|Year : 2015 | Volume
| Issue : 2 | Page : 73-78
Antihyperlipidemic activity of Hibiscus rosa-sinensis Linn. ethanolic extract fractions
Mukesh Singh Sikarwar1, MB Patil2
1 Department of Pharmacognosy and Phytochemistry, K.L.E University's College of Pharmacy, Belgaum, Karnataka, India; Unit of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, Kedah Darul Aman, Malaysia
2 Department of Pharmacognosy and Phytochemistry, K.L.E University's College of Pharmacy, Belgaum, Karnataka, India
|Date of Web Publication||10-Apr-2015|
Mukesh Singh Sikarwar
Unit of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia
Source of Support: None, Conflict of Interest: None
Aim: The study investigates the antihyperlipidemic effect of Hibiscus rosa-sinensis Linn. (Malvaceae) ethanolic extract fractions in triton and atherogenic diet-induced hyperlipidemic rats. Materials and Methods: Oral administrations of 500 mg/kg body weight of various fractions of selected plant were evaluated for possible antihyperlipidemic activity in triton and atherogenic diet-induced hyperlipidemic rats for duration of 48 h and 14 days respectively. In triton model, hyperlipidemia was induced by intra-peritoneal injection of a saline solution of triton 400 mg/kg in rats, whereas in diet induced model, rats were made hyperlipidemic by giving atherogenic diet for 20 days using oral route of administration. A comparative assessment was also made between the actions of selected drug with simvastatin, known antihyperlipidemic drug. Result and Discussion: The outcomes of this study were expressed as mean ± standard error and data were evaluated by using analysis of variance followed by Dunnett's t-test for multiple comparisons. Oral administration of 500 mg/kg body weight of ethanolic extract residual fraction of Hibiscus rosa-sinensis Linn. flowers exhibited a significant reduction (P < 0.01) in serum lipid parameters such as triglycerides, total cholesterol, low density lipoprotein (LDL), very LDL and increase in high density lipoprotein in hyperlipidemic rats when compared with hyperlipidemic control in both models. Conclusion: Our results demonstrated that ethanolic extract fraction of Hibiscus rosa-sinensis Linn. possessed significant antihyperlipidemic activity.
Keywords: Ethanolic extract fraction, Hibiscus rosa-sinensis Linn, hyperlipidemia, triton
|How to cite this article:|
Sikarwar MS, Patil M B. Antihyperlipidemic activity of Hibiscus rosa-sinensis Linn. ethanolic extract fractions. Int J Health Allied Sci 2015;4:73-8
|How to cite this URL:|
Sikarwar MS, Patil M B. Antihyperlipidemic activity of Hibiscus rosa-sinensis Linn. ethanolic extract fractions. Int J Health Allied Sci [serial online] 2015 [cited 2020 May 26];4:73-8. Available from: http://www.ijhas.in/text.asp?2015/4/2/73/153618
| Introduction|| |
Increased plasma lipid levels namely total cholesterol; triglycerides and low-density lipoprotein (LDL) along with decrease in high-density lipoprotein (HDL) are known to cause hyperlipidemia, which is core in initiation and progression of atherosclerosis impasse. Hyperlipidemia with increased concentration of cholesterol, triglycerides carrying lipoproteins is considered to be the cause of arteriosclerosis with its dual squeal of thrombosis and infraction. 
Hibiscus rosa-sinensis Linn. is an erect, much-branched, glabrous shrub, 1-4 m high. It is a bushy, evergreen shrub or small tree growing 2.5-5 m (8-16 ft) tall and 1.5-3 m (5-10 ft) wide, with glossy leaves and solitary, brilliant red flowers in summer and autumn. The 5-petaled flowers are 10 cm (4 inches) in diameter, with prominent orange-tipped red anthers. It is commonly cultivated as a garden ornamental shrub from sea level to 500 m. It grows on its own in sub-tropic and tropic regions of the world, ranging from 30° North latitude to 30° South latitude. It is a common house plant in places such as Europe, USA and in many Asian countries. It is also called as China rose and is a beautiful, showy flower native to East Asia, mainly to China. It is cultivated primarily as an ornamental plant. Plant breeders around the world have generated several varieties and hybrids of China rose giving rise to an enormous array of colors and patterns within the species. Hibiscus can't tolerate cold winters and dies if left outside.
Some of the chemical constituents isolated from this plant are cyanidin, quercetin, hentriacontane, calcium oxalate, thiamine, riboflavin, niacin, ascorbic acid, and flavonoids.  The flowers contains apigenidin, citric acid, cyanidin diglucoside, cyanin, fructose, gentisic acid, glucose, pelargonidin, quercetin, sucrose and tartaric acid.
Hibiscus rosa-sinensis petal infusion is widely used in Ayurvedic medicine in India as a demulcent refrigerant drink in fever and decoction is given in bronchial catarrh.
Previous studies showed that the plant possesses anti-complementary, anti-diarrhetic, anti-phologistic activity. It has been reported that the plant flower possesses anti-spermatogenic and androgenic, anti-tumor and anticonvulsant activities. ,,,,, It helps in inducing abortion, provide treatment for headache.  Young leaves are sometimes used as a substitute for spinach. It also reported to possess anti-implantation, anti-inflammatory, anti-pyretic, anti-spasmodic, anti-spermatogenic and anti-viral activities.  A decoction of the roots is used for coughs and colds. An infusion of the petals of the flowers soothes and protects the alimentary tract and relieves inflammation. In fevers, an infusion of the flowers helps to reduce body temperature. 
There is no scientific evidence to prove antihyperlipidemic activity of Hibiscus rosa-sinensis Linn. except its traditional use cited in Ayurvedic treatise. 
| Materials and methods|| |
Flowers of Hibiscus rosa-sinensis Linn. were collected in July 2012 around local forest area of Ankola in Western Ghats, Karnataka and authenticated by the Botanist Prof. G. S. Naik, Department of Botany, G. C. Science and Art College, Ankola. A voucher herbarium specimen number GCSAC/HRS/01 was also preserved in the same college. The collected flowers were dried and coarsely powdered. The powder was passed through 40 # mesh and stored in an airtight container at room temperature for future use.
Atherogenic diet and chemicals
Experimental hyperlipidemic diet
A well pulverized mixture of cholesterol (2%), cholic acid (1%), peanut oil (10%), sucrose (40%) and normal laboratory diet (47%) consisted the experimental diet. It was in the form of pellet diet.
Experimental hyperlipidemic agent
Triton WR-1339 (S D Fine chemicals) suspension in 0.15 M NaCl was administered in experimental rats for inducing hyperlipidemia.
Simvastatin (Dr. Reddy's Laboratories, Hyderabad), Diagnostic kits for assessment were procured from Merck Diagnostics India Ltd. All other chemicals were of analytical grade.
Adult Albino rats of Wistar strain (150-200 g) of either sex were selected and housed in the animal house with 12 h light and 12 h dark cycles. Standard pellets diet was used as a basal diet during the experimental period. The animals were provided food and drinking water ad libitum. Experiments were conducted by following ethical norms suggested by CPCSEA, Ministry of Social Justice and Empowerment, Government of India.
Extraction and fractionation of plant material
Powdered crude drug (2.5 kg of the fresh air-dried) of Hibiscus rosa-sinensis Linn. flowers were extracted with 95% ethanol and water by following the simple maceration procedure at room temperature for 1 week with occasional shaking and stirring. 
Once the extraction was done it was filtered and evaporated to dryness by using rota-evaporator. This dried total ethanolic extract was stored in a desiccator for future use. A total of 5 g of polar extract was dissolved in 100 ml of distilled water which was further fractionated with 200 ml of chloroform by using separating funnel. The chloroform soluble components were extracted by gentle shaking. The chloroform fraction was filtered and evaporated to dryness under reduced pressure by using rota-evaporator.
A total of 5 g polar extract was dissolved 100 ml of distilled water which was further fractionated with 200 ml of ethyl acetate by using separating funnel. The ethyl acetate soluble components were extracted by gentle shaking. The ethyl acetate fraction thus extracted, filtered and evaporated to dryness. The residue remaining after the fractionation with chloroform and ethyl acetate was filtered and evaporated to dryness and labeled as a residual fraction. 
Preparation of dose for dried extracts and standard drugs
Fractions were formulated as the suspension in distilled water using Tween 80 as suspending agent. The suspension strength was as per the administered dose and was expressed as weight of dried extract.  Simvastatin 10 mg/kg was used as reference standard drug.
Acute oral toxicity studies
The acute oral toxicity studies of extracts were carried out as per the OECD guidelines. One-tenth of upper limit dose was considered as the levels for investigation of antihyperlipidemic activity. 
Triton-induced hyperlipidemic model
Animals were kept for fasting for 12 h and were injected a saline solution of triton 400 mg/kg intra-peritoneally. The solvent fractions, at the dose of 500 mg/kg, were administered by using an oral route through gastric intubation. The first dose was given instantly after triton injection, followed by second dose after 20 h as per the treatment protocol. After 4 h of the second dose, the animals were used for various biochemical parameters. Blood collection was done by orbital plexus of the rat under ether anesthesia. Blood was centrifuged at 2000 rpm for 30 min in order to get serum. 
Animals were distributed into seven different groups with six animals in each group. Group I considered as normal control. Group II labeled as the hyperlipidemic control and did not receive any treatment except pellet diet. Group III was a positive control and given standard drug simvastatin (10 mg/kg/day p.o.). Groups IV, V and VI received different fractions of Hibiscus rosa-sinensis Linn. ethanolic extract at dose of 500 mg/kg/day, p.o. Lipid parameters in blood serum was studied at 6, 24 and 48 interval.
Diet-induced hyperlipidemic model
In this group, rats were made hyperlipidemic by giving atherogenic diet for 20 days using an oral route of administration. The rats were then given plant fractions suspended in 0.2% Tween 80 at the dose of 500 mg/kg once daily through gastric intubation for 14 successive days. The control animals received vehicle and hyperlipidemic diet. There were normal, negative (hyperlipidemic control) and positive (Simvastatin) control in this model assigned as Groups I, II and III, respectively. Blood collection was done by orbital plexus of the rat under ether anesthesia. Blood was centrifuged at 2000 rpm for 10 min in order to get serum. 
Treatment periods for all these groups were 14 days in atherogenic diet-induced hyperlipidemia and 48 h in case of triton-induced hyperlipidemia.
Collection of blood
Blood was collected by giving retro-orbital sinus puncture using mild ether anesthesia. The collected samples were centrifuged for 10 min. 
The serum was assayed for triglycerides, phospholipids, total cholesterol, LDL, very LDL (VLDL) and HDL using standard protocol method. 
The outcomes of this study were stated as mean ± standard error and data was analyzed by using one-way analysis of variance test, followed by Dunnett's t-test for multiple comparisons. P < 0.05 were considered as significant.  Statistical significant differences between the hyperlipidemic control groups and the different treatment groups were studied.
| Results|| |
The antihyperlipidemic effect of ethanolic extract fractions of Hibiscus rosa-sinensis Linn. flowers were assessed on triton induced, and diet induced hyperlipidemic rats. Results are expressed as change in serum lipid parameters.
In acute toxicity study, animals were observed at regular time intervals at least once during the first 30 min of initial dosing during the first 24 h. In all the cases, no death was observed within first 24 h. Additional observations like behavioral changes in skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behavior pattern were also found to be normal. Attention was also given to observation of tremors and convulsions. Overall results suggested the LD 50 value as 5000 mg/kg. Hence, therapeutic dose was calculated as 1/10 th (500 mg/kg) of the lethal dose for the purpose of antihyperlipidemic investigations.
As anticipated, the elevation of serum lipid and lipoprotein levels were observed after administration of triton WR-1339, which were sustained over a period of study in hyperlipidemic control group. These hyperlipidemic rats were given treatment with chloroform, ethyl acetate and residual fractions of ethanolic extract of Hibiscus rosa-sinensis Linn. The results were compared with reference drug simvastatin and were found to be moderately effective. Significant elevation in serum lipids and lipoproteins in the triton-induced hyperlipidemic control (P < 0.01) rats were observed when compared with normal control.
Hibiscus rosa-sinensis Linn. ethanolic extracts fraction
In triton induced model, the residual fraction of ethanolic extract were able to reduce serum lipid level significantly (P < 0.01). It significantly reduced total cholesterol, triglycerides, phospholipids, LDL, VLDL and increased HDL level in comparison to hyperlipidemic control total cholesterol, triglycerides, phospholipids, LDL, VLDL and HDL at 48 th h of study [Table 1] and [Table 2].
|Table 1: Effect of Hibiscus rosa-sinensis Linn. ethanolic extract fractions on serum total cholesterol, triglycerides and phospholipids level in triton induced hyperlipidemic rat|
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|Table 2: Effect of Hibiscus rosa-sinensis Linn. ethanolic extract fractions on LDL, VLDL and HDL level in triton induced hyperlipidemic rats|
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In diet-induced model, the residual fraction of formulations of ethanolic extract showed significant serum lipid-lowering effects in hyperlipidemic rats, which reduced total cholesterol 70 ± 2.854, triglycerides 68.66 ± 1.240, phospholipids 71 ± 3.287, LDL 45 ± 2.606, VLDL 26.5 ± 1.356 and increased level of HDL 29.16 ± 2.365 in comparison of diet-induced hyperlipidemic control total cholesterol 101.16 ± 2.613, triglycerides 86 ± 2.280, phospholipids 107.66 ± 2.642, LDL 81 ± 2.556, VLDL 35 ± 1.141 and HDL 21.08 ± 1.172 at 14 th day [Figure 1].
|Figure 1: Effect of Hibiscus rosa-sinensis Linn. ethanolic extract fractions on serum lipids and lipoproteins levels in diet-induced hyperlipidemic rats|
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| Discussion|| |
The biphasic nature of triton-induced hyperlipidemia is helpful in understanding the mode of action of the hypolipidemic agents. Drugs interfering with lipid biosynthesis or uptake will be active in the synthesis phase while drugs interfering with lipid excretion and metabolism will be active in the excretory phase. 
Nutrition plays a significant role in the etiology of hyperlipidemia and atherosclerosis. Cholesterol feeding has been often used to elevate serum or tissue cholesterol levels to assess the hypercholesterolemia related metabolic disturbances in animals. Cholesterol feeding alone however does not affect the serum thyroglobulin (TG) level. Groundnut oil is used in addition to cholesterol for significantly elevate serum TG level in the rat model. 
There was a marked increase in the level of serum cholesterol and lipoprotein level whereas decrease in HDL in the animals treated with triton and atherogenic diet. Treatment with Hibiscus rosa-sinensis Linn. flowers ethanolic extract fraction (500 mg/kg body weight) significantly decreased this level and increased HDL level as compared to control. This outcome may be due to the better activity of lecithin: Cholesterol acetyltransferase, which combines free cholesterol, free LDL into HDL and transferred back to VLDL and intermediate density lipoprotein. Reduction in triglyceride level may be due to inhibition of lipolysis and fatty acids do not get converted to triglyceride. 
Results of chloroform and ethyl acetate fraction were less significant in comparison to residual fraction. This may be due to higher concentrations of polyphenols, especially flavonoids in the residual fraction as compared to the other two methods of extraction. Future research on isolation and characterization of these fractions are needed, however phytochemical screening of ethanolic extract confirmed the presence of flavonoids and other poly-phenolic compounds. 
| Conclusion|| |
Residual fraction of ethanolic extract of Hibiscus rosa-sinensis Linn. flowers has significant antihyperlipidemic activity. Hence, it can be utilized as a therapeutic agent or in supportive treatment to existing therapy for the treatment of hyperlipidemia. Further research on isolation and characterization of fractionated compound is future scope of this research.
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[Table 1], [Table 2]