Antidiabetic effects of scoparic acid D isolated from Scoparia dulcis in rats with...

Natural Product ResearchVol. 23, No. 16, 10 November 2009, 1528–1540

Antidiabetic effects of scoparic acid D isolated from Scoparia dulcisin rats with streptozotocin-induced diabetes

Muniappan Lathaa*, Leelavinothan Paria, Kunga Mohan Ramkumarb,Palanisamy Rajagurub, Thangaraj Sureshc, Thangavel Dhanabalc, Sandhya Sitasawadd

and Ramesh Bhonded

aDepartment of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608002,Tamil Nadu, India; bDepartment of Biotechnology, Bharathidasan Institute of Technology,Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India; cDepartment of Chemistry,Bharathiar University, Coimbatore 641046, Tamil Nadu, India; dNational Centre for Cell Science,NCCS Complex, Ganeshkhind Road, Pune 411007, Maharashtra, India

(Received 26 August 2008; final version received 22 December 2008)

We evaluated the antihyperglycaemic effect of scoparic acid D (SAD),a diterpenoid isolated from the ethanol extract of Scoparia dulcis instreptozotocin (STZ)-induced diabetic male Wistar rats. SAD was administeredorally at a dose of 10, 20 and 40mgkg�1 bodyweight for 15 days. At the endof the experimental period, the SAD-treated STZ diabetic rats showeddecreased levels of glucose as compared with diabetic control rats.The improvement in blood glucose levels of SAD-treated rats was associatedwith a significant increase in plasma insulin levels. SAD at a dose of20mg kg�1 bodyweight exhibited a significant effect when compared with otherdoses. Further, the effect of SAD was tested on STZ-treated rat insulinomacell lines (RINm5F cells) and isolated islets in vitro. SAD at a dose of20mgmL�1 evoked two-fold stimulation of insulin secretion from isolatedislets, indicating its insulin secretagogue activity. Further, SAD protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells.The present study thus confirms the antihyperglycaemic effect of SAD andalso demonstrated the consistently strong cytoprotective properties of SAD.

Keywords: Scoparia dulcis; scoparic acid D, insulin secretion; RINm5F cells;diabetes; natural products

1. Introduction

Diabetes is a metabolic disease and its incidence is considered to be high all over the world(Devendra & Eisenbarth, 2003). Epidemiological and clinical studies strongly support thenotion that hyperglycaemia is the principal cause of complications in diabetes. Effectiveblood glucose control is the key for preventing or reversing diabetic complicationsand improving quality of life in patients with diabetes (Zimmet, Alberti, & Shaw, 2001).Thus, sustained reduction in hyperglycaemia will decrease the risk of developing

*Corresponding author. Email: drlathamuniappan@rediffmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2009 Taylor & Francis

DOI: 10.1080/14786410902726126

http://www.informaworld.com

microvascular complications and most likely reduce the risk of macrovascular complica-tions (Brownlee, 2001).

A wide variety of the traditional herbal remedies are used by diabetic patients,especially in third world war countries (Day, 1998), and this may, therefore, represent newavenues in the search for alternative hypoglycaemic drugs. Results of in vitro (Latha, Pari,Sandhya, & Bhonde, 2004a, 2004b) and in vivo (Latha et al., 2004b; Latha & Pari, 2003,2004; Pari & Latha, 2004) animal studies support the claim that the whole plant ofScoparia dulcis possesses antihyperglycaemic, antioxidant, cytoprotective and antiapopto-tic activities. The active components of S. dulcis are considered to be mainly diterpenoids(Hayashi, Kawasaki, Miwa, Taga, & Morita, 1990; Hayashi et al., 1987, 1991, 1993).A number of different principles include scoparic acid A, scoparic acid B (Hayashi et al.,1987), scopadulcic acid A and B, scopadulciol (Hayashi et al., 1993) and scopadulin(Hayashi et al., 1990) that have been identified as contributors to the observed medicinaleffects of the plant. Among them, scopadulcic acid B and scopadulciol were found to beunique biomolecules with inhibitory effects on replication of herpes simplex virus type-1(Hayashi et al., 1990), gastric proton pump and bone resorption stimulated by theparathyroid hormone. In addition, scopadulcic acid B showed antitumour promotingactivities. Because of their unique carbon skeleton and many-sided biological activities,they were paid much attention as chemical synthetic targets by organic synthetic chemists.Diterpenoids are distributed in many parts of the S. dulcis plant, including the root andaerial parts, and the whole plant has different pharmacological activities. The identi-fication of compounds from S. dulcis with antihyperglycaemic activity may also provide anopportunity to develop a new class of antidiabetic agent.

This study sought to determine whether a new diterpenoid isolated from S. dulcis,scoparic acid D (SAD), normalises hyperglycaemia and further we explored themechanism responsible for antihyperglycaemic activity of the same. In addition, weexamined the cytoprotective role of SAD in RINm5F cells and isolated islets.

2. Materials and methods

2.1. Plant material

Whole plants of S. dulcis L. (40–60 cm in height) were collected from Neyveli, CuddaloreDistrict, Tamil Nadu, India in September 2001. The plant was identified and authenticatedat the Herbarium of Botany Directorate in Annamalai University. A voucher specimen(No. 3412) was deposited in the Department of Botany, Annamalai University.

2.2. Isolation, extraction and separation

Dried whole plants of S. dulcis were ground and the coarse powder was (3 kg) extractedwith 80% ethanol. The extract was concentrated in vacuum and the residue still containingwater was freeze dried to give a brown powder (800 g). The TLC analysis of the powdershows the presence of a single major compound along with impurities. The residue,which was obtained from the chloroform extract of the powder, was purified by usingsilica gel column chromatography and eluted in CHCl3 :MeOH (95 : 5), which yieldedan amorphous dark brown coloured compound of 1.1 g which melted at 198�C.A diterpenoid has been identified by using the preliminary tests for terpenoids.The newly isolated compound was similar to the previously isolated scoparic acid A

Natural Product Research 1529

(Ahmed, 1990; Hayashi et al., 1993), i.e. it had the same basic carbon skeleton. The onlydifference is the side chain at C5 and C8 positions. This is evidenced by spectroscopicaland analytical data. In the 1H-NMR spectrum, no peak was observed between 6.8 and8.0, which indicates the absence of a phenyl ring at C8 position. Further, the elementalanalysis (Calculated: C% 68.06,H% 9.22,N% 0.00; Anal. Calcd: C% 68.24,H%9.28,N% 0.02) and the mass spectral analysis exactly attributed with the proposedstructure for the SAD.

2.3. Structural elucidation

The melting point was determined on a Boetius Microheating Table and Mettler-FP5melting apparatus. The isolated compound was subjected to spectral analysis. Infrared(IR) spectra were recorded on a Shimadzu–8201 FT instrument in KBr disc and onlynoteworthy absorption levels are listed. Nuclear magnetic resonance (1H-NMR and13C-NMR) spectra were recorded in an AMX-400MHz spectrometer in CDCl3 solution;chemical shifts are expressed in parts per million (�) relative to Tri-methyl silane (TMS),and coupling constants (J) in Hz. Gas chromatography–mass spectral data were recordedon a JEOL AX-505-HA mass selective detector.

In its IR spectrum, IR (KBr) (�max cm�1) bands at 3404 (–OH, hydroxyl), 2923.9,

2845.5 (–CH3) methyl and (–CH2–) methylene, 1618 (C¼C), 1650–1750 (–COOH) showthat the corresponding functional groups may be present in its structure. The molecularweight indicated by its molecular ion peak at m/z¼ 282 and the elemental analysis showedthe molecular formula of the compound to be C16H26O4.

1H-NMR (CDCl3) �(ppm): 1.2–1.3 (m, 2H, –CH2–CH2–OH), 0.91 (d, 1H,C8a–H, J¼ 7.3Hz), 2.03 (d, 2H,C7H, J¼ 8.0Hz), 1.10 (s, 3H,C4a–CH3), 1.38 (s, 3H,C1–CH3), 1.51 (p, 2H,C3–CH2), 1.72 (t, 2H,C4–CH2, J¼ 8.0 Hz), 2.79 (q, 1H,C8–H),1.9–2.1 (m, 3H, C2 and C5 protons), 3.88 (m, 2H,CH2–CH2–OH), 4.75 (s, 2H,¼CH2),4.70 (bs, 1H,C8–OH), 11.02 (s, 1H, –COOH), 4.23 (bs, 1H,CH2–CH2–OH).

13C-NMR (CDCl3) �(ppm): 17.41 (C3), 19.64 (C1–CH3), 20.45 (C4a–CH3), 21.98 (C4a),22.72 (C1), 24.09 (–CH2–CH2–OH), 24.43 (C2), 26.66 (C4), 34.34 (C7), 44.42 (C5), 53.12(C8a), 60.17 (–CH2–CH2–OH), 63.24 (CH–OH), 105.51 (¼CH2), 155.21 (C6), 181.23(COOH).

GC-MS: m/z¼ 282 [M] þ, [M-H] 281, 211, 168, 140, 111, 97, 83, 69, 57, with the massfragmentation values 69, 83, 97, 111 having the intervals 14. This indicates clearly thecleavage of the –CH2– fragment. The above spectral and analytical data led to thedefinition of the structure of the compound as 8-hydroxy-5-(2-hydroxy-ethyl)-1,4a-dimethyl-6-methylene-decahydro-naphthalene-1-carboxylic acid, and it was newly namedSAD (Figure 1).

2.4. Animals and diets

Adult male albino Wistar rats (8 weeks), weighing 180–200 g and bred in the CentralAnimal House, Rajah Muthiah Medical College, Annamalai University, India, were used.All animal experiments were approved by the Ethical Committee, Annamalai University(Vide. No: 73) and were maintained in accordance with the guidelines of the NationalInstitute of Nutrition, Indian Council of Medical Research, Hyderabad, India.The 6–8-week-old Balb/c male mice were obtained from the National Centre for Cell

1530 M. Latha et al.

Sciences (NCCS), Pune, India. Mice were used to isolate the islets and to study the insulin

secretagogue effect of SAD in vitro. The animals were housed in polycarbonate cages in

a room with a 12 h day-night cycle, at a temperature of 22� 2�C, humidity of 45–64%.

During the experimental period, the animals were fed with a balanced commercial diet

(Hindustan Lever Ltd., Mumbai, India) and water ad libitum.

2.4.1. Induction of experimental diabetes

Experimental animals (30 rats) received a freshly prepared solution of STZ (Sigma

Chemical Co., St Louis, MO), (45mgkg�1) in 0.1M sodium citrate buffer, pH 4.5, injected

intraperitoneally in a volume of 1mLkg�1 (Siddique, Sun, Lin, & Chien, 1987). Normal

rats (six rats) received 1mL citrate buffer as a vehicle. Two days after STZ administration,

rats showing moderate diabetes with glycosuria and hyperglycaemia (i.e. blood glucose

levels of 200–300mgdL�1) were used for the experiment. The animals were fasted

overnight and blood samples were collected from the tail vein (lmL).

2.4.2. Experimental design

In the experiment, a total of 30 rats (24 diabetic surviving rats, 6 normal rats) were used.

The rats were divided into five groups of six rats each. Group 1: Normal untreated rats;

Group 2: Diabetic control rats; Group 3: Diabetic rats receiving SAD (10mgkg�1 body

weight) in aqueous solution daily by gavage for 15 days; Group 4: Diabetic rats receiving

SAD (20mgkg�1 body weight) in aqueous solution daily by gavage for 15 days and Group

5: Diabetic rats receiving SAD (40mgkg�1 body weight) in aqueous solution daily by

gavage for 15 days.All doses were started after 2 days of STZ injection. No detectable irritation or

restlessness was observed after each drug or vehicle administration. No noticeable adverse

effect (i.e. respiratory distress, abnormal locomotion or catalepsy) was noticed in any

animals after the drug administration. Blood glucose levels were estimated at 0, 5 and

15 days to ascertain the diabetes status in different groups of rats. At the end of 15 days,

all the rats were killed by decapitation (pentobarbitone sodium) anaesthesia (60mg kg�1).

Blood was collected in two different tubes with the intention of one with whole blood for

OH

HOOC

OH

1

2

3

4

4a

5

6

7

8

8a

Figure 1. Structure of SAD.

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glucose estimation and another with anticoagulant – potassium oxalate and sodiumfluoride – for plasma insulin assay.

2.4.3. Measurement of blood glucose and plasma insulin

Blood glucose was determined by an enzymatic calorimetric glucose oxidase assaycommercially available from Roche (formally Boehringer Mannheim). Plasma insulin was

assayed by enzyme-linked immunosorbent assay, using the Boehringer–Mannheim Kitwith a Boehringer model ES300 analyser (Boehringer, Germany) (Anderson, Dinesen,Jorgensen, Poulsen, & Roder, 1993).

2.5. Isolation and cultivation of islets

Groups of two or four Balb/c mice were killed by cervical dislocation and the splenic

pancreas was removed aseptically without ductal injection and distention. The isletisolation was carried out by using the method of Shewade, Umrani, and Bhonde (1999)developed at NCCS. In short, the pancreas was cut into small pieces and was subjected to

enzymatic digestion for 20min at 37�C on a magnetic stirrer (Variomag ElectronicMultipoint, Germany). The dissociation medium consisted of Dulbecco’s modifiedminimum essential medium (DMEM) supplemented with Collagenase type V (C-9263 –Sigma, 1mgmL�1), Soyabean Trypsin Inhibitor, 2mgmL�1 (STI) (Type II-5 (T-9128),

Sigma) and BSA (2%) fraction V (Sigma). The tissue digested was then centrifuged at1000 rpm for 10min, washed twice in 1X Phosphate Buffered Saline (PBS) (pH 7.2) andseeded in culture flasks (25 cm2 Nunc, Denmark) containing RPMI-1640 supplemented

with 10% FCS and incubated at 37�C in a CO2 in air. After 48 h of incubation, islets wereseparated from exocrine pancreas by hand picking using a binocular dissecting microscope(�25) with a background of green illumination and white side lighting. This methodconsistently yielded more than 1500 islets from a single mouse pancreas.

2.5.1. In vitro islet STZ and SAD treatment

Islets cultured for 48 h as described above were washed twice with PBS and then countedunder an inverted microscope (Olympus, Tokyo, Japan). Islets were then transferred into

35mm plastic petri dishes with 1X PBS and given STZ (5mM) and SAD (20, 40 and80 mgmL�1) treatment for 1 h at 37�C. A STZ solution was prepared in PBS for in vitroexperimentation.

2.5.2. Insulin secretion in vitro

Isolated islets were cultured at 37�C in a humidified atmosphere of 5% CO2 in air inRPMI-1640 medium containing 11.1mM glucose, 10% FCS and antibiotics (50, 000 IU/I

penicillin and streptomycin). Islets were seeded at a concentration of 50 islets per wellin 24-well plates (Falcon, New Jersey, USA) and allowed to attach overnight prior toacute tests. Wells were washed three times with Krebs–Ringer bicarbonate buffer(KRB; 115mM NaCl, 4.7mM KCl; 1.3mM CaCl2, 1.2mM KH2PO4, 1.2mM MgSO4,

24mM NaHCO3, 10mM HEPES, 1 gL�1 BSA, 1.1mM glucose; pH7.4)and preincubated for 1 h at 37�C. Unless otherwise stated, wells were then incubatedfor 1 h with 1mL KRB at 5.5mM, 16.7mM glucose and SAD (20 mg, 40 mg and

1532 M. Latha et al.

80 mgmL�1). Aliquots were removed from each well, centrifuged (700� g, 5min, 4�C) andstored at �20�C for insulin assay.

2.6. Cell culture

A rat insulinoma cell line (RINm5F) that was used in all the experiments was a generousgift from Prof. J.W. Yoon, Julia McFurlane Diabetes Research Centre, University ofCalgary, Calgary, Canada. The cells were grown at 37�C under a humidified, 5% CO2

atmosphere in RPMI-1640 medium (GIBCO) supplemented with 10% foetal calf serum

(FCS) (GIBCO) and 2mM glutamine, 10,000 unitsmL�1 of penicillin, 10mgmL�1 ofstreptomycin (Hindusthan Antibiotics, India), and 2.5 mgmL�1 of amphotericin B. Allreagents and chemicals were purchased from Sigma Chemical Co. (St Louis, MO) unless

otherwise designated.

2.6.1. Culture of RINm5F cells and treatment with STZ and SAD

RINm5F cells were cultured in 25 cm2 tissue culture flasks and on attaining 75–80%

confluence, the cells were washed with RPMI-1640 medium and treated for 1 h at 37�Cwith 5mM STZ dissolved in citrate buffer (pH 4.5) and in L-15 medium with 20, 40 and80 mg of SAD. The cultured flasks were incubated at 37�C for 24 h.

2.6.2. Cytotoxicity assay

The viability of RINm5F cells after treatment with SAD was assayed by the reductionof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, asdescribed previously (Plumb, Milroy, & Kaye, 1989). Briefly, cells were seeded in 96-well

micro titer plates (1� 104 cells per well in 200 mL of medium) and left to adhere to theplastic plates overnight before being exposed to STZ and SAD (dissolved in RPMI-1640medium without FCS). In each experiment, 5mM STZ and 20 mg, 40 mg and 80 mg of SAD

were tested in three separate wells and the cytotoxicity curve was constructed from at leastthree different experiments. After 24 h of exposure to SAD, 50 mL of (5mg/5mL) MTTsolution was added to each well, and the cells were incubated in the dark at 37�C for anadditional 4 h. Thereafter, the medium was removed, the formazan crystals were dissolved

in 200 mL of DMSO and the absorbance was measured at 570 nm in a microplate reader(Molecular Devices, Spectra MAX 250). The data of the survival curves are expressedas the percentage of untreated controls.

2.6.3. Griess nitrite assay

Nitric oxide (NO) produced during STZ and SAD treatment was estimated spectro-photometrically as a formed nitrite (NO2). After 24 h of 5mM STZ and SAD (20, 40 and

80 mg) treatment, 100 mL of the culture medium was taken from each well. To measurethe nitrite content, 100 mL of the culture medium was incubated with 100 mL of Griessreagent (1% sulfanilamide in 0.1mL�1 HCl and 0.1% N-(1-naphthyl) ethylenediaminedihydrochloride) at room temperature for 10min. Then the absorbance was measured

at 540 nm using a microplate reader. The nitrite content was calculated based ona standard curve constructed with NaNO2 (Ignarro, Buga, Wood, Byrns, & Chaushury,1987).

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2.7. Statistical analysis

Samples were assayed at least three times for each determination (N¼ 7) and results weregiven as the mean� SD. One-way ANOVA with a Student Newman–Keuls post hoccomparison was used for statistical significance. Values of p5 0.0001 were consideredstatistically significant.

3. Results

3.1. Effect of SAD on STZ-induced hyperglycaemia

Prior to streptozotocin (STZ) administration, basal blood glucose levels did not differsignificantly between groups, whereas two days after STZ administration they weresignificantly higher in diabetic rats. Euglycaemic was normal throughout the course of thestudy.

Figure 2 shows the effect of treatment with SAD on blood glucose levels. Two daysafter the induction of the diabetes, the blood glucose was significantly higher in diabeticrats. In all the SAD-treated groups (10, 20 and 40mgkg�1), decrease in blood glucose wasmaximum on completion of the fifteenth day ( p5 0.001) in the group receiving 20mgkg�1

per day of SAD. On the basis of these studies, the dose of 20mgkg�1 SAD per day wasselected for further evaluation. The plasma insulin was significantly decreased in diabeticrats (Figure 3). Administration of SAD to diabetic rats significantly increased plasmainsulin levels.

3.2. Insulin secretion in vitro

SAD had a stimulatory effect on insulin secretion from islets. Incubation of islets with20 mgmL�1 of SAD increased the insulin secretion to one-fold as compared to 16.7mMglucose (positive control) and two-fold when compared with 5mM STZ (negative control)(Figure 4). The other two doses (i.e. 40 and 80 mgmL�1) did not show significant insulinstimulatory effect when compared with the positive control, but when compared withnegative control, 40 and 80 mgmL�1 potentiated the secretion of insulin, though notsignificantly with 20 mg of SAD.

Day 0 Day 2 Day 15

Blo

od g

luco

se (

mg

dL−1

)

Normal Diabeticcontrol

SAD10 mgkg−1

** * *

**

** ** **

SAD20 mgkg−1

SAD40 mgkg−1

0

100

200

300

400

Figure 2. Effect of SAD on fasting blood glucose in normal and experimental rats. Values are givenas mean� SD for six rats in each group. Experimental groups were compared with correspondingvalues after STZ injection (second day). *Significantly different from normal ( p5 0.0001).**Significantly different from STZ-treated group ( p5 0.0001).

1534 M. Latha et al.

3.3. Prevention of STZ-induced cell death by SAD

The RINm5F cells were cultured to near confluence. Using 5mM STZ, RINm5F

cells were treated with or without SAD at a dose of 20, 40 and 80 mgmL�1 for 24 h, at

which time the cells were harvested and their viability was analysed. A single treatment

of RINm5F cells with 5mM STZ decreased the percentage of live cells

*

**

**

**

0

1

2

3

4

5

6

Positivecontrol

Negativecontrol

20 µgSAD

40 µgSAD

80 µgSAD

Insu

lin (µ

Um

L−1

)

Figure 4. Effect of SAD on insulin secretion from isolated Balb/c splenic pancreatic islets. Insulinsecretion induced by glucose (16.7mM/5.5mM) served as positive control, negative control (5mMSTZ) and SAD (20, 40 and 80mg SAD) was measured. Duplicate batches of 50 islets wereincubated for 60min. Results are represented as means� SD of six replicate experiments in eachgroup. *Significantly different from positive control ( p5 0.0001). **Significantly different fromnegative control ( p5 0.0001).

*

**

** **

0

3

6

9

12

15

Normal Diabeticcontrol

Plas

ma

insu

lin (

µUm

L−1

)

SAD10 mgkg−1

SAD20 mgkg−1

SAD40 mgkg−1

Figure 3. Effect of SAD on plasma insulin in normal and experimental rats. Values are given asmean� SD for six rats in each group. Experimental groups were compared with correspondingvalues after STZ injection (second day). *Significantly different from normal ( p5 0.0001).**Significantly different from STZ-treated group ( p5 0.0001).

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(45� 2.0%) (Figure 5). Treatment of 5mM STZ-treated RINm5F cells along with SADincreased the cell viability. SAD (20 mgmL�1) increased the viability of 5mM STZRINm5F cells to 85� 4% (Figure 5). SAD alone did not affect the viability at thisconcentration (20mg mL�1), but a higher concentration of SAD (above 20 mg to1mgmL�1 of SAD), by itself, was cytotoxic to RINm5F cells (data not shown).

3.4. Effect of SAD on STZ induced nitrite formation by RINm5F cells

As shown in Figure 6, incubation of RINm5F cells with 5mM STZ for 24 h resulted insignificant nitrite formation (a stable oxidised product of NO) by these cells. RINm5F cellsstimulated with 5mM of STZ showed significant increase in nitrite production.The presence of SAD diminished the STZ-mediated nitrite formation by the cells at 20,40 and 80 mgmL�1. Near complete inhibition of nitrite formation is observed at a SADconcentration of 20 mgmL�1 (Figure 6). Treatment of SAD alone did not show nitriteproduction (data not shown). SAD significantly reduced the STZ-mediated NOproduction. SAD was able to quench the NO produced by STZ and was well correlatedwith its decreased cytotoxicity (Figures 5 and 6).

4. Discussion

In the present investigation, we have shown that STZ-induced diabetic rats treated withSAD for 15 days exhibited reduction in blood glucose with significant increase in plasmainsulin. Higher concentrations of SAD were found to be without any beneficial effect orwith detrimental effects, adding to more oxidative stress already induced by the diabeticstate (data not shown). Our present results support those of our previous report whereinwe demonstrated the antihyperglycaemic action of crude extracts of S. dulcis in STZ-induced diabetic animals. Our studies also show that the antihyperglycaemic effect of SAD

*

**** **

0

20

40

60

80

100

Control 5mM STZ 5mM STZ +20 µg SAD

5mM STZ +40 µg SAD

5mM STZ +80 µg SAD

Cel

l via

bilit

y (%

)

Figure 5. Effect of SAD on the percentage of viability in the STZ-treated RINm5F cells.The columns represent the percent viability RINm5F cells. Results are represented as means� SDof six replicate experiments in each group. *Significantly different from control ( p5 0.0001).**Significantly different from STZ-treated cells ( p5 0.0001).

1536 M. Latha et al.

is observed only at lower doses, whereas higher doses are non-effective or evendetrimental, as evidenced by high levels of lipid peroxidation (data not shown).

In the present study, we observed an increase in insulin release when islets wereincubated with various concentrations of SAD (20, 40 and 80 mgmL�1). A two-foldincrease was observed in insulin release with 20 mg SAD. The effect of SAD on insulinsecretion was more than that caused by 16.7mM glucose, suggesting that �-cell glucosemetabolism is able to augment the insulin secretagogue activity. This finding agrees withthe in vivo results displayed in Figure 3, thus strengthening the evidence that SAD acts asa stimulator of insulin secretion. Similar to in vivo studies, even in vitro studies showthat higher doses did not produce potentiation of insulin secretion. Insulin secretiondecreased beyond 20 mgmL�1 of SAD. The higher doses could not potentiate the secretionof insulin, maybe due to the toxic effect of the compound beyond the optimum level. Suchpancreatic action would prove to be an important advance on existing therapies used totreat and control diabetes, such as oral hypoglycaemic drugs (which act either byenhancing insulin secretion or by improving the action of insulin). Terpenoids arepromising compounds with the potential to be developed into new drugs for diabetes(Li, Zheng, Bukuru, & De kimpe, 2004). Several triterpenoids such as ginsenoside(Yamasaki, 1995), senticoside A (from Boussingaultia baselloides) (Ni et al., 1998),oleanolic acid (from Ligustrum lucidum), tormentic acid (from Eriobotrya japonica), ursolicacid (from Punica granatum and Cornus officinalis) (Li et al., 2004); diterpenoids such asstevioside, salvin, salvicin and savifolin (from Salvia japonica Thunb.) (Jeppesen,Gregersen, Alstrup, & Hermann, 2002; White, Kramer, Cammpbell, & Bernsten, 1994);monoterpenoids, mainly iridoid glycosides – catalpol (Nishimura, Ogino, Morota, &Sasaki, 1991), were all reported to possess antidiabetic effects. Similarly, terpenoids fromSambuscus nigra (Gray, Abdel-Wahab, & Flatt, 2000), Clausena anisata (Ojewole, 2002)and Cyclocarya paliurus (Kurihara et al., 2003) were reported to possess significantantihyperglycaemic activity.

*

**

** **

0

3

6

9

12

15

Control 5mM STZ 5mM STZ +20 µg SAD

5mM STZ +40 µg SAD

5mM STZ +80 µg SAD

Nitr

ite (

µM/1

04 cel

ls)

Figure 6. Effect of SAD on the levels of nitrite generation in RINm5F cells. The columns representthe mM of NO, in terms of NO2 formed in 104 RINm5F cells. Results are represented as means� SDof six replicate experiments in each group. * Significantly different from control ( p5 0.0001).**Significantly different from STZ treated cells ( p5 0.0001).

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We further show that the concomitant exposure of RINm5F cells to SAD protectsRINm5F cells from the detrimental effect of STZ. The protective effect is exhibitedin RINm5F cells at 20 mg of SAD with no ill effect on cell viability (MTT). STZ is a potentnitric oxide donor in pancreatic cells (Spinas, 1999). NO is an important destructor and/ormediator for insulitis during type 1 diabetic development (Raza, Ahmed, John, & Sharma,2000). In the present study, 5mM STZ caused destruction in RINm5F cells that wasprevented by SAD. These findings indicate that NO is an indispensable componentof STZ-induced toxicity of RINm5F cells, and the protective effect of SAD at 20 mgmL�1

against STZ-mediated killing is due to the inhibition of NO generation. Concomitantexposure of RINm5F cells to STZ and SAD protects RINm5F cells from the detrimentaleffect of STZ as assessed by MTT assay (Figures 5 and 6). The protective effect is exhibitedin RINm5F cells at 20 mgmL�1 of SAD with no ill effect on cell viability.

Moreover, antiapoptotic action of SAD observed in RINm5F cells is of paramountimportance for the protection of residual �-cell mass in the diabetic pancreas. These resultssupport our earlier observations on S. dulcis plant extracts, which documented itscytoprotective action. Although previous studies have demonstrated that STZ is cytotoxicto RINm5F cells (Sandhya, Shewade, & Bhonde, 2000), no studies to date have elucidatedthe protection of cells by SPEt. Treatment of RINm5F cells with SAD (27%) was effectivein abrogating apoptosis induced by STZ, resulting in a cytogram with similar profile tocontrol cells.

Our data suggests that SAD has a beneficial effect on a diabetic individual, whichis exerted through its antihyperglycaemic and cytoprotective action. Thus, our studiesstrongly support the notion that SAD would help in achieving good glycaemic andmetabolic control due to the protection offered by its cytoprotective action; probablypreserving �-cell mass without further loss.

This is potentially the first report depicting the antihyperglycaemic and cytoprotectiverole of SAD, suggesting its utility in control of a diabetic status. In addition, theidentification of the antihyperglycaemic activity of SAD may provide an opportunity todevelop a novel class of antidiabetic agent.

Acknowledgements

The authors wish to thank the University Grants Commission, New Delhi Project No. F.12-36/2001(SR-I), for the research grant. The authors are grateful to the Director of NCCS for his permissionto undertake part of the studies at NCCS.

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