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Inggris - Saponin
Inggris - Saponin
ABSTRACT
Bryonia laciniosa Linn. (Cucurbitaceae) seed is used in traditional medicine for a number of ailments
including metabolic disorders. This investigation was carried out to investigate the anti-hyperglycemic and
anti-hyperlipidemic potential of the ethanolic extract of seeds of B. laciniosa Linn. and its saponin fraction in
streptozotocin-induced diabetic rats. The ethanolic extract (250 and 500 mg/kg; p.o.) and saponin fraction (100 and
200 mg/kg; p.o.) were administered to diabetic rats and standard drug insulin (5 IU/kg; i.p.) to the group serving
as a positive control. Effects of the ethanolic extract and saponin fraction on various biochemical parameters
were studied in diabetic rats. Data were statistically analysed by one-way ANOVA followed by Dunnett’s t-test.
Oral administration of the ethanolic extract and saponin fraction for 28 days to streptozotocin-induced diabetes
UDWVVLJQL¿FDQWO\P < 0.05) decreased the levels of blood glucose and improved the levels of plasma insulin. The
levels of triglycerides, cholesterol, high density lipoprotein, low density lipoprotein, very low density lipoprotein,
aspartate amino transferase and alanine amino transferase, urea, and creatinine were markedly altered in
streptozotocin-induced diabetic rats. Oral administration of the ethanolic extract and saponin fraction restored all
WKHVHELRFKHPLFDOSDUDPHWHUVWRQHDUFRQWUROOHYHOV7KLVVWXG\UHYHDOVWKHHI¿FDF\RIB. laciniosa seed extract
and its saponin fraction in the amelioration of diabetes and its associated complications.
be developed into new drugs for anti-diabetic.[2,3] Many approximately using the method described by Hiai et al. [17]
studies have demonstrated that saponins exert antioxidant %ULHÁ\WKHH[WUDFWVƬOZHUHPL[HGZLWKWKHYDQLOOLQ
action and alleviate the streptozotocin-induced toxicity and (8% w/v, 0.5 ml) and sulfuric acid (72% w/v, 5 ml). The
contribute to hypoglycemic and hypolipidemic effects.[4] mixture was incubated at 60°C for 10 min, cooled in an
ice water bath for 15 min, and the absorbance read at 538
Bryonia laciniosa Linn. Syn (Cucurbitaceae) plant is nm. Oleanolic acid was used as a reference standard, and
commonly used as a hypoglycemic herb by the native the content of total saponins was expressed as oleanolic
people of the Porbandar region. An ayurvedic literature DFLGHTXLYDOHQWV2$(ƬJPJH[WUDFW
survey indicated the use of entire plant is a bitter tonic,
hepatoprotective, anti-pyretic, laxative, and used to correct Animals
the metabolic abnormalities.[5-8] The occurrence of bitter
principle bryonin, saponin, punicic acid, goniothalamine, All experiments were performed on Swiss albino mice
and glucomannan has been reported in this plant.[9-12] The of either sex weighing about 20–25 g and female albino
various pharmacological activates of the extract of B. Wistar rats of body weight 200–250 g. Animals were
laciniosa is already explored.[13-16] The anti-hyperglycemic maintained under controlled conditions of humidity,
and anti-hyperlipidemic activities of the B. laciniosa seeds temperature (22 ± 2°C), and 12 h light and dark cycle. The
are remain unclear. Hence the present study was planned standard pelleted diet (Pranav Agro Industries limited,
to determine the antihyperglycemic and antihyperlipidemic Vadodara) and water were given ad libitum. The animals
activities of the B. laciniosa seed extract. experiments were conducted as per Institutional animal
ethical committee (910/ac/05/CPCSEA) instructions.
MATERIALS AND METHODS Acute toxicity studies
Materials The acute toxicity study was carried out in female Swiss
DOELQR PLFH XVLQJ À[HG GRVH SURGXFHU GHVFULEHG E\ WKH
Streptozotocin was purchased from Sisco Research Organisation for Economic Cooperation and Development
Laboratory Pvt. Ltd, India. Insulin was commercially (OECD). The compound was tested for maximum
purchased from Novo Nordisk India Pvt. Ltd. All recommended dose of 2000 mg/kg.[18] Based on the toxicity
other standard chemicals were obtained from common testing result the pharmacological experiment dose as
commercial suppliers. À[HGDWDQGPJNJIRU%/((DQGDQG
mg/kg for BLSF.
Collection of plant material and extraction
Experimental induction of diabetes in rats
The fruits of B. laciniosa were collected in October to
December from Anand and Jamnagar region of Gujarat, Hyperglycemia was induced in overnight fasted adult albino
India. The plant was authenticated by comparison with Wister rats weighing 200–250 g by a single intra-peritoneal
voucher specimen no. VSM502 and ARM 2174 at the Prof. injection of 50 mg/kg streptozotocin (dissolved in 0.1 M
G.L. Shah Herbarium of S.P. University, Vallabh Vidyanagar, ice-cold citrate buffer, pH 4.5, immediately before use) in
Anand, Gujarat, India. The seeds were separated from a volume of 1 ml/kg body weight. Hyperglycemia was
the fruits and washed with large quantity of water. The FRQÀUPHGE\WKHHOHYDWHGJOXFRVHOHYHOLQSODVPDGHWHUPLQHG
seeds were grinded mechanically to make powder. The at 48 h after injection. The rats found hyperglycemic were
powdered sample was extracted with ethanol by Soxhlet screened for the anti-hyperglycemic study.
to give B. laciniosa ethanol extract (BLEE). Total saponins
were prepared by the method described previously.[4] The Drug administration
alcohol extract was concentrated, suspended in distilled
water, and then partitioned successively with n-butanol The BLEE and BLSF suspended in 0.25% NaCMC
saturated with water. The n-butanol extract was combined and administered orally through orogastric tubes at the
and evaporated using a rotary evaporator at 60°C to yield following doses of 250 and 500 mg/kg body wt for BLEE
B. laciniosa saponin fraction (BLSF). and 100 and 200 mg/kg body wt for BLSF.
The total saponins content of each extract was determined Totally sixty-six rats (42 diabetic rats, 24 normal rats) were
172 Journal of Young Pharmacists Vol 4 / No 3
Patel, et al.: Anti-hyperglycemic and anti-hyperlipidemic effects of B. laciniosa seed extract in rats
used for the experiment. Diabetes was induced in rats 5 in glucose concentrations over 120 min during OGTT
days before starting the treatment. A study of effect of were expressed as AUCglucose (mg/dl 120 min).
BLEE and BLSF on type 1 diabetic rats involved two sets
of experiments. The groups of animals in two different Biochemical assays
sets were as follows:
Serum glucose, total cholesterol, triglycerides, high
Set 1: Effect of BLEE on type 1 diabetic rats density lipoprotein (HDL-C), urea, creatinine, aspartate
Group 1: Normal control rats were administered amino transferase (AST), and alanine amino transferase
vehicle (ALT) levels were determined using standard kits of
Group 2: Normal rats were administered BLEE Span Diagnostics Ltd., India. Low-density lipoprotein
(500 mg/ kg) cholesterol (LDL-C), very low density lipoprotein
Group 3: Diabetic control rats were administered cholesterol (VLDL-C) and atherogenic index (AI)
vehicle were calculated. [20] Serum insulin was measured by
Group 4: Diabetic rats were administered BLEE radioimmuno assay (Rat Insulin RIA Kit, Linco Research,
(250 mg/ kg) Inc., St. Charles, MO).
Group 5: Diabetic rats were administered BLEE
(500 mg/ kg) Statistical analysis
Group 6: Diabetic rats were administered Insulin
(5 IU/kg) Data were statistically analysed by utilizing one-way
ANOVA and expressed as mean ± SEM followed by
Set 2: Effect of BLSF on type 1 diabetic rats Dunnett’s t-test. A P ZDVFRQVLGHUHGDVVLJQLÀFDQW
Group 1: Normal control rats were administered
vehicle
RESULTS
Group 2: Normal rats were administered BLSF (0.2
g/kg) The total saponins content determination showed that
Group 3: Diabetic control rats were administered %/(( DQG %/6) SRVVHVV DQG ƬJPJ RI 2$(
vehicle respectively. In the study, the STZ treated diabetic rats
Group 4: Diabetic rats were administered BLSF developed uncontrolled type 1 diabetes mellitus. Diabetes
(100 mg/ kg) was associated with reduced body weight and increased food
Group 5: Diabetic rats were administered BLSF intake when compared with the normal control rats. Final
(200 mg/ kg) body weight (BW) and food intake of normal control and
experimental groups are shown in Tables 1 and 2. However,
All the studies were carried out for a period of 28 days. During treatment with BLEE and BLSF with all the doses appeared
the study standard food and water were provided ad libitum. to protect the diabetic rats from body weight loss.
Changes in body weight and food intake were recorded. At
the end of experimental period, the animals were deprived 7KHUHZDVVLJQLÀFDQWP < 0.05) elevation in serum glucose
of food overnight. Blood were collected from retro-orbital levels associated with hypoinsulinemia in the diabetic
plexus under light ether anesthesia for biochemical analyses. animals as compared to normal control animals. Treatment
with BLEE (250 and 500 mg/kg) and BLSF (100 and 200
Oral glucose tolerance test PJNJ VLJQLÀFDQWO\ P < 0.05) reduced glucose levels
associated with an increase in serum insulin levels. STZ-
At the end of the study, the oral glucose tolerance test LQGXFHG GLDEHWLF DQLPDOV VKRZHG VLJQLÀFDQW P < 0.05)
was performed after an overnight fasting.[19] The animals increase in AUCglucose as compared to normal animals.
were orally administered with 1.5 g/kg of glucose, and Treatment with BLEE and BLSF at all the doses prevented
blood samples were collected from the retro orbital the increase in AUCglucoseDWVLJQLÀFDQWP < 0.05) extent
plexus under light ether anesthesia before, i.e. 0, 30, 60, as compared to diabetic control animals [Tables 3 and 4].
and 120 min after oral glucose administration. Serum 67=LQUDWVSURGXFHGDVLJQLÀFDQWP < 0.05) increase in
was analyzed for glucose by the GOD–POD method by cholesterol, triglycerides, VLDL, and LDL while decrease
using diagnostic reagent kits (Span diagnostics Limited, in HDL levels as compared to nondiabetic control rats.
India). The results were expressed as integrated area under Treatment of rats with BLEE (250 and 500 mg/kg) and
the curve (AUC) for glucose. This was calculated by the %/6)DQGPJNJFDXVHGDVLJQLÀFDQWP < 0.05)
trapezoid rule [AUC = (C1 + C2)/2 × (t2 - t1)] and changes decrease in cholesterol, triglycerides, VLDL, and LDL
Journal of Young Pharmacists Vol 4 / No 3 173
Patel, et al.: Anti-hyperglycemic and anti-hyperlipidemic effects of B. laciniosa seed extract in rats
Table 3: Effect of BLEE on blood glucose, insulin, and Table 4: Effect of BLSF on blood glucose, plasma
AUCglucose of STZ-induced diabetic rats insulin, and AUCglucose of STZ- induced diabetic rats
Treatment Glucose Plasma AUCglucose Treatment Glucose Plasma AUCglucose
(mg/dl) insulin (μU/ml) (mg/dl per min) (mg/dl) insulin (μU/ml) (mg/dl per min)
Normal control 100.83 ± 3.86 4.98 ± 0.39 4.17 ± 0.14 Normal control 106.63 ± 6.47 5.55 ± 0.55 4.17 ± 0.14
Normal control 96.63 ± 6.47 5.20 ± 0.47 4.29 ± 0.21 Normal control 102.39 ± 7.69 6.10 ± 0.43 4.29 ± 0.21
+ BLEF + BLSF
(500 g/kg) (200 mg/kg)
Diabetic control 262.9 ± 17.03* 2.55 ± 0.19* 9.65 ± 0.42* Diabetic control 388.36 ± 19.46* 1.95 ± 0.29* 9.65 ± 0.42*
Diabetic + BLSF 200.16 ± 9.13** 4.19 ± 0.21** 6.47 ± 0.30** Diabetic + BLSF 263.26 ± 19.09 3.79 ± 0.24 7.47 ± 0.30**
(250 mg/kg) (100 mg/kg)
Diabetic + BLSF 177.16 ± 7.43** 5.39 ± 0.36** 5.50 ± 0.30** Diabetic + BLSF 201.19 ± 5.69 ± 0.56** 6.50 ± 0.30**
(500 mg/kg) (200 mg/kg) 18.46**
Values are mean ± SEM (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKH Values are mean ± SEM (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKH
QRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKH QRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKH
GLDEHWLFFRQWUROJURXSP < 0.05 GLDEHWLFFRQWUROJURXSP < 0.05
An oral glucose tolerance test is a more sensitive index for early hormone-sensitive lipase (HSL) which is resulted to enhance
detection of abnormalities in glucose regulation than fasting the release of free fatty acids from adipose tissue.[29] Thus,
glucose levels.[27],PSDLUHGJOXFRVHWROHUDQFHUHÁHFWVKHSDWLF excess fatty acids in the blood produced by the STZ-
gluconeogenesis and reduced uptake of glucose from blood induced diabetes promote the conversion of excess fatty
into skeletal muscle and adipose tissue following a meal.[28] acids into phospholipids and cholesterol in the liver. These
two substances, along with excess triglycerides formed in
In STZ-induced diabetes, the increase in elevated glucose the liver, may be discharged into the blood in the form
levels is usually associated with an increase in plasma of lipoproteins.[29] Therefore, the marked hyperlipidemia
cholesterol, triglycerides, LDL, and VLDL and decreases in that characterizes the diabetic state may be regarded as
+'/7KHLQVXOLQGHÀFLHQWVWDWHWXUQVLQWRWKHDFWLYDWLRQRI a consequence of the uninhibited actions of lipolytic
7DEOH(IIHFWRI%/((RQOLSLGSUR¿OHVRI67=LQGXFHGGLDEHWLFUDWV
Treatment Triglycerides (mg/dl) Total Cholesterol (mg/dl) HDL-C (mg/dl) VLDL-C (mg/dl) LDL-C (mg/dl) Atherogenic index
Normal control 103.83 ± 4.12 107.16 ± 3.85 37.58 ± 2.05 20.76 ± 0.82 57.56 ± 3.86 3.67 ± 0.25
Normal control + BLEE 86.59 ± 6.37 103.59 ± 12.08 39.33 ± 3.11 17.31 ± 1.27 46.94 ± 10.59 2.20 ± 0.32
(500 mg/kg)
Diabetic control 198.40 ± 17.11* 143.8 ± 8.07* 22.0 ± 1.30* 39.68 ± 3.42* 82.12 ± 10.67* 9.01 ± 1.12*
Diabetic + BLEE 149.66 ± 4.09** 132.50 ± 2.04 29.33 ± 1.20** 29.93 ± 0.81** 71.06 ± 3.26 4.78 ± 0.17**
(250 mg/kg)
Diabetic + BLEE 136.16 ± 3.60** 124.66 ± 4.73** 35.50 ± 1.40** 27.23 ± 0.72* 68.10 ± 5.06 4.64 ± 0.29**
(500 mg/kg)
Diabetic + Insulin 127.15 ± 5.39** 88.69± 4.80** 33.27 ± 1.94** 25.43 ± 1.08** 62.86 ± 5.90** 3.82 ± 0.33**
(5 IU/kg)
Values are mean ± SEM of 6 animals (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHQRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKH
GLDEHWLFFRQWUROJURXSP < 0.05
7DEOH(IIHFWRI%/6)RQEORRGJOXFRVHDQGOLSLGSUR¿OHVRI67=LQGXFHGGLDEHWLFUDWV
Treatment Triglycerides (mg/dl) Total cholesterol (mg/dl) HDL-C (mg/dl) VLDL-C (mg/dl) LDL-C (mg/dl) Atherogenic index
Normal control 86.07 ± 5.49 111.16 ± 6.10 36.58 ± 2.05 17.21 ± 1.10 57.03 ± 3.37 2.29 ± 0.11
Normal control + BLSF 98.26 ± 7.49 109.12 ± 8.14 35.27 ± 3.11 19.25 ± 1.79 54.22 ± 5.69 2.78 ± 0.41
(200 mg/kg)
Diabetic control 167.97 ± 8.05* 167.49 ± 5.01* 25.77 ± 1.30* 33.59 ± 1.61* 105.88 ± 5.13* 6.23 ± 0.65*
Diabetic + BLSF 132.72 ± 7.45** 130.98 ± 4.96 35.27 ± 1.94** 26.54 ± 1.49** 69.19 ± 5.08** 3.30 ± 0.46**
(100 mg/kg)
Diabetic + BLSF 105.65 ± 3.43** 116.63 ± 8.75 44.55 ± 1.87** 21.13 ± 0.68** 50.94 ± 8.14** 2.38 ± 0.10**
(200 mg/kg)
Values are mean ± SEM (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHQRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHGLDEHWLFFRQWURO
JURXSP < 0.05
Table 7: Effect of BLEE on creatinine, urea, AST, and ALT of STZ-induced diabetic rats
Treatment Creatinine (mg/dl) Urea (mg/dl) AST (U/ml) ALT (U/ml)
Normal control 0.555 ± 0.03 27.66 ± 2.56 26.00 ± 2.44 42.83 ± 2.05
Normal control +BLEE (500 mg/kg) 0.503 ± 0.05 32.02 ± 1.70 38.66 ± 4.86 40.50 ± 2.48
Diabetic control 0.816 ± 0.03 * 58.80 ± 8.85* 87.60 ± 15.25* 72.60 ± 2.69*
Diabetic + BLEE (250 mg/kg) 0.490 ± 0.04** 37.66 ± 2.83** 54.66 ± 4.99** 46.00 ± 6.27**
Diabetic + BLEE (500 mg/kg) 0.463 ± 0.03** 35.33 ± 2.75** 42.16 ± 4.75** 39.00 ± 4.26**
Diabetic + insulin (5 IU/kg) 0.565 ± 0.03** 33.00± 2.63** 40.83 ± 2.70** 42.83 ± 4.53**
Values are mean ± SEM (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHQRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHGLDEHWLFFRQWURO
JURXSP < 0.05
Table 8: Effect of BLSF on creatinine, urea, AST, and ALT of STZ-induced diabetic rats
Treatment Creatinine (mg/dl) Urea (mg/dl) AST (U/ml) ALT (U/ml)
Normal control 0.977 ± 0.03 29.85 ± 2.18 23.16 ± 1.95 24.33 ± 2.77
Normal control + BLSF (200 mg/kg) 0.503 ± 0.05 32.02 ± 1.70 38.66 ± 4.86 40.50 ± 2.48
Diabetic control 2.017 ± 0.19* 59.07 ± 2.94* 83.16 ± 5.16* 69.83 ± 2.69*
Diabetic + BLSF (100 mg/kg) 1.633 ± 0.08** 35.92 ± 1.69** 60.83 ± 2.70** 42.83 ± 4.53**
Diabetic + BLSF (200 mg/kg) 1.180± 0.08** 32.94 ± 3.04** 48.00 ± 2.33** 35.16 ± 2.27**
Values are mean ± SEM (n
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHQRUPDOFRQWUROJURXSP
6WDWLVWLFDOVLJQL¿FDQFHLQFRPSDULVRQZLWKWKHGLDEHWLFFRQWURO
JURXSP < 0.05
hormones on fat depots. However, treatment with the pharmaceutical saponin molecules of seeds of Bryonialaciniosa. IUPAC.
Internationational conference on biodiversity, natural product chemistry
BLEE and BLSF normalized the serum lipid status, which and medicinal applications, New Delhi;2004 Jan 26-31. p. 368.
was presumably mediated by a control of lipid metabolism. 10. Gowrikumar G, Mani VV, Chandrasekhararao T, Kaimal TN,
Lakshminarayana G. Diplocyclospalmatus L: A new seed source of punicic
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The increased activity of ALT in diabetes represents the 11. Mosaddik MA, Ekramul Haque M, Abdur Rashid M. Goniothalamin
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lipid metabolism in the liver of diabetic rats. 2009;75:534-7.
15. Gupta M, Mazumdar UK, Sivakumar T, Vamsi ML, Karki SS, Sambathkumar
R, et al (YDOXDWLRQ RI DQWLLQÁDPPDWRU\ DFWLYLW\ RI FKORURIRUP H[WUDFW
The elevated blood glucose levels in diabetes leads to of Bryonialaciniosa in experimental animal models. Biol Pharm Bull
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16. Karageuzyan K, Vartanyan G, Agadjanov M. Restoration of disordered
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OHYHORI XUHDDQGFUHDWLQLQH7KHLUOHYHOVUHÁHFWWKHUHQDO 17. Hiai S, Oura H, Nakajima T. Color reaction of some sapogenins and
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18. Parasuraman S. Toxicological screening. J Pharmacol Pharmacother
and BLEF brought back their levels to the near normal 2011;2:74-9
OHYHO 7KHVH UHVXOWV LQGLFDWH WKH HIÀFDF\ RI WKH Bryonial 19. Olefsky JM. Insulin resistance and insulin action. An in vitro and in vivo
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CONCLUSION
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Source of Support: Nil, &RQÀLFWRI,QWHUHVW None declared.
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