|Year : 2015 | Volume
| Issue : 2 | Page : 31-36
Anti-hyperlipidemic activity of Crataeva nurvala Buch-Hum ethanolic extract fractions
Mukesh Singh Sikarwar1, MB Patil2
1 Department of Pharmacognosy and Phytochemistry, Karnatak Lingayat Education University College of Pharmacy, Belgaum, Karnataka, India; Unit of Pharmaceutical Chemistry, Faculty of Pharmacy, Asian Institute of Medicine, Science and Technology University, Kedah, Malaysia
2 Department of Pharmacognosy and Phytochemistry, Karnatak Lingayat Education University College of Pharmacy, Belgaum, Karnataka, India
|Date of Web Publication||25-Feb-2015|
Dr. Mukesh Singh Sikarwar
Faculty of Pharmacy, Asian Institute of Medicine, Science and Technolog University, Semeling - 08100 Bedong, Kedah, Malaysia
Source of Support: None, Conflict of Interest: None
Aim: This study investigates the anti-hyperlipidemic effect of Crataeva nurvala Buch-Hum ethanolic extract fractions in triton and atherogenic diet-induced hyperlipidemic rats. Methods: Oral administrations of 500 mg/kg body weight of various fractions of selected plant were evaluated for possible hyperlipidemic activity in triton and atherogenic diet-induced hyperlipidemic rats for duration of 48 hours and 14 days, respectively. A comparative assessment was also made between the actions of selected drug with known anti-hyperlipidemic drug simvastatin. The outcomes of the study were expressed as mean± standard error (SE) and data was evaluated by using analysis of variance (ANOVA), followed by Dunnett's t-test for multiple comparisons. Results and Discussion: Ethanolic extract Ethyl acetate fraction of Crataeva nurvala stem bark 500 mg/kg body weight on oral administration exhibited a significant reduction (P < 0.01) in serum lipid parameters like triglycerides, total cholesterol, low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and increase in high-density lipoprotein (HDL) in hyperlipidemic rats as compared with hyperlipidemic control in both models. Conclusion: Our results demonstrated that Crataeva nurvala Buch-Hum ethanol extract ethyl acetate fractions possessed significant anti-hyperlipidemic activity.
Keywords: Crataeva nurvala, ethanolic extract fraction, hyperlipidemia, triton
|How to cite this article:|
Sikarwar MS, Patil M B. Anti-hyperlipidemic activity of Crataeva nurvala Buch-Hum ethanolic extract fractions. Sifa Med J 2015;2:31-6
|How to cite this URL:|
Sikarwar MS, Patil M B. Anti-hyperlipidemic activity of Crataeva nurvala Buch-Hum ethanolic extract fractions. Sifa Med J [serial online] 2015 [cited 2019 Mar 23];2:31-6. Available from: http://www.imjsu.org/text.asp?2015/2/2/31/152114
| Introduction|| |
Increased plasma lipid levels mainly 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 sequel of thrombosis and infraction. Hyperlipidemia in the modem age is caused by over-ingestion of alcohol or foods. 
Crataeva nurvala Buch-Hum (Capparidaceae) is a deciduous tree with palmately compound leaves each typically bearing three leaflets. Leaves are trifoliolate, leaflets 7.5-15.5 cm long, 3.5-7.5 cm wide, ovate or ovate-lanceolate, acuminate, entire, glabrous, subcoriaceous pale beneath. Thickness of bark varies usually 1-1.5 cm according to the age and portion of the plant from the bark is removed, outer surface, grayish to grayish brown with ash gray patches, at place surface rough due to number of lenticels, shallow fissures and new vertical or longitudinal ridges, innermost surface smooth and creamy white in color, fracture tough and short, odor, indistinct, taste slightly bitter. Root bark contains saponins and tannins particularly lupeol, lupeol acetate, d-spinasterol acetate, taraxterol, 3 epilupeol, β- sitosterol, lupenone, betulenic acid diosgenin. Fruits found to have glucocapparin. The drug is bitter, stomachic, tonic, laxative, vesicant, anthelmintic, in treatment of chest and blood related diseases. It promotes appetite, increase the secretion of bile and removes urinary disorders. It has diuretic and anti-lithic properties and is largely employed in urinary gravel and calculi. Bark is especially useful in treatments of urinary complaints hypercrystalluria, hyperoxaluria, hypercalciurea, bladder stones, fever, relieve vomiting and symptoms of gastric irritation. ,,
Wal et al. 2010 study revealed the most active compound 3a, 4a and 5a exhibited minimum inhibitory concentration (MIC) 10 μg/ml. Activity of lupeol improved to a level of 10 μg/ml. This evidently exhibited that fluorine group introduction in quinoline augmented the activity while other substituent in quinoline ring did not show any activity as compared to lupeol.  Haque et al. 2008 separated two triterpenoids using repeated chromatography over silica gel from an ethyl acetate extract of the stem bark.  Agarwal et al. 2010 study on Varuna (Crataeva nurvala) suggested that the decoction of Varuna is effective in the management of urolithiasis.  Varalakshmi et al. 2000 study reported the anti-oxidant property of lupeol. Lipid peroxides level showed a significant decrease indicating nephro protective action of the drug. 
Scientific data on the anti-hyperlipidemicpotential of Crataeva nurvala ethanolic extract fractions is not available except in Ayurvedic treatise.  Hence, the present study was carried out in order to evaluate its anti-hyperlipidemic effect.
| Materials and Methods|| |
Crataeva nurvala Buch-Hum stem bark was collected in July 2012 from local forest area in Western Ghats, Karnataka. Plant was authenticated by the Botanist Prof. G. S. Naik, Department of Botany, G. C. Science and Art College, Ankola and a herbarium voucher accession number GCSAC/CN/01 was submitted. The selected crude drug stem bark were dried and coarsely powdered which was stored in an airtight container at room temperature.
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.
Experimental hyperlipidemic agent
Triton-WR 1339 (SDFine 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 (150-200g) of either sex were selected and housed in the animal house with 12-hour light and 12-hour 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. All the animal experiments were conducted according to the ethical norms approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of social justice and empowerment, Government of India and ethical clearance was granted by institutional ethical committee in resolution no. 1/18/2007 held on 23rd November 2007 at J N Medical college, Belgaum (Ethical committee IAEC reg. no.: 627/02/a/CPCSEA).
Extraction and fractionation of plant material
Crataeva nurvala dried stem bark powder (2.5 kg) were extracted with 95% ethanol and water by following simple maceration procedure at room temperature for 1 week with occasional shaking and stirring. 
For chloroform fraction, the required quantity of polar extract was dissolved in water and 5 volume of dissolved extract 10 volume of chloroform were added. The chloroform soluble components were extracted by gentle shaking. The chloroform fraction was filtered and evaporated to dryness.
For ethyl acetate fraction, the required quantity of polar extract was dissolved in water and 5 volumes of dissolved extract 10 volumes of ethyl acetate were added. 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 residual fraction. 
High-performance thin layer chromatography (HPTLC) study
Pre-coated thin layer chromatography (TLC) layer Silica gel 60F254 + 366 plates, size of the plate: 20 × 20 cm.
Support material: Aluminium sheet (0.1 mm thickness).
Application of sample
The application of sample extract in form of band by using CAMAG LINON, an automatic sample application device by keeping band width-9 mm, space-9 mm, sec/μl l-15. Application of sample: 5 μl to 10 μl.
The development has done by keeping the plate in the pre-saturated or preconditioned tank (12 cm height) with mobile phase for 2 hours. Dry the plate by allowing the plate to evaporate the solvent either by keeping in outside atmosphere at room temp or by spraying hot air by air dryer.
Instrument: CAMAG TLC SCANNER IV, densitometric evaluation system with Camag Win CAT software (CAT) software instrument was used for scanning of thin layer chromatogram objects in reflectance or transmission mode by absorbance or by fluorescence at 254 or 366 nm, respectively.
Preparation of dose for dried extracts and standard drugs
Fractions were formulated as 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 the reference standard drug.
Acute oral toxicity studies
The acute oral toxicity studies of extracts were carried out as per the Organization for Economic Co-operation and Development (OECD) guidelines. One-tenth of upper limit dose were considered as the levels for investigation of anti-hyperlipidemic activity. 
Triton-induced hyperlipidemic model
Animals were kept for fasting for 24 hours and were injected a saline solution of Triton 400 mg/kg intra-peritoneally. The plant fractions, at the dose of 500 mg/kg, were administered by using oral route through gastric intubation. The first dose was given instantly after triton injection followed by second dose after 20 hours. After 4 hour of second dose, the animals were used for various biochemical parameters. Blood collection was done by orbital plexus of rat under ether anesthesia. Blood was centrifuged by using centrifuge 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 whereas Group II was positive control and given standard drug simvastatin (10 mg/kg/day p.o.). Group III labeled as hyperlipidemic control and did not receive any treatment except pellet diet. Group IV, V, and VI received different fractions of Crataeva nurvala ethanolic extract at dose of 500 mg/kg/day, p.o.
Diet-induced hyperlipidemic model
In this group, rats were made hyperlipidemic by giving atherogenic diet for 20 days using 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. Blood collection was done by orbital plexus of rat under ether anesthesia. Blood was centrifuged by using centrifuge at 2,000 rpm for 30 min in order to get serum. 
Treatment periods for all these groups were 14 days in atherogenic diet-induced hyperlipidemia and 48 hours 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, VLDL, and HDL using standard protocol method.
The outcomes of the study were stated as mean ± standard error of mean (SEM) and data was analyzed by using one-way analysis of variance test (ANOVA), followed by Dunnett's t-test for multiple comparisons. Values with P < 0.05 were considered significant. 
| Results and Discussion|| |
HPTLC chromatograph of ethyl acetate fraction is shown in [Figure 1] which shows the presence of phytoconstituents partitioned after fractionation. The effect of various fractions, obtained from ethanolic extract of Crataeva nurvala Buch-Hum stem bark were investigated on serum lipids and lipoproteins level of triton induced hyperlipidemic rats and outcomes are expressed as change in serum lipid and lipoprotein levels.
|Figure 1: Effect of Crataeva nurvala Buch-Hum ethanolic extract fractions on serum lipids and lipoproteins levels in diet-induced hyperlipidemic rats|
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As anticipated, the elevation of serum lipid and lipoprotein levels were observed after administration of triton WR1339 which were sustained over a period of study in hyperlipidemic control group and these rats, were given treatment with chloroform, ethyl acetate, and residual fractions of ethanolic extract of Crataeva nurvala Buch-Hum. The results were similar but lees effective with reference drug simvastatin. Significant elevation in serum lipids and lipoproteins in triton-induced hyperlipidemic control (P < 0.01) rats were observed when compared with normal control.
In triton induced model, the ethyl acetate fraction of formulations of ethanolic extract were able to reduce serum lipid level significantly (P < 0.01) as compare to hyperlipidemic control. It significantly reduced total cholesterol; triglycerides, phospholipids, LDL, VLDL, and increased HDL level in comparisons of hyperlipidemic control group. However, results were not equivalent to standard drug. [Table 1] and [Table 2]
|Table 1: Effect of Crataeva nurvala Buch-Hum ethanolic extract fractions on serum total cholesterol, triglycerides, and phospholipids level in triton induced hyperlipidemic rat|
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|Table 2: Effect of Crataeva nurvala Buch-Hum ethanolic extract fractions on LDL, VLDL, and HDL level in triton-induced hyperlipidemic rats|
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In diet-induced model, the ethyl acetate fraction of formulations of ethanolic extract showed significant serum lipid lowering effects in hyperlipidemic rats which brought down total cholesterol; triglycerides, phospholipids, LDL, VLDL, and increased HDL level in comparisons of hyperlipidemic control group. However, results were not equivalent to standard drug. [Figure 2]
|Figure 2: High-performance thin layer chromatography (HPTLC) chromatogram of ethanolic extract ethyl acetate fraction of Crataeva nurvala Buch-Hum at 254 nm (a) Rf values and peak details of HPTLC chromatograph|
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There was 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 Crataeva nurvala Buch-Hum stem bark ethanolic extract fraction (500 mg/kg b.w.) significantly decreased this level and increased HDL level as compared to control. This outcome may be due to the better activity of lecithin: Cholesterol acetyl transferase which combines free cholesterol, free LDL into HDL and transferred back to VLDL and intermediate density lipoprotein. Reduction in triglyceride level may be due inhibition of lipolysis so that fatty acids do not get converted to triglyceride.
Results of chloroform and residual fraction were less significant in comparison of ethyl acetate fraction it may due to high concentration of polyphenols especially flavonoids in ethyl acetate fraction which needs further confirmation by future research on isolation and characterization of these fractions however phytochemical screening of ethanolic extract validated the presence of flavonoids and other poly-phenolic compounds. 
| Conclusion|| |
Ethyl acetate fraction of ethanolic extract of Crataeva nurvala Buch-Hum stem bark has significant anti-hyperlipidemic activity. Hence, it can be utilized as anti-hyperlipidemic therapeutic agent or supportive treatment to existing therapy for the treatment of hyperlipidemia. Further research on isolation and characterization of fractionated compound is future scope of research in this direction.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]