Isolation Pharmacological Evaluation and Formulation Development of a Bio-active Principle from Crude Oryza sativa Bran Oil

Abstract

In recent years, naturally occurring phytochemicals with antioxidant capacity have generated surmount interest for their therapeutic usage against a wide range of pathophysiological conditions, most of which involve oxidative deterioration induced by free radicals. Therefore, the present study was designed to isolate oryzanol (OZ), a bio-active natural antioxidant from crude Oryza sativa bran oil (cRBO) using an optimized technique, followed by its pharmacological investigation, strategic formulation development and subsequent in vivo pharmacokinetic evaluation. The physicochemical properties, fatty acid profile, and tocopherol/tocotrienol composition of the commercially obtained cRBO were comparable to those reported in the literature. The isolated OZ from cRBO by a two-step crystallization process was identified by several analytical techniques. In the present study, oral administration of OZ (50 and 100 mg/kg, p.o.) reduced the blood glucose level in normal and in streptozotocin (STZ)-induced diabetic rats in both single and multidose study (14 days). Oxidative stress produced by STZ was found to be significantly ameliorated by OZ when compared to the control rats. In addition, chronic treatment with OZ for 8 weeks significantly improved the renal function and attenuated the behavioral as well as biochemical changes associated with diabetic nephropathy and neuropathy respectively in a dose-dependent manner. Histopathological observations also evidenced regression in renal pathological alterations with OZ and were comparable to glibenclamide (Gl) (10 mg/kg, p.o.). Further, OZ strongly inhibited the proliferation of colon and liver cancer cells in-vitro by a mechanism that involved concentration dependent cytotoxicity. The predominant form of cell death was likely by the induction of apoptosis. In-vivo findings suggested that chronic administration of OZ (100 mg/kg, p.o.) throughout an entire period of 32 weeks to carcinogenexposed Balb/c mice ameliorated the deleterious effects of 1, 2 dimethyl hydrazine (DMH) mediated aberrant crypt foci (ACF) development, tumor occurrence, and oxidative stress during three different phases (initiation, post-initiation and entire period) of colon carcinoma. Similarly, administration of OZ to N-nitrosodiethylamine (NDEA) induced Balb/c mice for 16 and 32 weeks reduced the liver injury markers, suppressed the hepatic nodular incidence and Abstract vi multiplicity, restored the levels of liver tumor markers, and favorably modulated the liver antioxidant status in a time dependent manner. Histologically, no obvious signs of neoplasia in the colonic and liver tissues were observed in the OZ supplemented DMH/NDEA induced mice and the effects were comparable to that of 5-fluorouracil (5-FU) (25 mg/kg, p.o.). Moreover, in various experimental animal models of immunity, OZ (50 and 100 mg/kg, p.o.) augmented cellular and humoral immune response, and overcame the side effects of cyclophosphamide (CP)-induced myelosuppression. To enhance gastro-intestinal absorption by oral administration, liposome-encapsulated oryzanol (LEO) was prepared by reverse evaporation phase (REV) technique and optimized based on its physicochemical properties. The optimized LEO was stable in suspension form at 4ºC for 90 days and was subsequently examined for its effect on oral bioavailability in rats by comparison of various pharmacokinetic parameters to those of free OZ at 100 mg/kg. Enhanced bioavailability of OZ, coupled with better absorption was evident in case of LEO, thus highlighting its potential as a novel nutrient delivery system.