*Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

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#### *Functional Foods - Phytochemicals and Health Promoting Potential*

Gamma oryzanol was unable to react with hydroxyl radicals it was therefore unable to interrupt the reaction with pnitrosodimethylaniline (PNDA). The rate of reduction of nitroblue tetrazolium (NBT) was unaffected by *γ* oryzanol addition. It revealed that in the experimental conditions the compound was unable to scavenge superoxide radicals. Although weaker than alpha tocopherol, *γ* - oryzanol demonstrated dose dependent DPPH scavenging activity. Whether *γ*- oryzanol was pre-existing in the suspension or added exogenously as ethanolic solution, it was ineffective in inhibiting lipid peroxidation. In the light of the above findings, it was confirmed that *γ*-oryzanol is unable to compartmentalize into liposomes. During the evaluation avocado and castor oils were found to be resistant to heat induced lipid peroxidation. Particularly, castor oil proved to be impossible to evaluate in terms of antioxidant activity. In contrast rosa mosqueta oil and grape seed oil were very sensitive to oxidation. The remaining oils displayed average sensitivity to lipid peroxidation. An increase in *γ*- oryzanol concentration showed an increase in AI values of all the samples. AI is the antioxidative index calculated by ITs induction period of oil with the addition of antioxidant and ITo induction period of oil alone.

Free radical scavenging action of *γ*- oryzanol as well as its preventative nature against lipoperoxidation offer it as a viable contender for natural use as an antioxidant. *γ*- oryzanol offers a dose-dependent increase in induction time (of maximum capacity) while simultaneously lending protection from lipid peroxidation brought about by means of heat and O2 exposition. This particular trait was most notable in cases of oils rich with polyunsaturated fatty acids (Rosa mosqueta, linoleic acid, grape seed oil).

Although its individual use as an antioxidant proved to be unimpressive, the use of another natural antioxidant along with *γ*- oryzanol can lead to enhancement of antioxidant property. The array of benefits offered in terms of pharmaceutical, cosmetic and food use "of rice bran oil which is rich in Gamma-oryzanol" suggest that *γ*- oryzanol can be studied further as an antioxidant component in complex lipophilic formulations such as ointments/emulsions or as an excipient for topical use.

#### **3.2 Anti-hypercholesteremic activity**

Multiple studies conducted among human beings and animals have shown that oils which constitute saturated fatty acids raise serum total cholesterol (TC) levels as well as low density lipoprotein levels. Wilson *et al*. conducted a study in Golden

#### *Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

Syrian Hamsters by feeding a non-purified hypercholesterolemia diet which comprised of 10% coconut oil and 0.1% cholesterol for a duration of 2 weeks, and separated into 4 groups of 12 in accordance to plasma cholesterol concentrations [7]. Blood samples were withdrawn at the 2 and 8 week marks from food deprived hamsters. At the 10 week mark (time of sacrifice) the aortic tissue was collected by administration of anesthesia. The fecal samples, were obtained towards the last 3 days of their exposure. Following procurement, the fecal samples were freeze dried and grinded prior to observation/analysis.

All the hamsters survived the complete course of the experiment. Hamsters that were fed rice bran oil (RBO) *γ* - oryzanol diets and hamsters that were fed coconut oil, ferulic acid diets displayed no discernable difference in terms of plasma triglyceride (TG) plasma *γ* tocopherol and alpha tocopherol concentrations. Although insignificant, hamsters administered RBO diet exhibited higher plasma lipid hydroperoxides (LPH) concentrations in contrast to hamsters administered ferulic acid and *γ* - oryzanol diets. In spite of having increased vitamin E concentrations and laminating high levels of cholesterol from their body through feces, the Coconut oil fed hamsters were found to have higher levels of aortic TC and free cholesterol in contrast to hamsters fed RBO, Ferulic acid and *γ*- oryzanol diets. The ratio of aortic free cholesterol to the ratio of esters were higher in RBO fed hamsters in contrast to hamsters fed the coconut oil and ferulic acid diets. RBOs contain multiple components, first being plant sterols and *γ*- oryzanol (unsaponifiable component) which contribute greatly to cholesterol lowering. The other component of RBOs i.e., tocotrienols assist in inhibition of cholesterol synthesis.

It was observed that both *γ*- oryzanol and ferulic acid both in concentrations of 0.5% each, lower plasma total cholesterol and non-high-density lipoprotein cholesterol (HDL-C) when compared to control hamsters. Increased excretion of cholesterol and its metabolic products could be the major mechanism utilized by RBOs in lowering of blood cholesterol levels. Although hamsters fed RBO, *γ*- oryzanol and ferulic acid visibly lowered cholesterol accumulation, of the 3, RBO and *γ*- oryzanol displayed a more significant decrease in ester accumulation in comparison with control. The experiment and its observations imply that at uniform dietary levels *γ*oryzanol has better impact on both increase of plasma HDL-C and decrease of plasma non HDL-C in when compared to ferulic acid.

#### **3.3 Anti-diabetic activity**

An individual with type 2 Diabetes mellitus is likely to experience an increased rate of mortality as the result of cardiovascular diseases. Studies conducted at random within controlled environment have suggested that lipid lowering substances significantly reduce risk of cardiovascular diseases.

*γ*- oryzanol induces hypolipidemic action as well as influences reduction of aortic fatty streak formation. Palm oil was observed to significantly reduce plasma cholesterol as well as trigger growth of aortic cholesterol in relation to coconut oil within hamsters. Palm oil also tends to reduce serum lipids within healthy individuals as well as oxidative stress in rats.

The study conducted by Cheng *et al.* evaluated the impact of an effective component in RBO and *γ* oryzanol on insulin resistance and lipid metabolism within rats induced with type 2 Diabetes and treated with palm oil [8]. Diabetes was induced in Wistar rats by means of intraperitoneal injection consisting of streptozotocin, 15 minutes followed by another injection of nicotinamide. The rats are divided in three groups of 8, first group being the control, the second, Palm oil group (PO) and third group was treated with Palm Oil and *γ*- oryzanol (POO). After administration of diet for 5 weeks the diabetic rats were withheld from

consumption of food overnight (12 hours) and anesthetized by ether. The rats were then sacrificed by exsanguination from abdominal aorta. The plasma was then isolated by means of collection and centrifugation of blood. Plasma glucose level, triglycerides, HDL-C, LDL-C, non-esterified fatty acid (NEFA) concentration were evaluated by spectrophotometric means.

The diet had no impact on weight gain and neither did it display any side effects (diarrhea/death). No rats were dead as a result of T2DM induced by means of injection. The LDL-C concentration increased in PO groups instead of control. LDL-C in POO groups decreased when compared to PO groups. HDL-C increased in POO groups more than that of PO groups. Total cholesterol TC/HDL-C ratio was lower in POO groups than the other groups. Triglyceride concentrations were observed to increase in PO groups whereas TG concentrations decreased in POO group compared to PO group. At the end of week 5, fecal neutral sterol and bile acid content was notably higher in POO groups in contrast to control and PO groups.

The results gathered imply that PO could impair lipid metabolism in T2DM rats and *γ* - oryzanol a predominant component of RBO stabilizes irregular lipid status. Animals treated with *γ* oryzanol also displayed a 25% reduction in cholesterol absorption in comparison to control group. Secretion of acid and neutral sterols was notably increased in RBO administered animals. The AUC value of insulin in POO group observed prominent reduction compared to PO group. The result dictates that *γ* - oryzanol has tendency to increase sensitivity towards insulin in T2DM rats.

Increase of TG in plasma and liver increases output of glucose while decreasing clearance of insulin thereby promoting gluconeogenesis ultimately resulting in hyperinsulinemia and insulin resistance. Hypotriglyceridemic effect of *γ*- oryzanol positively impacted insulin resistance in T2DM rats. In summary, the plasma LDL-C, TG and hepatic TG all showed a decrease in concentration. The AUCs for glucose and insulin decreased in minimal concentrations within rats. Addition of *γ*oryzanol to PO group minimized the negative impact of PO on lipid metabolism within T2DM rats.

#### **3.4 Effect on male gonads**

Testicular degeneration is a condition prevalent in males of domestic species; it is characterized by reduced fertility as a result of many animals being withheld in unfavorable atmospheric conditions. An increase in temperature levels promotes testicular cellular metabolism which in turn is not met with an increase in oxygen levels, thereby resulting in tissue hypoxia.

Escobar *et al.* conducted an experiment consisting of 8 rams with an average age of 10 months and weight of 35 kg bound in a surrounding with a mean temperature of 26°*C* to attain insulation [9]. The animals were administered a 10% solution of *γ*oryzanol within soybean oil. The animals were divided into two groups. The first being control group that was only administered soybean oil (33 mg/ body weight) orally per day for a month. The second or the test group was administered 10% solution of *γ* - oryzanol in soy bean oil orally. Semen samples were collected weekly by an electroejaculator for 11 weeks and analyzed.

In case of testicular consistency and plasma levels of testosterone, there was no apparent difference between the two treatment groups. After the completion of experimental phase the animals were orchidectomized and samples were utilized to evaluate oxidative stress. The test group was observed to have a significant decline in reactive oxygen species (ROS) levels within their testes (by about 26%) when compared to the control group. In general, between week 5 and week 11, more defects were identified within the sperm of the test group as opposed to the control

#### *Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

group. In case of sperm motility, the largest difference was observed in week 1, with the test group displaying increased motility.

It was observed that during week 2, the test group displayed a decrease in lipid peroxidation (TBARS) levels whereas control group displayed an increase. Simultaneously there was a decrease in total anti-oxidant potential (FRAP) levels in the control group. The group receiving *γ* - oryzanol experienced a decrease in TRAP levels and an increase in the ROS levels in weeks 3 and 9. During weeks 10 and 11, there was an increase in FRAP and TBARS levels respectively in both groups.

The study did help in making the effects of heat stress on the testes and semen of the rams evident as well as reported changes that occurred throughout the duration of the experiment. Though partial protection within oxidative parameters of semen and testes were achieved by administration of *γ*- oryzanol, the experiment did not assist in improving the negative impact of heat stress among the other parameters. In fact, the administration of *γ*- oryzanol resulted in an increase in morphological abnormalities in ram on the whole.

#### **3.5 Hepatoprotective activity**

#### *3.5.1 Acetaminophen induced hepatic injury*

Liver injury because of drug abuse is termed as hepatotoxicity. Acetaminophen (APAP) which is used as an anti-pyretic as well as an analgesic when overdosed can cause acute liver injury, furthermore can lead to liver failure. Natural compounds extracted from food substances such as rice bran oil used as a source of *γ*- oryzanol are utilized for treatment of autonomic dysfunction and menopause syndrome. *γ* - oryzanol is shown to have modulatory effects on metabolic syndrome, while inhibiting oxidative stress and delaying cell aging (senescence).

Shu *et al.* performed experiment in male Kunming mice, aged 6-8 weeks. For assessment of hepatoprotective activity, 40 mice divided into 4 groups of 10 each [21]. First group served as normal, while the second received 300 mg/kg of APAP intraperitoneally, the third group was administered the same dose of APAP combined with 7 mg/kg*γ* -Oryzanol orally daily for a week, lastly the fourth group was administered the same dose of APAP with twice the dose of *γ* -oryzanol given in the third group.

*γ*- Oryzanol showed an undetectable cytotoxic effect on HL-7702. The viability of HL-7702 cells was decreased by APAP. Oryzanol was able to inhibit activation of Caspase-3 by APAP which leads to cell apoptosis. The intracellular accumulation of ROS plays an important role in APAP hepatotoxicity. Oryzanol decreased ROS levels in HL-7702 cells and indicated that oryzanol is capable of reversing APAP induced hepatotoxicity. Nrf2 is a crucial part of signaling pathway in anti-oxidative effect. Oryzanol aided the nuclear translocation of Nrf2, increased mRNA levels and downstream protein levels of Nrf2 like H0-1, NQ01, GCL and GCLM. Key upstream signals AMPK and GSK3B regulate Nrf2 activity, oryzanol upregulated the phosphorylation of both AMPK and GSK3B.

AMPK phosphorylation is one of the essential preceding steps in the nuclear translocation of Nrf2 and AMPK depends on phosphorylation of its substrate GSK3B. To confirm the action through AMPK/GSK3B, the test drug was challenged with the inhibitor of AMPK by compound Compund C (CC). It was observed that CC revoked oryzanol mediated phosphorylation of GSK3B eventually, obstructing the transcription of Nrf2 responsive gene. As a net effect, CC abolished the protective effect of oryzanol in APAP model. This established the fact that activation of AMPK accounts for oryzanol mediated upregulation of Nrf2 in its hepatoprotective action.

Histoarchitecture of liver remained unchanged after treatment with *γ*oryzanol. AMPK/GSK3B/Nrf2 cascade can be activated by *γ*- oryzanol without hepatotoxicity. The liver index and serum levels of ALT, AST and LDH increased due to APAP treatment. *γ*- Oryzanol was able to reduce these parameters on pretreatment. APAP led to loss of hepatocyte architecture, intra-tissue hemorrhage and infiltration of inflammatory cells which were prevented by *γ*- oryzanol.

The number of apoptotic cells in liver increased when exposed to APAP in TUNEL and Hoechst 33258 staining assay and these were reversed by *γ*- oryzanol preadministration. The paracetamol intoxication increased hepatic activities of Caspase 3, 8 and 9. A dose dependent decrease in caspases was observed with the treatment of *γ* - oryzanol in mice liver of APAP. Bcl-2 is an anti-apoptotic protein while Bax is a pro-apoptotic protein. Acetaminophen treatment leads to upregulation of Bax levels and downregulation of Bcl-2 levels. The effect on Bax and Bcl-2 levels was inverted by *γ* - oryzanol.

Exposure of liver to APAP led to increase in MDA and decrease of GSH, total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC). These were enhanced by *γ*- oryzanol. Intrahepatic inflammation is a significant part of hepatotoxicity of APAP. Intrahepatic inflammatory contents- TNF-α, IL-1β, IL-6, and NO significantly increased by APAP. The inflammatory markers were restricted by *γ* - oryzanol. Acetaminophen increased nuclear translocation of p65 of NFκB in the liver. COX-2 and iNOS levels increased after paracetamol intoxication which in turn were suppressed by *γ* - oryzanol.

#### *3.5.2 Ethanol induced liver toxicity*

Ethanol consumption leads to liver injury by inducing hepatotoxicity, oxidative stress and a decrease in antioxidant levels. A therapeutic approach for treating ethanol induced hepatotoxicity is fairly sought after since the liver is among the most essential organs for metabolism of chemical compounds to obtain energy, as well as for detoxification. Trans-ferulic acid and *γ* - oryzanol exhibit certain physiological activities such as inhibition of tumor promotion, reduction of serum cholesterol levels, as well as antioxidant properties in several models.

Chotimarkorn and Ushio conducted a study to evaluate the effect of *γ*- oryzanol on ethanol induced liver injury in male C57BL mice. The investigation was carried out by administering *γ*- oryzanol in ethanol at the dose of 5.0 g/kg, p.o. for 30 days [11]. The experiment consisted of six groups, each group containing 15 mice. Group 1 served as a normal control and received distilled water (5.0 g/kg); group 2, negative control received ethanol (5.0 g/kg); test groups 3 and 4 were treated with trans-ferulic acid and *γ*- oryzanol respectively at the concentration of 0.025 mmol with ethanol (5.0 g/kg). The positive control groups 5 and 6 received trans-ferulic acid and *γ*- oryzanol respectively at the dose of 0.025 mmol alone. At the end of the treatment period animals were sacrificed, livers were removed and homogenated for the estimation of AST, ALT, GSH, protein, SOD, TBARS and lipid hydroperoxide by fluorescent imaging. Coadministration of trans-ferulic acid or *γ*- oryzanol with ethanol exhibited potent inhibition of ethanol stimulated lipid peroxidation or oxidative stress in liver. High increase in 3- PeDPPO in ethanol treated C57BL mice liver reflected high levels of lipid peroxidation. Low intensities of 3-PeDPPO was observed in *γ* - oryzanol treated group indicating low levels of lipid peroxidation. A significant decrease in lipid peroxide level in hepatic tissue of ferulic acid or *γ*- oryzanol treated mice was observed. Similarly, a significant decrease in TBARS level was seen. This demonstrated antioxidant effect of *γ*- oryzanol. However, the mechanism is unclear.

*Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

Gamma oryzanol or trans-ferulic acid maintain GSH levels. The coadministration significantly rose levels of GSH and SOD activity. A similar increase in SOD activity in macrophage cell line RAW 264.7 cells is reported [22]. Abnormally high level of serum aspartate and alanine transaminases in ethanol treated mice was reduced by trans-ferulic acid and *γ*- oryzanol. In the earlier studies, *γ*oryzanol has exhibited antioxidant properties in *in-vitro* model systems namely – in cholesterol oxidation by 2,20 -azobis 2-methylpropionamidine, porcine retinal homogenate oxidation accelerated by ferric ion, pyrogallol autooxidation and pharmaceutical oils [12, 23–25]. In short, *γ*- oryzanol showed high hepatoprotective effect by preserving the livers from chemically induced injury.

Administration of daily dose of ethanol to mice resulted in visible increase in serum enzymes AST and ALT with reference to normal control, trans-ferulic acid, *γ* - oryzanol, co-administration of trans ferulic acid and *γ* - oryzanol with ethanol for 30 days. Co-administration of Trans-ferulic acid/*γ* - oryzanol to mice with ethanol for 30 days showed potent inhibition of ethanol stimulated lipid peroxidation and oxidative stress in the liver.

Trans-ferulic acid and *γ* - oryzanol reduced AST and ALT activities of ethanol. The observed significant decrease in the activity of these enzymes suggests that trans-ferulic acid and *γ* - oryzanol protects against liver injury resulting from the toxic effect of daily dose of ethanol. Furthermore, Trans-ferulic acid and *γ* - oryzanol treatment improved the antioxidative response of the liver defense system. Mechanisms for activation or induction of SOD were investigated. The study demonstrated that oral administration of trans-ferulic and *γ* - oryzanol exerted a protective action on liver injury induced by chronic ethanol ingestion.
