharides, etc. No research claiming a specific group of chemicals present in TC is reported for antidiabetic action or any mechanism responsible for hypoglycemic activity. In the present study, we evaluated the mechanism of action of AFTC and isolated alkaloids. A sulfonylurea compound (tolbutamide) was used as a positive standard. It stimulates insulin secretion by blocking ATP-sensitive K+ channels (K+ -ATP channels) of the ␤-cell membrane, thereby causing depolarization, Ca2+ influx, and rise in cytoplasmic Ca2+ concentration (Mariot et al. 1998). Insulin plays an important role in type 2 diabetes resulting from defects in both insulin secretion and insulin action. Thus, we determined the insulin releasing effect of AFTC and its major components in RINm5F cell line and in fasted or glucose loaded rats. These rat insulinoma cell cultures were initiated from a transplantable islet cell tumor, induced by high-dose x-irradiation in an inbred NEDH (New England Deaconess Hospital) rat (Gazdar et al. 1980). When tested with tolbutamide, RINm5F cells showed significant rise in insulin secretion both in absence and presence of glucose. In contrast, AFTC did not exhibited much rising effect on insulin secretion in absence of glucose but a dose dependent insulin secreting activity was observed at hyperglycemic condition, i.e., 16.7 mM glucose. This study revealed that the mechanism by which AFTC act as insulin secretogogue may not be exactly same as that of tolbutamide. AFTC may remain in active in hypoglycemic condition and show its effect only in hyperglycemic environment. Palmatine, unlike AFTC produced insulin secreting activity in the same manner as of tolbutamide. In absence of glucose, jatrorrhizine and magnoflorine were effective on RINm5F cells at the concentration 20 ␮g/ml and above where as in presence of 16.7 mM glucose, it produced significant insulin secretion at all studied concentra-

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tions. Result of in vivo studies strongly justified the hypoglycemic effect of AFTC and isolated alkaloids in rats. The increase in insulin levels in AFTC or palmatine or jatrorrhizine or magnoflorine treated diabetic rats attribute to the stimulation of the surviving beta cells, which in turn exerts an antihyperglycemic action. One of the hallmarks of diabetes is the inability of insulin to inhibit hepatic glucose production. It has been suggested that increased gluconeogenesis is a main source of increased hepatic glucose production and that the ability of insulin to regulate transcription of the rate-controlling gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6phosphatase may contribute to this problem. This point is underscored by the observation that in several animal models of type II diabetes and obesity, PEPCK mRNA levels are increased 2–3-fold over that observed in non-diabetic animals, despite the higher circulating insulin levels observed in the diabetic animals (Hofmann et al. 1992; Noguchi et al. 1993; Shafrir et al. 1994). In the study performed on rat hepatocytes, insulin produced significant inhibition of dexamethasone and pCPT-cAMP induced glucose production. The effects of AFTC, palmatine, jatrorrhizine and magnoflorine in the studied concentration range were not as potent as insulin but it shows promising activity. Thus, being effective as hypoglycemic, activity of AFTC and isolated alkaloids are insulin secreting rather than insulin mimicking. The present investigation thus proves the hypoglycemic activity of mixture of alkaloids act through various mechanisms. As describe by Wagner and Ulrich-Merzenich (2009) the effect of phytomedicines can be better evaluated by studying their synergistic effects. In the present case these can be synergistic multi-target effects or effects on pharmacokinetic or physicochemical properties via solubility improvement, resorption rate and enhanced bioavailability. In conclusion, AFTC has the antidiabetic effects which may relate to several mechanisms such as insulin releasing, insulin sensitizing and gluconeogenesis inhibitory activities. Thus, it is worthwhile to evaluate further the effective components of AFTC and three of its alkaloids. Acknowledgements One of the authors, Mr. Mayurkumar Patel, would like to thank All India Council for Technical Education, New Delhi, India, for providing the financial assistance (National Doctoral Fellowship) to carryout this work. References Anonymous, 1976. Wealth of India: Raw Materials, vol. X. CSIR, New Delhi, p. 251. Atal, C.K., Sharma, M.L., Kaul, A., Khajuria, A., 1986. Immunomodulating agents of plant origin. I: preliminary screening. J. Ethnopharmacol. 18, 133–141. Badar, V.A., Thawani, V.R., Wakode, P.T., Shrivastava, M.P., Gharpure, K.J., Hingorani, L.L., Khiyani, R.M., 2005. Efficacy of Tinospora cordifolia in allergic rhinitis. J. Ethnopharmacol. 96, 445–449. Bahekar, R.H., Jain, M.R., Jadav, P.A., Prajapati, V.M., Patel, D.N., et al., 2007. Synthesis and antidiabetic activity of 2,5-disubstituted-3-imidazol-2-yl-pyrrolo[2,3b]pyridines and thieno[2,3-b]pyridines. Bioorg. Med. Chem. 15, 6782–6795. Barbosa-Filho, J.M., Da-Cunha, E.V.L., Corni-Lio, M.L., Dias, C.D.S., Gray, A.I., 1997. Cissaglaberrimine, an aporphine alkaloid from Cissampelos glaberrima. Phytochemistry 44, 959–961. Benjamin, B.D., Kelkar, S.M., Pote, M.S., Kaklij, G.S., Sipahimalani, A.T., Heble, M.R., 1994. Catharanthus roseus cell cultures: growth, alkaloid synthesis and antidiabetic activity. Phytother. Res. 8, 185–186. Bishayi, B., Roychowdhury, S., Ghosh, S., Sengupta, M., 2002. Hepatoprotective and immunomodulatory properties of Tinospora cordifolia in CCl4 intoxicated mature albino rats. J. Toxicol. Sci. 27, 139–146. Bisset, N.G., Nwaiwu, J., 1983. Quaternary alkaloids of Tinospora species. Planta Med. 48, 275–279. Chattopadhyay, R.R., 1999. A comparative evaluation of some blood sugar lowering agents of plant origin. J. Ethnopharmacol. 67, 367–372. Chintalwar, G., Jain, A., Sipahimalani, A., Banerji, A., Sumariwalla, P., Ramakrishnan, R., Sainis, K., 1999. An immunologically active arabinogalactan from Tinospora cordifolia. Phytochemistry 52, 1089–1093.

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