Antidyslipidemic and Antioxidant Activities of <em>Hibiscus rosa sinensis</em> Root Extract in Alloxan Induced Diabetic Rats
Abstract
The antidyslipidemic activity of Hibiscus rosa sinensis (Malvaceae) root extract has been studied in alloxan induced diabetic rats. In this model, oral administration of root extract (500 mg/kg bw. p.o.) for 15 days resulted in significant decreased in the levels of blood glucose, plasma lipids and reactivated post heparin lipoprotein lipase activity in alloxan induced diabetic rats. Furthermore, the root extract (50–500 μg) when tested for its antioxidant activity, inhibited the generation of super oxide anions and hydroxyl radicals, in both enzymic and non enzymic systems in vitro. The results of the present study demonstrated antidyslipidemic and antioxidant activities in root extract of H. rosa sinensis which could be used in prevention of diabetic-dyslipidemia and related complications.
Introduction
Hibiscus rosa sinensis Linn (known as Gudhal in Hindi, Japa in Sanskrit, and Shoe Flower in English) is mentioned in ancient medical literature to possess anti-tumor, detoxifier, antifertility, and wound healing activities [1]. Recent researches have found a variety of pharmacological effects of almost all the parts of this plant [2].
Diabetes mellitus with an alarmingly rising incidence, is a cluster of abnormal metabolic paradigm having a common feature of hyperglycemia and dyslipidemia, has been a leading cause of death in both industrialized and developing nations. Apart from hyperglycemia, disorders of lipid metabolism following oxidative stress are the prime risk factors for initiation and progression of diabetic complications in patients and thus there is an urgent need for a simultaneous treatment [3]. The known lipid lowering drugs such as fibrates, statins, bile acid sequestraints and antidiabetic drugs such as glibenclamide, metformin are not so effective against diabetic-dyslipidemia and also cause many side effects in patients [4]. Therefore, research and development of a lipid lowering drug with antidiabetic and antioxidant potential altogether, from natural products are the best option and also are in great demand. We had earlier reported lipid lowering effect of ethanol extract of root of this plant in two different models of hyperlipidemia in rats [5]. In continuation the present study was designed to investigate possible antidyslipidemic and antioxidant activity of the root in alloxan induced diabetic rats.
Materials and Methods
Preparation of Root Extract
Preparation of root extract was done as described in our earlier paper [5]. The extract was stored in refrigerator and used for in vivo and in vitro experiments. The root extract was not soluble in water. One gram of dried mass was crushed and a paste was prepared in distilled water containing 2 % w/v gum acacia. The paste was diluted with continuous triturating with above vehicle to a final volume of 20 ml. This homogenous suspension contained 50 mg drug/ml. A dose of 1 ml/100 g bw of rat i.e. 500 mg/kg bw was fed orally once daily for 15 days by feeding canula. Similarly glibenclamide suspension (60 μg/ml) was also prepared and administered as above. The freshly prepared drugs were used every day.
Antidyslipidemic activity in Alloxan induced diabetic rats. The rats were maintained exactly as in our previous study [5]. Diabetes was induced by a single intraperitoneal injection of alloxan monohydrate 150 mg/kg bw. After three days of injection, diabetes was confirmed by glucometer. The rats with serum glucose level 280–320 mg/dl were taken for the study.
Experimental Design
The rats were divided in four groups having six animals in each as follows: Group 1: control rats (on normal saline); Group 2, Alloxan treated diabetic rats (on normal saline); Group 3, Alloxan treated diabetic rats + H. rosa sinensis (500 mg/kg b.w); Group 4, Alloxan treated diabetic rats + glibenclamide (600 μg/kg b.w). After 15 days of feeding rats were fasted overnight, anaesthetized with thiopental solution, and injected (ip) with 1 ml/kg bw of 10 mg/ml solution of heparin. After 15 min blood was withdrawn from the retro-orbital plexus and collected in EDTA coated tubes. The blood was used for the estimation of glucose, simultaneously plasma was separated and used for the estimations of total cholesterol: TC, phospholipids: PL, triglyceride: TG, free fatty acids: FFA, plasma posts heparin lipolytic activity (PHLA) and lipid peroxide levels by standard spectrophotometric methods mentioned in our earlier paper [5].
In Vitro Anti-oxidant Activity
Enzymic and Non Enzymic Generation of Superoxide Anions
The effect of H. rosa sinensis root extract on the generation of superoxide anions (O) in vitro, in an enzymic system of xanthine–xanthine oxidase was investigated [6]. Xanthine oxidase activity in system (A) containing xanthine and different concentrations of root extract (50–500 μg) added with 0.03 U/ml of xanthine oxidase in phosphate buffer, was assayed spectrophotometrically at 295 nm. The change in optical density corresponding to amount of uric acid formed was compared with reaction mixture which did not include with their test substance. The influence of root extract on nitro blue tetrazolium (NBT) reduction by O anions, was measured in a reaction mixture (B) containing xanthine oxidase and NBT in absence or presence of extract (50–500 μg). After incubation, the amount of formazone formed was measured at 560 nm on spectrophotometer. Another system employed for non enzymic generation of O anions was comprised of phenazine methosulphate, NADH and NBT [7]. After 90 s incubation in absence or presence of test extract 50–500 μg, the amount of formazone formed was read at 560 nm against respective reagent blank.
Enzymic and Non Enzymic Generation of Hydroxyl Radical
Hibiscus rosa sinensis root extract (50–500 μg) was tested against the formation of hydroxyl radicals (OH) in vitro in an enzymic system composed of sodium salicylate, FeSO4, hypoxanthine and xanthine oxidase, assayed for 2,3-dihydroxybenzoate formed by OH mediated hydroxylation of salicylate on spectrophotometer at 510 nm [8]. In another set of experiment, OH was generated non-enzymically by FeSO4, sodium ascorbate, H2O2 and deoxyribose. After reaction in the absence or presence of root extract (50–500 μg), incubation mixture was assayed for malondialdehyde formed [9].
Statistical Analysis
The statistical analysis was done as before [5]. Similarly, the generations of oxygen free radicals with different concentrations of A. indicus root extract were compared with that of their formation without extract. The values were tested for significance at P < 0.05.
Preparation of Root Extract
Preparation of root extract was done as described in our earlier paper [5]. The extract was stored in refrigerator and used for in vivo and in vitro experiments. The root extract was not soluble in water. One gram of dried mass was crushed and a paste was prepared in distilled water containing 2 % w/v gum acacia. The paste was diluted with continuous triturating with above vehicle to a final volume of 20 ml. This homogenous suspension contained 50 mg drug/ml. A dose of 1 ml/100 g bw of rat i.e. 500 mg/kg bw was fed orally once daily for 15 days by feeding canula. Similarly glibenclamide suspension (60 μg/ml) was also prepared and administered as above. The freshly prepared drugs were used every day.
Antidyslipidemic activity in Alloxan induced diabetic rats. The rats were maintained exactly as in our previous study [5]. Diabetes was induced by a single intraperitoneal injection of alloxan monohydrate 150 mg/kg bw. After three days of injection, diabetes was confirmed by glucometer. The rats with serum glucose level 280–320 mg/dl were taken for the study.
Experimental Design
The rats were divided in four groups having six animals in each as follows: Group 1: control rats (on normal saline); Group 2, Alloxan treated diabetic rats (on normal saline); Group 3, Alloxan treated diabetic rats + H. rosa sinensis (500 mg/kg b.w); Group 4, Alloxan treated diabetic rats + glibenclamide (600 μg/kg b.w). After 15 days of feeding rats were fasted overnight, anaesthetized with thiopental solution, and injected (ip) with 1 ml/kg bw of 10 mg/ml solution of heparin. After 15 min blood was withdrawn from the retro-orbital plexus and collected in EDTA coated tubes. The blood was used for the estimation of glucose, simultaneously plasma was separated and used for the estimations of total cholesterol: TC, phospholipids: PL, triglyceride: TG, free fatty acids: FFA, plasma posts heparin lipolytic activity (PHLA) and lipid peroxide levels by standard spectrophotometric methods mentioned in our earlier paper [5].
In Vitro Anti-oxidant Activity
Enzymic and Non Enzymic Generation of Superoxide Anions
The effect of H. rosa sinensis root extract on the generation of superoxide anions (O) in vitro, in an enzymic system of xanthine–xanthine oxidase was investigated [6]. Xanthine oxidase activity in system (A) containing xanthine and different concentrations of root extract (50–500 μg) added with 0.03 U/ml of xanthine oxidase in phosphate buffer, was assayed spectrophotometrically at 295 nm. The change in optical density corresponding to amount of uric acid formed was compared with reaction mixture which did not include with their test substance. The influence of root extract on nitro blue tetrazolium (NBT) reduction by O anions, was measured in a reaction mixture (B) containing xanthine oxidase and NBT in absence or presence of extract (50–500 μg). After incubation, the amount of formazone formed was measured at 560 nm on spectrophotometer. Another system employed for non enzymic generation of O anions was comprised of phenazine methosulphate, NADH and NBT [7]. After 90 s incubation in absence or presence of test extract 50–500 μg, the amount of formazone formed was read at 560 nm against respective reagent blank.
Enzymic and Non Enzymic Generation of Hydroxyl Radical
Hibiscus rosa sinensis root extract (50–500 μg) was tested against the formation of hydroxyl radicals (OH) in vitro in an enzymic system composed of sodium salicylate, FeSO4, hypoxanthine and xanthine oxidase, assayed for 2,3-dihydroxybenzoate formed by OH mediated hydroxylation of salicylate on spectrophotometer at 510 nm [8]. In another set of experiment, OH was generated non-enzymically by FeSO4, sodium ascorbate, H2O2 and deoxyribose. After reaction in the absence or presence of root extract (50–500 μg), incubation mixture was assayed for malondialdehyde formed [9].
Enzymic and Non Enzymic Generation of Superoxide Anions
The effect of H. rosa sinensis root extract on the generation of superoxide anions (O) in vitro, in an enzymic system of xanthine–xanthine oxidase was investigated [6]. Xanthine oxidase activity in system (A) containing xanthine and different concentrations of root extract (50–500 μg) added with 0.03 U/ml of xanthine oxidase in phosphate buffer, was assayed spectrophotometrically at 295 nm. The change in optical density corresponding to amount of uric acid formed was compared with reaction mixture which did not include with their test substance. The influence of root extract on nitro blue tetrazolium (NBT) reduction by O anions, was measured in a reaction mixture (B) containing xanthine oxidase and NBT in absence or presence of extract (50–500 μg). After incubation, the amount of formazone formed was measured at 560 nm on spectrophotometer. Another system employed for non enzymic generation of O anions was comprised of phenazine methosulphate, NADH and NBT [7]. After 90 s incubation in absence or presence of test extract 50–500 μg, the amount of formazone formed was read at 560 nm against respective reagent blank.
Enzymic and Non Enzymic Generation of Hydroxyl Radical
Hibiscus rosa sinensis root extract (50–500 μg) was tested against the formation of hydroxyl radicals (OH) in vitro in an enzymic system composed of sodium salicylate, FeSO4, hypoxanthine and xanthine oxidase, assayed for 2,3-dihydroxybenzoate formed by OH mediated hydroxylation of salicylate on spectrophotometer at 510 nm [8]. In another set of experiment, OH was generated non-enzymically by FeSO4, sodium ascorbate, H2O2 and deoxyribose. After reaction in the absence or presence of root extract (50–500 μg), incubation mixture was assayed for malondialdehyde formed [9].
Statistical Analysis
The statistical analysis was done as before [5]. Similarly, the generations of oxygen free radicals with different concentrations of A. indicus root extract were compared with that of their formation without extract. The values were tested for significance at P < 0.05.
Results
Effect of H. rosa sinensis in Alloxan Induced Hyperglycemia
The acute administration of alloxan caused marked increase in their plasma levels of blood glucose 260 %, TC 57 %, TG 84 %, PL 45 %, FFA 49 % and lipid peroxide 229% following decrease in PHLA by 31 %. However, treatment with H. rosa sinensis root extract caused reversal in these levels of blood glucose by 30 %, TC by 24 %, PL by 17 %, TG by 23 %, FFA by 10 %, lipid peroxide by 36 % and reactivation of PHLA by 17 %. The anti-diabetic and lipid lowering activities of H. rosa sinensis root extract was comparatively less to that of glibenclamide (Table 1).
Table 1
Effect of H. rosa sinensis roots extract on blood glucose and serum lipid levels in alloxan induced diabetic rats
| Groups | Blood glucose (mg/dl) | Total cholesterol (mg/dl) | Triglyceride (mg/dl) | Phospholipids (mg/dl) | FFA (μmol/l) | PHLA (nmol FFA released/h/l) | Lipid peroxide (nmol MDA/dl) |
|---|---|---|---|---|---|---|---|
| Control | 83.22 ± 9.67 | 83.98 ± 6.31 | 81.00 ± 7.77 | 69.19 ± 8.23 | 1.58 ± 0.17 | 16.00 ± 1.58 | 2.73 ± 0.49 |
| Alloxan treated | 300.23*** ± 32.20 (+260) | 132.83*** ± 11.90 (+57) | 149.29*** ± 9.27 (+84) | 100.00*** ± 8.20 (+45) | 2.36*** ± 0.30 (+49) | 11.12*** ± 1.13 (−31) | 8.98** ± 1.40 (+229) |
| Alloxan + Hibiscus rosa sinensis extract (500 mg/kg bw) | 220.41*** ± 20.40 (−30) | 100.83*** ± 11.84 (−24) | 24.43* ± 7.93 (−17) | 76.29** ± 7.74 (−23) | 2.12 NS ± 0.25 (−10) | 13.00* ± 0.83 (+17) | 5.97*** ± 0.49 (−36) |
| Alloxan + glibenclamide (600 μg/kg bw) | 175.38*** ± 11.67 (−45) | 98.36*** ± 17.05 (−26) | 121.51* ± 12.35 (−19) | 90.33 NS ± 7.86 (−8) | 1.98** ± 0.29 (−20) | 14.09*** ± 1.27 (+27) | 5.55*** ± 0.99 (−39) |
Alloxan treated diabetic group is compared with normal rats and alloxan + drug treated groups with diabetic group. Values in the parenthesis are % change
Values are expressed as mean ± SD of six rats *P < 0.05, **P < 0.001, rest NS not significant
Effect of H. rosa sinensis on Generation of Super Oxide Anions
The data in Table 2 showed that enzymic oxidation of xanthine to uric acid (A) as well as the generation of O anions in xanthine–xanthine oxidase system, as measured by reduction of NBT to formazone (B) were inhibited to varying extents by root extract in a concentration dependent manner and this effect was maximum by 55 and 51 % respectively at 500 μg/ml of test sample. The root extract also trapped the O anions generated by non enzymic system of NADH–phenozine–methosulphate and were responsible for reduction of NBT in the reaction mixture. The effect was dose dependent and was highest by 58 % at 500 μg/ml of test substance.
Table 2
Effect of H. rosa sinensis roots extract on generation of oxygen free radicals in vitro
| Concentration of H. rosa sinensis roots extract (μg/ml) | Generation of O anions | Generation of OH radicals | |||
|---|---|---|---|---|---|
| Enzymic system | Non enzymic system (NADH–PMS–NBT-system)b | Enzymic system (sodium salicylate–FeSO4 hypoXn–XnOD-system)c | Non enzymic system (FeSO4–EDTA–H2O2–sodium ascorbate–deoxy ribose-system)d | ||
| (Xn–XnOD-system)a | (Xn–XnOD–NBT-system)b | ||||
| None | 46.44 ± 1.3 8 | 118.05 ± 21.3 0 | 333.00 ± 16.8 2 | 540.09 ± 43.8 6 | 26.31 ± 2.22 |
| 50 | 42.95 NS ± 0.95 (−8) | 104.12 NS ± 11.82 (−12) | 286.23 ± 21.43* (−14) | 531.98 NS ± 10.28 (−1) | 25.68 NS ± 1.26 (−2) |
| 100 | 40.11 * ± 1.22 (−14) | 100.59 * ± 8.41 (−15) | 239.99 ± 7.44** (−28) | 500.69 ± 14.93* (−7) | 21.00 ± 1.97** (−20) |
| 200 | 36.44 ** ± 0.98 (−22) | 87.99 ** ± 3.89 (−25) | 164.88*** ± 12.86 (−50) | 419.33** ± 24.67 (−22) | 17.44*** ± 0.78 (−34) |
| 250 | 32.18*** ± 0.56 (−31) | 75.85*** ± 4.76 (−36) | 160.99*** ± 4.98 (−52) | 340.99*** ± 18.32 (−37) | 15.98*** ± 1.34 (−39) |
| 300 | 28.34*** ± 0.46 (−39) | 70.44*** ± 2.64 (−40) | 151.78*** ± 11.87 (−54) | 323.69*** ± 18.53 (−40) | 15.11*** ± 2.44 (−42) |
| 400 | 23.88*** ± 2.66 (−49) | 66.66*** ± 3.44 (−44) | 148.88*** ± 12.69 (−55) | 300.87*** ± 12.77 (−44) | 14.96*** ± 0.59 (−43) |
| 500 | 20.98*** ± 1.22 (−55) | 57.68*** ± 5.22 (−51) | 139.48*** ± 12.13 (−58) | 270.65*** ± 13.97 (−50) | 13.65*** ± 1.33 (−48) |
Values are mean ± SD of four separate observations. The systems added with H. rosa sinensis root extract were compared with those without adding Hibiscus rosa sinensis root. Values in the parenthesis are % change *P < 0.05, **P < 0.01; ***P < 0.001
nmol uric acid formed/min
nmol formazon formed/min
nmol 2,3 dihydroxy benzoate formed/h
nmol malondialdehyde formed/h
Effect of H. rosa sinensis on Generation of Hydroxyl Radicals
The data in Table 2 also showed that H. rosa sinensis root extract inhibited the formation of OH by enzymic system of hypoxanthine–xanthine oxidase and Fe. Addition of extract (50–500 μg) inhibited the OH mediated formation of 2,3 dihydroxybenzoate in concentration dependant manner which was 50 % at 500 μg/ml of test extract. Furthermore, this preparation, when added with reaction mixture containing Fe–sodium ascorbate–H2O2 employed for nonenzymic generation of OH inhibited fragmentation of deoxyribose into MDA and this effect was maximum by 48 % at peak concentration (500 μg/ml) of root extract.
Effect of H. rosa sinensis in Alloxan Induced Hyperglycemia
The acute administration of alloxan caused marked increase in their plasma levels of blood glucose 260 %, TC 57 %, TG 84 %, PL 45 %, FFA 49 % and lipid peroxide 229% following decrease in PHLA by 31 %. However, treatment with H. rosa sinensis root extract caused reversal in these levels of blood glucose by 30 %, TC by 24 %, PL by 17 %, TG by 23 %, FFA by 10 %, lipid peroxide by 36 % and reactivation of PHLA by 17 %. The anti-diabetic and lipid lowering activities of H. rosa sinensis root extract was comparatively less to that of glibenclamide (Table 1).
Table 1
Effect of H. rosa sinensis roots extract on blood glucose and serum lipid levels in alloxan induced diabetic rats
| Groups | Blood glucose (mg/dl) | Total cholesterol (mg/dl) | Triglyceride (mg/dl) | Phospholipids (mg/dl) | FFA (μmol/l) | PHLA (nmol FFA released/h/l) | Lipid peroxide (nmol MDA/dl) |
|---|---|---|---|---|---|---|---|
| Control | 83.22 ± 9.67 | 83.98 ± 6.31 | 81.00 ± 7.77 | 69.19 ± 8.23 | 1.58 ± 0.17 | 16.00 ± 1.58 | 2.73 ± 0.49 |
| Alloxan treated | 300.23*** ± 32.20 (+260) | 132.83*** ± 11.90 (+57) | 149.29*** ± 9.27 (+84) | 100.00*** ± 8.20 (+45) | 2.36*** ± 0.30 (+49) | 11.12*** ± 1.13 (−31) | 8.98** ± 1.40 (+229) |
| Alloxan + Hibiscus rosa sinensis extract (500 mg/kg bw) | 220.41*** ± 20.40 (−30) | 100.83*** ± 11.84 (−24) | 24.43* ± 7.93 (−17) | 76.29** ± 7.74 (−23) | 2.12 NS ± 0.25 (−10) | 13.00* ± 0.83 (+17) | 5.97*** ± 0.49 (−36) |
| Alloxan + glibenclamide (600 μg/kg bw) | 175.38*** ± 11.67 (−45) | 98.36*** ± 17.05 (−26) | 121.51* ± 12.35 (−19) | 90.33 NS ± 7.86 (−8) | 1.98** ± 0.29 (−20) | 14.09*** ± 1.27 (+27) | 5.55*** ± 0.99 (−39) |
Alloxan treated diabetic group is compared with normal rats and alloxan + drug treated groups with diabetic group. Values in the parenthesis are % change
Values are expressed as mean ± SD of six rats *P < 0.05, **P < 0.001, rest NS not significant
Effect of H. rosa sinensis on Generation of Super Oxide Anions
The data in Table 2 showed that enzymic oxidation of xanthine to uric acid (A) as well as the generation of O anions in xanthine–xanthine oxidase system, as measured by reduction of NBT to formazone (B) were inhibited to varying extents by root extract in a concentration dependent manner and this effect was maximum by 55 and 51 % respectively at 500 μg/ml of test sample. The root extract also trapped the O anions generated by non enzymic system of NADH–phenozine–methosulphate and were responsible for reduction of NBT in the reaction mixture. The effect was dose dependent and was highest by 58 % at 500 μg/ml of test substance.
Table 2
Effect of H. rosa sinensis roots extract on generation of oxygen free radicals in vitro
| Concentration of H. rosa sinensis roots extract (μg/ml) | Generation of O anions | Generation of OH radicals | |||
|---|---|---|---|---|---|
| Enzymic system | Non enzymic system (NADH–PMS–NBT-system)b | Enzymic system (sodium salicylate–FeSO4 hypoXn–XnOD-system)c | Non enzymic system (FeSO4–EDTA–H2O2–sodium ascorbate–deoxy ribose-system)d | ||
| (Xn–XnOD-system)a | (Xn–XnOD–NBT-system)b | ||||
| None | 46.44 ± 1.3 8 | 118.05 ± 21.3 0 | 333.00 ± 16.8 2 | 540.09 ± 43.8 6 | 26.31 ± 2.22 |
| 50 | 42.95 NS ± 0.95 (−8) | 104.12 NS ± 11.82 (−12) | 286.23 ± 21.43* (−14) | 531.98 NS ± 10.28 (−1) | 25.68 NS ± 1.26 (−2) |
| 100 | 40.11 * ± 1.22 (−14) | 100.59 * ± 8.41 (−15) | 239.99 ± 7.44** (−28) | 500.69 ± 14.93* (−7) | 21.00 ± 1.97** (−20) |
| 200 | 36.44 ** ± 0.98 (−22) | 87.99 ** ± 3.89 (−25) | 164.88*** ± 12.86 (−50) | 419.33** ± 24.67 (−22) | 17.44*** ± 0.78 (−34) |
| 250 | 32.18*** ± 0.56 (−31) | 75.85*** ± 4.76 (−36) | 160.99*** ± 4.98 (−52) | 340.99*** ± 18.32 (−37) | 15.98*** ± 1.34 (−39) |
| 300 | 28.34*** ± 0.46 (−39) | 70.44*** ± 2.64 (−40) | 151.78*** ± 11.87 (−54) | 323.69*** ± 18.53 (−40) | 15.11*** ± 2.44 (−42) |
| 400 | 23.88*** ± 2.66 (−49) | 66.66*** ± 3.44 (−44) | 148.88*** ± 12.69 (−55) | 300.87*** ± 12.77 (−44) | 14.96*** ± 0.59 (−43) |
| 500 | 20.98*** ± 1.22 (−55) | 57.68*** ± 5.22 (−51) | 139.48*** ± 12.13 (−58) | 270.65*** ± 13.97 (−50) | 13.65*** ± 1.33 (−48) |
Values are mean ± SD of four separate observations. The systems added with H. rosa sinensis root extract were compared with those without adding Hibiscus rosa sinensis root. Values in the parenthesis are % change *P < 0.05, **P < 0.01; ***P < 0.001
nmol uric acid formed/min
nmol formazon formed/min
nmol 2,3 dihydroxy benzoate formed/h
nmol malondialdehyde formed/h
Effect of H. rosa sinensis on Generation of Hydroxyl Radicals
The data in Table 2 also showed that H. rosa sinensis root extract inhibited the formation of OH by enzymic system of hypoxanthine–xanthine oxidase and Fe. Addition of extract (50–500 μg) inhibited the OH mediated formation of 2,3 dihydroxybenzoate in concentration dependant manner which was 50 % at 500 μg/ml of test extract. Furthermore, this preparation, when added with reaction mixture containing Fe–sodium ascorbate–H2O2 employed for nonenzymic generation of OH inhibited fragmentation of deoxyribose into MDA and this effect was maximum by 48 % at peak concentration (500 μg/ml) of root extract.
Discussion
In the present study, root of H. rosa sinensis was tested for its anti-diabetic, anti-dyslipidemic and anti-oxidant activities in alloxan induced diabetic rats. Alloxan causes reversible damage to insulin-producing β-cells found in the pancreas, and that is why this animal model have been used for primary screening of test drugs for antidiabetic activity [10]. We found that intoxication with alloxan caused increased levels of plasma glucose in rats and their reversal by the treatment with H. rosa sinensis root. Furthermore root extract also reduced lipid peroxide levels in above diabetic rats following inhibition of ROS generation in vitro.
The phytochemical studies showed that H. rosa sinensis contains a variety of sterols, carbohydrates, glycosides, tannins, and flavonoids [11]. In addition to above mentioned constituents, the root bark of H. rosa sinensis also contains aliphatic enone ethers : oclade(−1)-yn-1-oic acid methyl ester 10-oxa, dec-9-ynoic acid methyl ester, non-8-ynoic acid methyl ester, nonadec-trans-10-enoic acid, 11-methoxy-9-oxo-methyl ester, octadec-11-ynoic acid, 10-oxo methyl ester, octadec-9-ynoic acid, 8-oxo methyl ester octadecanoic acid, 10-methylene-9-oxo methyl ester and cyclopropenoids [12]. It is suggested that all or some of these bioactive compounds may be responsible for hypoglycemic, antidyslipidemic and antioxidant effects of this herb. Recently, Mandade and Sreenivas [13] have reported hypoglycemic activity of aqueous ethanolic extract of aerial parts H. rosa sinensis in streptozotocin induced diabetic rats. Also Bhuvana et al. [14] showed that treatment with H. rosa sinensis flower petals (1,000 mg/kg of body weight) mixed with normal chow diet and administrated for 4 weeks, exhibited protective effects by reactivation of the antioxidant enzymes following a decrease in lipid peroxidation in 10 % d-glucose-induced oxidative stress in rat heart tissues. However, there are hardly any pharmacological investigations on root of this herb done so far. The present work is anther report on new properties of H. rosa sinensis root to exert hypoglycemic, antidyslipidemic as well as antioxidant activities in in vivo and in vitro models. Further work on drug metabolism and to assess the biological activity of H. rosa sinensis root and its fraction is under progress to substantiate the present findings.
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