Aop01512.indd

Revista Brasileira de Farmacognosia Brazilian Journal of Pharmacognosy Anti-hyperlipidemic activity of oryzanol,
isolated from crude rice bran oil, on Triton
WR-1339-induced acute hyperlipidemia in
Somsuvra B. Ghatak, Shital J. Panchal*
Department of Pharmacology, Institute of Pharmacy, Nirma University, India. Abstract: Experimental studies carried out for evaluating the anti-hyperlipidemic
Received 15 Jul 2011 Accepted 1 Nov 2011 properties of rice bran components have given interesting but often contrasting results. Therefore, the current study was initiated to investigate the anti-hyperlipidemic activity of oryzanol (OZ), a commercially-important bioactive phytochemical, isolated from crude rice bran oil (cRBO). OZ was isolated by a two-step solvent crystallization process from cRBO, which was extracted from fresh rice bran by hexane mediated soxhlet extraction. Subsequently, OZ (50 and 100 mg/kg, p.o.) was evaluated for anti-hyperlipidemic activity in Triton WR-1339-induced acute hyperlipidemic albino rats by estimating serum triacylglyceride (TG), total cholesterol (TC), very low density lipoprotein- cholesterol (VLDL-C), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) levels with atorvastatin as the reference standard. The degree of protection was also assessed by measuring the levels of various hepatic anti- oxidant enzymes. OZ evoked a signifi cant decrease in the levels of serum cholesterol, triacylglycerides, LDL, VLDL and a signifi cant increase in the level of serum HDL and hepatic anti-oxidant enzymes. It also showed a signifi cant ameliorative action on elevated atherogenic index (AI) and LDL/HDL-C ratios. These fi ndings indicate that OZ possesses the potential to lower plasma lipid concentrations and might be of therapeutic benefi t in hyperlipidemia and atherosclerosis.
prevention of cardiovascular diseases, that a global interest has been generated in recent years pertaining to Experimental and epidemiological studies have the benefi cial nutritive effects of bioactive phytochemicals suggested that hyperlipidemia is a highly predictive risk like OZ, obtained from crude rice bran oil (cRBO) (Cicero factor for atherosclerosis, coronary artery disease (CAD), & Gaddi, 2001).
and cerebral vascular diseases, the primary causes of RBO has been the focus of surmount attention mortality in the developing countries like India (Ghatak because of its balanced fatty acid profi le and rich source & Asthana, 1995). The allopathic hypolipidemic drugs, of bioactive phytoceuticals such as, OZ, tocopherols, although available at large in the market, their popularity phytic acid, lecithin, inositol, waxes etc. OZ is an has been marred by numerous side effects, severe important natural constituent of cRBO with its content contraindications and exuberant cost and this has further ranging from 1.5 to 2.9% (Seetharamaiah & Prabhakar, necessitated the search for alternatives (Speight, 1987). 1986). Its fundamental molecular structure is the Furthermore, published reports have ascertained the ferulic acid aromatic phenolic nucleus esterifi ed to isolation of certain nutrients that can be used as dietary cyclopentanperihydrophenanthrene. OZ, although initially supplements to meet up with the dietary imbalances in presumed to be a single component, it was shown to be order to reduce the cardiovascular disease risk associated a mixture of ferulic acid (4-hydroxo-3-methoxycinnamic with hyperlipidemia (Cicero & Derosa, 2005). For acid) ester with phytosterol or triterpene alcohols. instance, there are ample evidences elucidating the anti- Major portions of OZ include cycloartenyl ferulate, 24- hypercholesterolemic effect of fi xed oils that are rich methylene cycloartanyl ferulate and campesteryl ferulate in polyunsaturated fatty acids, especially linolenic and (Metwally et al., 1974). OZ is an anti-oxidant compound that has been associated with decreasing platelet It is in this context of developing alternative, aggregation, increasing the muscle mass and treating effective and better anti-hyperlipidemic drugs for the nerve imbalances and the disorders of menopause. It has Anti-hyperlipidemic activity of oryzanol, isolated from crude rice bran oil,
on Triton WR-1339-induced acute hyperlipidemia in rats
Somsuvra B. Ghatak and Shital J. Panchal
also been shown to exhibit anti-aging effect similar to published previously (Ghatak & Panchal, 2010). tocopherols, anti-dandruff and anti-itching properties and to cause an improvement of capillary action of blood Isolation of OZ from cRBO vessels (Patel & Naik, 2004). In the last couple of decades, numerous studies The isolation of OZ from cRBO was achieved have been carried out to elucidate the effect of RBO and by a two-step crystallization process previously described OZ activity on lipid metabolism and oxidation in rats, by Zullaikah et al. (2009) with some modifications. rabbits, hamsters, monkeys and humans. While most of In the first step of isolation, the OZ-rich product was these studies have confirmed the OZ component of RBO concentrated in the liquid phase by solvent crystallization to be responsible for the cholesterol lowering property using methanol/acetone (7:3, v/v). In the second step, the in chronic hyperlipidemia models using high fat diet, the OZ-rich product was kept at ambient temperature for 24 h. studies carried out on the rats have yielded interesting After that, hexane was added as an anti-solvent and kept but often contrasting results (Cicero & Derosa, 2005). at 5±1 ºC for 48 h. Under optimal operational conditions, Furthermore, there is paucity of reports on the potential supplemented by considerable savings of both time and effects of OZ on acute hyperlipidemia using Triton WR- solvents, white OZ crystals with a purity of 94.07% and 1339, a non-ionic detergent (oxyethylated tertiary octyl recovery of 55.61% were obtained.
phenol formaldehyde polymer) that has been widely used to produce acute hyperlipidemia in animal models in Experimental animals order to screen natural or chemical drugs and to study cholesterol and triacylglycerol metabolism (Zeniya & Adult wistar rats of either sex weighing 250- 300 g were procured from the central animal facility In light of the above perspective, the current of the Institute of Pharmacy, Nirma University, investigation was carried to assess the anti-hyperlipidemic Ahmedabad. The animals were maintained at controlled activity of OZ in triton WR-1339-induced acute temperature as well as humidity and fed with standard hyperlipidemia in rats. diet and water provided ad libitum. The experimental protocol was approved by Institutional Animal Ethics Materials and methods
Committee (IAEC) of Institute of Pharmacy, Nirma University, as per the guidance of committee for the Drugs and chemicals purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Rice bran was procured from Suryodaya Rice Empowerment, Government of India. Test doses of OZ Mills, Ahmedabad, Gujarat through their milling process. were selected for rats (50 and 100 mg/kg b. w., p.o.) Triton WR-1339 was purchased from Sigma-Aldrich based on the published literature reports (Sakamoto et Chemie GMBH. Atorvastatin calcium was obtained as a al., 1987; Nakayama et al., 1987).
gift sample from Troikaa Pharmaceuticals Limited, India. Thiobarbituric acid was purchased from Spectrochem Treatment protocols Pvt. Ltd., India. All diagnostic kits were purchased from Lab Care Diagnostics Pvt. Ltd., India. All solvents and Rats (n=36) were randomized into the following reagents were either of HPLC grade or of analytical groups: reagent grade and were obtained from commercial 1) Normal Control (NC): Received no treatment. 2) Vehicle Control (VC): Administered with 4% Tween- 80 solution per day for 21 days. Extraction of RBO 3) Triton WR 1339 treated group (TRIT-C): 4% Tween- 80 solution per day for 21 days and on the 21st day intra- Fresh rice bran obtained from the local rice mill peritoneal injection of Triton WR 1339 (400 mg/kg in was stored in a refrigerator before use. Crude RBO was saline) was given.
extracted from rice bran (50 g) by soxhlet extraction for 4) Oryzanol 50 mg/kg+Triton WR 1339 (OZ-50): 3 h using hexane as the solvent. The extracted crude oil Oryzanol 50 mg/kg/day, p.o in 4% Tween-80 solution was stored at -5 ºC and subsequently analyzed for the was administered for 21 days and on the 21st day intra- various physicochemical parameters, such as organoleptic peritoneal injection of triton WR 1339 (400 mg/kg in characters, specific gravity, viscosity, moisture content, saline) in saline was given.
saponification value, unsaponifiable matter, wax content, 5) Oryzanol 100 mg/kg+Triton WR 1339 (OZ-100): iodine value, acetyl value, acid value, hydroxyl value, Oryzanol 100 mg/kg/day, p.o in 4% Tween-80 solution ester value and peroxide value using various standard was administered for 21 days and on the 21st day intra- official methods, the results of which have already been peritoneal injection of triton WR 1339 (400 mg/kg in Rev. Bras. Farmacogn. / Braz. J. Pharmacogn. Anti-hyperlipidemic activity of oryzanol, isolated from crude rice bran oil,
on Triton WR-1339-induced acute hyperlipidemia in rats
Somsuvra B. Ghatak and Shital J. Panchal saline) in saline was given.
The activities of anti-oxidant enzymes like 6) Atorvastatin 2 mg/kg+Triton WR 1339 (Ator): reduced glutathione and the extent of lipid peroxidation Atorvastatin (2 mg/kg/day, orally in 0.5% CMC) was were assayed in the liver homogenate as per previously administered for 21 days and on 21st day intra-peritoneal established methods (Moron et al., 1979; Ohkawa et al., injection of triton WR 1339 (400 mg/kg in saline) was 1979; Lowry et al., 1951). After 24 h of triton injection or in case of control Statistical analysis after 24 h of vehicle or drug treatment, blood samples were collected from the retro-orbital plexus of rat.
All the values are expressed as mean±SEM. Statistics was applied using Graph Pad Prism version 5.0 Estimation of biochemical parameters for Windows, Graph Pad software, San Diego, California, USA. One way ANOVA followed by Tukey's multiple Collection of serum comparison test was used to determine the statistical significance between various groups. Differences were The blood samples were withdrawn from retro- considered to be statistically significant when p<0.05.
orbital plexus under light ether anesthesia without any anticoagulant and allowed to clot for 10 min at room Results
temperature. It was centrifuged at 2500 x g for 20 min. The serum obtained was kept at 4 °C until used.
Effects on serum lipid profile Isolation of liver The normal control and vehicle control animals showed nearly similar lipid profile. Treatment with triton All the animals were euthanasiously sacrificed by WR 1339 showed a significant increase in serum TC, cervical dislocation. Liver was collected and was blotted LDL-C, TG and VLDL-C when compared to the normal free of blood and tissue fluids. Then it was weighed on control animals. A significant decrease in HDL-C was balance and the relative weight was calculated.
observed in the triton WR 1339 control animals. A dose dependant reduction in the levels of TC, TG, LDL-C and Preparation of the tissue homogenate for enzyme VLDL-C was observed with 50 mg/kg and 100 mg/kg dose of OZ. A significant increase in HDL-C levels was seen in the animals pretreated with 100 mg/kg/day of OZ Liver, kept in cold conditions (pre-cooled in (Figure 1).
inverted petridish on ice) was removed. It was cross chopped with surgical scalpel into fine slices and was Effects on atherogenic index, HDL-C ratio and LDL-C/ chilled in the cold 0.25 M sucrose, quickly blotted on HDL-C ratio (Coronary disease risk factors) a filter paper. The tissue was minced and homogenized in 10 mM Tris-HCl buffer, pH 7.4 (10% w/v) with 25 Atherogenic index (A.I.), an indicator of the strokes of tight teflon pestle of glass homogenizer at a deposition of foam cells or plaque or fatty infiltration speed of 2500 x g. The clear supernatant was used for or lipids in aorta and liver, was found to be higher in other enzymes assays.
the Triton WR 1339 group as compared to the normal and vehicle control group. Other coronary disease risk Parameters assessed in serum predictor indices such as HDL ratio and LDL-C/HDL-C in the Triton WR 1339 group were also higher as compared In vitro quantitative determinations of the to the groups pre-treated with OZ (Table 1).
activity of TC, TG, LDL-C, HDL-C concentration in serum were carried out using standard enzymatic kits Effects on lipid peroxidation and anti-oxidant enzyme (Lab Care Diagnostics, India). The concentration of level VLDL-C was estimated as per the method proposed by Russell et al. (1990). Various other coronary disease Malondialdehyde level was measured as an risk factors such as atherogenic index (A.I.), HDL ratio index of lipid peroxidation in the liver homogenate. Triton and LDL-C/HDL-C ratio were also evaluated as per WR 1339 controlled animals showed a malondialdehyde previously reported methods (Sheela & Augusti; 1995; (MDA) level (nmol/mg protein) significantly higher Sharma et al., 1995).
than that observed in normal and vehicle control groups. Atorvastatin 2 mg/kg and OZ 50 and 100 mg/ Parameters assessed in the liver homogenate kg significantly reduced the MDA level. A remarkable depression was seen in the levels of antioxidant enzyme- Rev. Bras. Farmacogn. / Braz. J. Pharmacogn. Anti-hyperlipidemic activity of oryzanol, isolated from crude rice bran oil,
on Triton WR-1339-induced acute hyperlipidemia in rats
Somsuvra B. Ghatak and Shital J. Panchal
Figure 1. Effect of oryzanol pre-treatment on (a) total cholesterol levels (b) triacylglyceride levels (c) very low density lipoprotein
cholesterol (VLDL-C) levels (d) low density lipoprotein cholesterol (LDL-C) levels and (e) high density lipoprotein cholesterol
(HDL-C) levels; n=6; Values are expressed in mean±SEM; NC: Normal control (no treatment); VC: Vehicle control (4% Tween-
80), orally per day for 21 days; TRIT-C: 4% Tween-80 solution per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p.
injection on the 21st day; OZ 50 mg/kg: Oryzanol (50 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p.
injection on the 21st day; OZ 100 mg/kg: Oryzanol (100 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single
i.p. injection on the 21st day; Ator: Atorvastatin (2 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p.
injection on the 21st day. *p<0.05 versus NC, $p<0.05 versus VC, #p<0.05 versus TRIT-C; Values are obtained by one way ANOVA
followed by Tukey's multiple comparison test.
Rev. Bras. Farmacogn. / Braz. J. Pharmacogn. Anti-hyperlipidemic activity of oryzanol, isolated from crude rice bran oil,
on Triton WR-1339-induced acute hyperlipidemia in rats
Somsuvra B. Ghatak and Shital J. Panchal reduced glutathione (μg/mg protein) in the triton WR Discussion
1339 treated group as compared to the normal control. Pre-treatment with OZ 50 mg/kg and 100 mg/kg showed There are ample scientific evidences to a significant dose-dependent increase in the levels of demonstrate the hypocholesterolemic effects of rice reduced glutathione (Figure 2).
bran in experimental animals and humans in long term experimental models of hyperlipidemia, induced Table 1. Effect of oryzanol pre-treatment on atherogenic by high fat diet. Such effects may be attributed to the
index, high density lipoprotein cholesterol (HDL-C) ratio and unsaponifiable fraction of rice bran oil, primarily
low density lipoprotein cholesterol/high density lipoprotein phytosterol, tocols (tocopherols and tocotrienols), OZ,
cholesterol (LDL-C/HDL-C) ratio.
triterpene alcohol and other minor compounds (Sharma & Rukmini, 1986). Although the exact mechanism of action of rice bran and the derived oil on lipid metabolism is not completely evident as yet, the possible fundamental anti- atherosclerotic role rests on OZ (Kanbara et al., 1992).
The reduction of TC by OZ was associated with a decrease in its LDL fraction, which is a potentially modifiable risk factor of cardiovascular diseases and the target of several hypocholesterolemic therapies. The findings of the present study suggest that cholesterol- lowering activity of OZ can result from the enhanced n=6; Values are expressed in mean±SEM; NC: Normal control (no catabolism of LDL-C through hepatic receptors for final treatment); VC: Vehicle control (4% Tween-80), orally per day for 21 elimination in the form of bile acids (Khanna et al., days; TRIT-C: 4% Tween-80 solution per day for 21 days+Triton WR 2002).
1339 (400 mg/kg), single i.p. injection on the 21st day; OZ 50 mg/kg: Increased levels of HDL after administration Oryzanol (50 mg/kg), orally per day for 21 days+Triton WR 1339 (400 of OZ indicated its potent protective action against mg/kg), single i.p. injection on the 21st day; OZ 100 mg/kg: Oryzanol atherogenesis since an independent inverse relationship (100 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), between blood HDL-C levels and cardiovascular risk single i.p. injection on the 21st day; Ator: Atorvastatin (2 mg/kg), orally incidence has been documented widely (Malloy & Kan, per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p. injection 1994). The underlying mechanism may be attributed to on the 21st day. *p<0.05 versus NC, $p<0.05 versus VC, #p<0.05 versus the enhancement of lecithin cholesteryl acyl transferase TRIT-C; Values are obtained by one way ANOVA followed by Tukey's (LCAT), which plays a key role in incorporating the free multiple comparison test.
cholesterol into HDL and transferring back to VLDL Figure 2. Effect of oryzanol pre-treatment on A. lipid peroxidation (malondialdehyde level); B. anti-oxidant enzyme level (reduced
glutathione) n=6; Values are expressed in mean±SEM; NC: Normal control (no treatment); VC: Vehicle control (4% Tween- 80), orally per day for 21 days; TRIT-C: 4% Tween-80 solution per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p. injection on the 21st day; OZ 50 mg/kg: Oryzanol (50 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p. injection on the 21st day; OZ 100 mg/kg: Oryzanol (100 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p. injection on the 21st day; Ator: Atorvastatin (2 mg/kg), orally per day for 21 days+Triton WR 1339 (400 mg/kg), single i.p. injection on the 21st day. *p<0.05 versus NC, $p<0.05 versus VC, #p<0.05 versus TRIT-C; Values are obtained by one way ANOVA followed by Tukey's multiple comparison test. Rev. Bras. Farmacogn. / Braz. J. Pharmacogn. Anti-hyperlipidemic activity of oryzanol, isolated from crude rice bran oil,
on Triton WR-1339-induced acute hyperlipidemia in rats
Somsuvra B. Ghatak and Shital J. Panchal
or Intermediate Density Lipoproteins (IDL), which is be an effective scavenger of free radicals (Xu & Godber, taken back by the liver cells (Devi & Sharma, 2004). 2001).
Additional mechanism may involve an inhibition of It is possible that OZ's antihypercholesterolemic hepatic triglyceride lipase (HTL) on HDL which may lead effect is partially due to its sterol moiety, which is partly to a rapid catabolism of blood lipids through extrahepatic split off from the ferulic acid structure in the small tissues (Anila &Vijayalakshmi, 2002).
intestine by cholesterol esterase (Swell et al., 1954). The Moreover, OZ at the dose of 100 mg/kg/day; antiatherogenic action of OZ could also be based on the p.o. significantly suppressed the elevated levels of TG, inhibition of the accumulation of cholesterol-esters within thereby suggesting that OZ is able to restore, at least the macrophages or by the modulation of cholesterol partially, the catabolism of triacylglycerides.
acid esterase and acyl-CoA-cholesterol-acyltransferase Administration of OZ provides a beneficial effect (Rukmini & Raghuram, 1991).
on lipid metabolism in regard to the reduction of AI. In Therefore, the findings obtained from the current fact, the AI was decreased in all the treated groups, with study reinforce the potential anti-hyperlipidemic activity a significant reduction being observed in the OZ 100 mg/ of OZ in an acute hyperlipidemia model in rats, which kg/day, p.o. and atorvastatin 2 mg/kg, p.o. treated group. may be attributed partially to its anti-oxidant activity. Similar results have been reported while investigating the The findings are encouraging for further assessment to hypolipidemic effect of other natural products (Cherng elucidate a more detailed mechanism(s) of action on a & Shih, 2005). Such an ameliorative action may be cellular and molecular level for the anti-hyperlipidemic attributed to the lipid-lowering property of OZ. activity of OZ.
Furthermore, there exists a positive correlation between an increased LDL-C/HDL-C ratio and the Acknowledgement
development of atherosclerosis. Again, the administration of OZ 100 mg/kg/day, p.o. and atorvastatin 2 mg/kg, p.o. The authors wish to thank the Department of significantly suppressed the higher values of LDL-C/ Science & Technology, New Delhi, India for providing HDL-C ratio elucidating the beneficial effect of OZ in financial assistance (INSPIRE Fellowship: JRF preventing atherosclerosis incidence.
Professional) for carrying out this work.
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