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Journal of Ethnopharmacology 123 (2009) 499–503

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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ntihyperglycemic and hypolipidemic activity of aqueous extract of Cassiauriculata L. leaves in experimental diabetes

hipra Guptaa, Suman Bala Sharmaa,∗,urendra Kumar Bansalb, Krishna Madhava Prabhua

Department of Biochemistry, University College of Medical Sciences and GTB Hospital, Delhi 110095, IndiaDepartment of Biochemistry, Vallabhbhai Patel Chest Institute, Delhi 110007, India

r t i c l e i n f o

rticle history:eceived 6 June 2007eceived in revised form 3 August 2008ccepted 10 February 2009vailable online 21 February 2009

eywords:assia auriculataiabetesntihyperglycemic drug

a b s t r a c t

Ethnopharmacological relevance: Cassia auriculata L. (Caesalpiniaceae) is widely used from ancient periodto treat diabetes mellitus. The leaves of Cassia auriculata are having potential in the development of drugfor diabetes due to its antihyperglycemic and lipid-lowering activity.Aim of the study: The present study was to evaluate antihyperglycemic and hypolipidemic activity ofaqueous extract of Cassia auriculata leaves (CLEt) in streptozotocin (STZ)-induced mild diabetic (MD) andsevere diabetic (SD) rats.Materials and methods: Male Albino rats were rendered diabetic by STZ (45 mg/kg, intraperitoneally).CLEt was orally administered to MD and SD rats at 100, 200 and 400 mg/kg doses for 1 day to determineantihyperglycemic activity. The 400 mg/kg dose was administered daily for 3 weeks to assess glycemic

ypolipidemic drug control and hypolipidemic effect.Results: CLEt showed dose dependant fall in fasting blood glucose (FBG). After 5 h of extract administrationat 400 mg/kg dose, FBG was reduced by 13.9% and 17.4% in MD and SD rats respectively. After 3 weekstreatment, CLEt produced significant reduction in FBG and glycosylated haemoglobin (GHb) in both MDand SD rats. Serum lipid levels were reversed towards normal in extract fed MD and SD rats.Conclusions: The results demonstrate that CLEt possesses potent antihyperglycemic and hypolipidemic

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activity in both MD and S

. Introduction

Diabetes mellitus is a syndrome, characterized by hyper-lycemia together with impaired metabolism of glucose and othernergy-yielding fuels such as lipids and proteins (Scheen, 1997).n spite of the presence of known antidiabetic medicine in theharmaceutical market, there is growing interest in herbal reme-ies due to the fact that synthetic drugs lead to undesirable side

ffects (Davis and Granner, 1996). The search for effective and saferntidiabetic plant drugs is thus of great importance. The WHOas also recommended that this area warrants attention (WHO,980).

Abbreviations: CLEt, Cassia auriculata leaf extract; IDDM, insulin dependent dia-etes mellitus; NIDDM, non-insulin dependent diabetes mellitus; MD, mild diabetic;D, severe diabetic; STZ, streptozotocin; FBG, fasting blood glucose; GTT, glucose tol-rance test; GHb, glycosylated haemoglobin; TG, triglyceride; TC, total cholesterol;DL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density

ipoprotein.∗ Corresponding author. Tel.: +91 11 22582972; fax: +91 11 22590495.

E-mail address: drsbs08@yahoo.in (S.B. Sharma).

378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd.oi:10.1016/j.jep.2009.02.019

s.© 2009 Published by Elsevier Ireland Ltd.

Out of a large number of plants in the Ayurvedic system ofmedicine, Cassia auriculata L. of family Caesalpiniaceae (called Ten-ner’s cassia/Mature tea tree in English and Tarwar in Hindi) is beingwidely used in Indian folk medicine for the treatment of diabetesmellitus (Joshi, 2000). This shrub is a common plant in Asia. InAyurvedic medicine, Cassia auriculata is used as ‘Avarai PanchagaChoornam’ (mixture of five parts of the shrub, i.e. roots, leaves, flow-ers, bark and unripe fruits) which establishes good control on sugarlevels (Brahmachari and Augusti, 1961).

Till date, only limited preliminary studies are available show-ing antihyperglycemic and/or hypolipidemic activity of Cassiaauriculata. Pari and Latha (2002) reported the effect of Cassiaauriculata flowers on blood glucose and lipids in streptozotocin(STZ)-induced diabetic rats. The effect of hydro-ethanolic extract ofCassia auriculata leaves (CLEt) in lowering blood glucose was foundin alloxan-induced diabetic rats by Sabu and Subburaju (2002). Thehypolipidemic effect of Cassia auriculata leaves was reported in rats

with alcoholic liver injury (Kumar et al., 2002). However, none ofthe studies have reported hypolipidemic activity of Cassia auricu-lata leaves in experimental diabetes. In this paper, we described theantihyperglycemic and hypolipidemic effect of aqueous extract ofCLEt in STZ-induced diabetic rats. We studied the effect of CLEt in

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ifferent models of diabetes, i.e. severe diabetic (SD) rats (Type I orDDM) and mild diabetic (MD) rats (Type II or NIDDM), which hasot been reported in the literature.

. Materials and methods

.1. Experimental animals

Male Albino rats (150–225 g) were procured from Central Ani-al House, University College of Medical Sciences (UCMS), Delhi,

ndia. The animals were provided with standard pellet diet (Hin-ustan Lever Ltd., Mumbai, India) and water ad libitum. The animalsere kept in air-conditioned room (22 ± 2 ◦C) and acclimatized forperiod of 1 week. The experimental protocol was approved by

he Institutional Animal Ethical Committee (IAEC) of UCMS, India.earing up of animals and their upkeep in the experimental periodonformed to the norms of IAEC and ethical guidelines for inves-igations of experimental pain in conscious animals (Zimmerman,983).

.2. Induction of experimental diabetes

A freshly prepared solution of STZ (45 mg/kg in 0.1 M citrateuffer, pH 4.5) was injected intraperitoneally to overnight fastedats (Siddiqui et al., 1987). After 48 h of STZ administration, fast-ng blood glucose (FBG) levels were measured and again repeatedwice at intervals of 3 days. The rats with stabilized diabetes havingBG values between 150 and 250 mg/dl were considered as MD andhose with FBG values of 250 mg/dl or above as SD.

.3. Plant material

The leaves of Cassia auriculata were obtained from Nagercoil,amilnadu, India. The plant was identified and authenticated byr. Sudhanshu Shekhar Dash, a taxonomist in the Botanical Gar-en of Indian Republic (BGIR), Noida, India and the specimen waseposited in the Herbarium of BGIR, Noida (Voucher specimen no.605).

.4. Preparation of crude extract from leaves of Cassia auriculata

The shade dried powdered leaves (100 g) were suspended in00 ml of cold distilled water and allowed to stand overnight. Itas then filtered through several layers of muslin cloth. The wholerocedure was carried out at 4 ◦C. The filtrate (aqueous extract) was

yophilized. The yield of lyophilized aqueous extract was approxi-ately 10 g/100 g of Cassia auriculata leaves.

.5. Experimental procedure

The rats were divided into following groups of five animals each:

roup I Healthy untreated rats

roup II (a) Untreated mild diabetic rats(b) Untreated severe diabetic rats

roup III (a) CLEt (100 mg/kg) treated mild diabetic rats(b) CLEt (100 mg/kg) treated severe diabetic rats

roup IV (a) CLEt (200 mg/kg) treated mild diabetic rats(b) CLEt (200 mg/kg) treated severe diabetic rats

roup V (a) CLEt (400 mg/kg) treated mild diabetic rats(b) CLEt (400 mg/kg) treated severe diabetic rats

roup VI (a) Glibenclamide (600 �g/kg) treated mild diabetic rats(b) Glibenclamide (600 �g/kg) treated severe diabetic rats

The drugs, dissolved in 1 ml of distilled water, were administeredrally via a standard orogastric cannula. Initially, 1-day treatment

acology 123 (2009) 499–503

was given to both MD and SD rats. Antihyperglycemic activity in MDrats was assessed by fall in FBG and improvement in glucose toler-ance, while in SD rats by fall in FBG only because animals may dieduring glucose tolerance test (GTT). Blood samples were collectedfrom overnight fasted rats prior to and at 90 min, 3 h and 5 h aftergiven the drugs to determine blood glucose levels. Then glucose(2 g/kg in 1 ml of distilled water) was administered to MD rats forGTT and blood was withdrawn after 1 h of glucose load to determinepeak blood glucose. The antihyperglycemic effect of CLEt at a doseof 400 mg/kg was also studied at 24 h, 48 h and 72 h after extractadministration.

After assessment of antihyperglycemic activity with differentdoses of CLEt, maximum fall in blood glucose level was observedwith the dose of 400 mg/kg in both MD and SD rats. Therefore,further studies were carried out with 400 mg/kg dose adminis-tered daily for a period of 3 weeks in MD as well as SD rats. After3 weeks of treatment, blood samples were taken from overnightfasted rats.

Blood was drawn by retro-orbital venepuncture technique usingmicrocapillary (Sorg and Buckner, 1964). Blood samples were col-lected in anticoagulant (sodium fluoride and potassium oxalate)vials for plasma and in plain vials for serum. The blood wasallowed to clot and then centrifuged at 2500 rpm for 10 min toobtain clear serum. Blood glucose in plasma was estimated byglucose oxidase method (Barham and Trinder, 1972). All the lipidprofile parameters were done in serum. Triglycerides (TGs) weredetermined by the method of Fossati and Prencipe (1982). Totalcholesterol (TC) was estimated by the method as described byAllain et al. (1974). High density lipoprotein (HDL) was deter-mined by the method of Burstein et al. (1970). Low densitylipoprotein (LDL) and very low density lipoprotein (VLDL) werecalculated by using Friedewald et al. (1972) formula. Blood sam-ples, collected in EDTA vials, were used for the estimation ofglycosylated haemoglobin (GHb) by using Goldstein et al. (1986)method.

2.6. Acute toxicity study

The rats were given CLEt at doses of 1000 and 2000 mg/kg dailyfor a period of 3 weeks (five rats were taken for each dose). The ani-mals were observed for 1 h continuously and then hourly for 4 h andfinally after every 24 h up to 3 weeks for any physical signs of toxic-ity such as writhing, gasping, palpitation and decreased respiratoryrate or mortility.

2.7. Statistical analysis

Results were expressed as mean ± S.E.M. Statistical analysis wasdone by using repeated measure analysis of variance (ANOVA) fol-lowed by Dunnett’s test and two-way ANOVA followed by Tukey’stest at 5%.

3. Results

3.1. One-day treatment

3.1.1. Blood glucose and glucose toleranceTable 1 depicts antihyperglycemic effect of CLEt in MD and SD

rats at different intervals of time. No significant reduction in FBGwas observed from 90 min to 5 h after oral administration of theextract at a dose of 100 mg/kg. The 200 mg/kg dose produced sig-

nificant fall in FBG (p < 0.001) at 5 h after extract administration,while 400 mg/kg dose showed significant antihyperglycemic activ-ity (p < 0.001) from 3 h onwards. The dose of 400 mg/kg was alsofound effective in decreasing peak blood glucose by 15.5% duringGTT.

S. Gupta et al. / Journal of Ethnopharmacology 123 (2009) 499–503 501

Table 1Antihyperglycemic effect of aqueous extract of Cassia auriculata leaves (CLEt) at various time intervals in mild diabetic and severe diabetic rats.

Experimental groups Dose (per kg) Blood glucose (mg/dl) % fall in peak bloodglucose during GTT

0 h After treatment

90 min % fall 3 h % fall 5 h % fall

Mild diabeticControl 207 ± 9.5 210 ± 11.8 Nil 213 ± 13.0 Nil 210 ± 9.3 Nil NilGlibenclamide 600 �g 208 ± 8.7 191 ± 11.9 8.2 181 ± 12.0* 13.0 172 ± 12.4* 17.3 19.4

CLEt 100 mg 196 ± 11.2 201 ± 13.5 Nil 191 ± 11.7 2.6 183 ± 10.7 6.6 4.7200 mg 196 ± 12.1 190 ± 13.3 3.1 184 ± 11.7 6.1 176 ± 9.9* 10.2 13.2400 mg 202 ± 15.3 192 ± 20.9 5.0 184 ± 17.8* 8.9 174 ± 13.6* 13.9 15.5

Severe diabeticControl 302 ± 16.6 309 ± 17.7 Nil 305 ± 12.3 Nil 297 ± 5.7 NilGlibenclamide 600 �g 299 ± 20.6 278 ± 25.2* 7.0 270 ± 21.0* 9.7 258 ± 24.2* 13.7

CLEt 100 mg 291 ± 11.7 289 ± 15.0 0.7 293 ± 18.2 Nil 280 ± 17.3 3.8200 mg 300 ± 28.1 301 ± 26.4 Nil 285 ± 24.3 5.0 273 ± 21.7* 9.0400 mg 299 ± 16.7 289 ± 20.1 3.3 267 ± 15.0* 10.7 247 ± 8.2* 17.4

The data were expressed as mean ± S.E.M. (n = 5) and evaluated by repeated measure ANOVA followed by Dunnett’s test. The drugs were orally administered to rats accordingt eatme

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o their respective group and FBG levels were measured before (0 h) and after the tr* p < 0.001 compared to 0 h.

As shown in Fig. 1, the oral administration of single dose of CLEt400 mg/kg) exhibited a gradual decline in FBG level from 90 min

o 48 h and thereafter rise in FBG was noticed.

The antihyperglycemic effect of CLEt (400 mg/kg) was betterhan that of glibenclamide as shown in Table 1 and Fig. 1.

ig. 1. Effect of single dose of CLEt (400 mg/kg) on fasting blood glucose at differentime intervals in mild diabetic (A) and severe diabetic (B) rats. Effect of glibenclamide600 �g/kg) is shown for comparison. The rats were treated with a single dose ofrugs and FBG levels were measured at different time points, i.e. 90 min–72 h ineparate groups of animals. In CLEt treated group, FBG level gradually declined up to8 h, whereas in glibenclamide treated group, gradual fall in FBG level was observednly up to 5 h.

nt (90 min, 3 h and 5 h).

3.2. Long-term treatment (3 weeks)

3.2.1. Blood glucose, glycosylated haemoglobin and body weightTreatment with the extract exhibited remarkable glycemic con-

trol in both MD and SD rats as evident by significant decrease(p < 0.001) in the levels of FBG and GHb (Table 2). A significantincrease (p < 0.001) in body weight was also observed in CLEttreated MD and SD rats.

3.2.2. Serum lipid profileThe effect of CLEt on serum lipid profile in MD and SD rats

is summarized in Table 3. The levels of TG, TC, LDL and VLDLwere significantly reduced (p < 0.001), whereas HDL was signifi-cantly increased (p < 0.001) in CLEt treated MD as well as SD rats.Atherogenic index (TC/HDL ratio) was also significantly decreased(p < 0.001) in CLEt fed MD and SD rats. The hypolipidemic effect ofCLEt was comparable to that of glibenclamide, except for HDL whichwas not significantly increased by glibenclamide.

3.3. Toxicity study

The rats treated with 1000 and 2000 mg/kg doses of CLEt did notshow any drug-induced physical signs of toxicity during the wholeexperimental period and no deaths were registered.

4. Discussion and conclusion

The present study is a preliminary assessment of the antihyper-glycemic and hypolipidemic activity of aqueous extract of Cassiaauriculata leaves. The CLEt showed dose dependant fall in FBG with100, 200 and 400 mg/kg doses in both MD and SD rats.

In the present study, we observed that the antihyperglycemiceffect of CLEt starts to come from 90 min and gradually increasesup to 5 h (effect was not seen for more than 5 h due to restriction offasting period). In separate groups of animals, it was found that fallin FBG steadily occurs up to 48 h with the single 400 mg/kg dose andthereafter increases in both MD and SD rats. This might mean that

the drug takes time to reach the target tissues in the body or it getsmetabolized and the metabolite(s) is/are active. This also showedthat the antihyperglycemic effect of CLEt persists till 48 h with itssingle dose (400 mg/kg) in both models of diabetes, which was notobserved in glibenclamide treated animals.

502 S. Gupta et al. / Journal of Ethnopharmacology 123 (2009) 499–503

Table 2Glycemic control by CLEt (400 mg/kg) in mild and severe diabetic rats after 3 weeks treatment.

Experimental groups FBG (mg/dl) GHb (%) Body weight (g)

Initial Final

Healthy control 79 ± 2.2 3.49 ± 0.13 183 ± 6.0 201 ± 6.4

Mild diabeticControl 201 ± 9.7a 6.85 ± 0.14a 186 ± 1.9 167 ± 4.1a

Glibenclamide 144 ± 11.6* 5.19 ± 0.28* 180 ± 6.5 194 ± 8.3*

CLEt 115 ± 12.3* 4.48 ± 0.18* 183 ± 2.5 199 ± 1.9*

Severe diabeticControl 296 ± 7.4a 8.08 ± 0.15a 170 ± 6.5 143 ± 5.8a

Glibenclamide 198 ± 20.4* 5.55 ± 0.26* 177 ± 6.0 189 ± 5.3*

CLEt 156 ± 13.1* 4.98 ± 0.22* 176 ± 6.6 189 ± 9.3*

The data were expressed as mean ± S.E.M. (n = 5) and evaluated by two-way ANOVA followed by Tukey’s test. The drugs were given to rats daily for a period of 21 days. FBGand GHb were measured at the end of the experiment. Body weight was taken before and after the experimental period.

a p < 0.001 compared to healthy control.* p < 0.001 compared to diabetic control.

Table 3Hypolipidemic effect of CLEt (400 mg/kg) in mild and severe diabetic rats after 3 weeks treatment.

Experimental groups Serum lipid profile (mg/dl)

TG TC HDL LDL VLDL TC/HDL

Healthy control 62.6 ± 3.2 67.1 ± 2.2 33.3 ± 1.2 21.3 ± 1.6 12.5 ± 0.7 2.02 ± 0.07

Mild diabeticControl 97.7 ± 3.2a 93.3 ± 3.0a 25.7 ± 1.1a 48.0 ± 4.4a 19.5 ± 0.6a 3.67 ± 0.26a

Glibenclamide 76.1 ± 2.2* 80.2 ± 2.6* 28.1 ± 1.5 36.8 ± 3.3* 15.2 ± 0.4* 2.89 ± 0.22*

CLEt 71.9 ± 2.1* 73.3 ± 1.6* 29.8 ± 1.1* 29.1 ± 1.2* 14.4 ± 0.4* 2.47 ± 0.07*

Severe diabeticControl 121.0 ± 7.3a 105.6 ± 4.9a 21.1 ± 0.9a 60.3 ± 4.7a 24.2 ± 1.4a 5.03 ± 0.22a

Glibenclamide 82.2 ± 5.2* 89.8 ± 3.0* 23.5 ± 0.7 49.8 ± 3.4* 16.4 ± 1.0* 3.84 ± 0.19*

CLEt 74.3 ± 5.3* 80.0 ± 2.5* 25.4 ± 0.6* 39.7 ± 2.2* 14.9 ± 1.0* 3.15 ± 0.04*

The data were expressed as mean ± S.E.M. (n = 5) and evaluated by two-way ANOVA followed by Tukey’s test. The drugs were administered to rats daily for 21 days. Seruml

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ipid parameters were estimated at the end of the experiment.a p < 0.001 compared to healthy control.* p < 0.001 compared to diabetic control.

As the maximum antihyperglycemic activity of CLEt was foundith the dose of 400 mg/kg, the detailed studies were carried out

or the period of 3 weeks with the same dose. The CLEt producedighly significant reduction in blood glucose level in both MD andD rats after 3 weeks treatment.

STZ, a highly cytotoxic agent of pancreatic �-cells (Elsner et al.,000), induces diabetes by damaging the cells that causes reduction

n insulin release. It is reported that treatment of diabetic animalsith medicinal plant extracts resulted in activation of �-cells and

ranulation returned to normal, showing an insulinogenic effectKedar and Chakrabarti, 1982). The possible mechanism throughhich CLEt exerts its antihyperglycemic effect might have beenue to the increased release of insulin from remnant �-cells and/oregenerated �-cells. In this context, a number of other plants haveeen reported to have antihyperglycemic activity with a stimu-

atory effect on insulin release (Esmaeili and Yazdanparas, 2004;harma et al., 2006). Since the extract produced highly significantntihyperglycemic effect even in SD rats in which most of the �-ells are damaged, it is likely that CLEt might have extrapancreaticechanism of action.CLEt administration to MD and SD rats exhibited significant

eduction in the level of GHb. As the amount of increase in GHbs directly proportional to the FBG level (Jackson et al., 1979), theecrease in GHb might have been due to the improved glycemic

ontrol produced by the extract. CLEt treated MD and SD rats alsohowed gain in body weight. The ability of CLEt to recover bodyeight loss seems to be due to its antihyperglycemic effect.

In diabetes, hyperglycemia is accompanied with dyslipidemiaBierman et al., 1966; Garber, 2002), i.e. characterized by increase

in TC, LDL, VLDL, TG and fall in HDL. This altered serum lipidprofile was reversed towards normal after treatment with theextract. CLEt exhibited hypocholesterolemic and hypotriglyceri-demic effects, while increased the levels of HDL in both MD andSD rats. However, CLEt was found to be more effective in reducingthe levels of TG and LDL as compared to its effect on TC and HDL.The elevated atherogenic index, i.e. TC/HDL ratio, which is a usefuldeterminant of cardiovascular risk (Grover et al., 1999), was alsoshifted towards normal after CLEt treatment.

Since CLEt up to 2000 mg/kg dose did not reveal any physicalsigns of toxicity or mortility even after 3 weeks of treatment, it canbe considered relatively safe.

It is concluded from the data that CLEt possesses potent anti-hyperglycemic and hypolipidemic activity and it may prove to beeffective for the treatment of both types of diabetes, i.e. IDDM andNIDDM. Further studies to isolate, identify and characterize theactive principle(s) are in the progress.

Acknowledgement

The authors thank the Secretary, Department of Science andTechnology, New Delhi (India) for providing financial assistance.

References

Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W., Fu, P.C., 1974. Enzymatic determina-tion of total serum cholesterol. Clinical Chemistry 20, 470–475.

Barham, D., Trinder, P., 1972. An improved color reagent for the determination ofblood glucose by the oxidase system. Analyst 97, 142–145.

opharm

B

B

B

D

E

E

F

F

G

G

G

S. Gupta et al. / Journal of Ethn

ierman, E.L., Amaral, J.A.P., Belknap, B.H., 1966. Hyperlipidemia and diabetes mel-litus. Diabetes 15, 675–679.

rahmachari, H.D., Augusti, K.T., 1961. Hypoglycaemic agents from Indian indigenousplants. Journal of Pharmacy and Pharmacology 13, 381–385.

urstein, M., Scholnick, H.R., Morfin, R., 1970. Rapid method for isolation of lipopro-tein from human serum by precipitation with polyanions. Journal of LipidResearch 11, 583–595.

avis, S.N., Granner, D.K., 1996. Insulin, oral hypoglycemic agents and the pharmacol-ogy of the endocrine pancreas. In: Hardman, J.G., Limbird, L.E. (Eds.), Goodman& Gilman’s The Pharmacological Basis of Therapeutics, 10th ed. McGraw-HillCompanies Inc., New York, pp. 1679–1714.

lsner, M., Guldbakke, B., Tiedge, M., Munday, R., Lenzen, S., 2000. Relativeimportance of transport and alkylation for pancreatic beta-cell toxicity of strep-tozotocin. Diabetologia 43, 1528–1533.

smaeili, M.A., Yazdanparas, R., 2004. Hypoglycemic effect of Teucrium polium:studies with rat pancreatic islets. Journal of Ethnopharmacology 95, 27–30.

ossati, P., Prencipe, L., 1982. Serum triglycerides determined colorimetrically withan enzyme that produces hydrogen peroxide. Clinical Chemistry 28, 2077–2080.

riedewald, W.T., Levy, R.I., Fredrickson, D.S., 1972. Estimation of the concentrationof low-density lipoprotein cholesterol in plasma without use of the preparativeultracentrifuge. Clinical Chemistry 18, 499–502.

arber, A.J., 2002. Attenuating CV risk factors in patients with diabetes: clinicalevidence to clinical practice. Diabetes, Obesity and Metabolism 4, S5–S12.

oldstein, D.E., Little, R.R., Weidmeyer, H.M., England, J.D., McKenzie, E.M., 1986. Gly-cated haemoglobin methodologies and clinical applications. Clinical Chemistry32, B64–B70.

rover, S.A., Levington, C., Paquet, S., 1999. Identifying adults at low risk for signifi-cant hyperlipidemia: a validated clinical index. Journal of Clinical Epidemiology52, 49–55.

acology 123 (2009) 499–503 503

Jackson, R.L., Hess, R.L., England, J.D., 1979. Haemoglobin A1c values in childrenwith overt diabetes maintained in varying degrees of control. Diabetes Care 2,391–395.

Joshi, S.G., 2000. Medicinal Plants. Oxford and IBH Publishing Company, New Delhi,p. 119.

Kedar, P., Chakrabarti, C.H., 1982. Effects of bittergourd (Momordica charantia) seedand glibenclamide in streptozotocin induced diabetes mellitus. Indian Journalof Experimental Biology 20, 232–235.

Kumar, R.S., Ponmozhi, M., Viswanathan, P., Nalini, N., 2002. Effect of Cassia auriculataleaf extract on lipids in rats with alcoholic liver injury. Asia Pacific Journal ofClinical Nutrition 11, 157–163.

Pari, L., Latha, M., 2002. Effect of Cassia auriculata flowers on blood sugar levels,serum and tissue lipids in streptozotocin diabetic rats. Singapore Medical Journal43, 617–621.

Sabu, M.C., Subburaju, T., 2002. Effect of Cassia auriculata Linn. on serum glucoseutilization by isolated rat hemidiaphragm. Journal of Ethnopharmacology 80,203–206.

Scheen, J.A., 1997. Drug treatment of non-insulin dependent diabetes mellitus in the1990s. Achievements and future development. Drugs 54, 355–368.

Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., 2006. Antihyperglycemic effect ofthe fruit-pulp of Eugenia jambolana in experimental diabetes mellitus. Journalof Ethnopharmacology 104, 367–373.

Siddiqui, O., Sun, Y., Liu, J.C., Chien, Y.W., 1987. Facilitated transdermal transport ofinsulin. Journal of Pharmaceutical Sciences 76, 341–345.

Sorg, D.C., Buckner, B., 1964. A simple method of obtaining venous blood from small

laboratory animals. Proceedings of the Society for Experimental Biology andMedicine 115, 1131–1132.

WHO Expert Committee on Diabetes Mellitus, 1980. Second Report, Technical ReportSeries 646. World Health Organisation, Geneva.

Zimmerman, M., 1983. Ethical guidelines for investigations of experimental pain inconscious animals. Pain 16, 109–110.