*3.5.3 CCl4 induced liver damage*

An organism when exposed to chemical agents or undergoes an infection initiates a process of hepatic fibrosis which ultimately leads to chronic liver damage. Carbon tetrachloride is utilized as a hepatotoxin to induce hepatic fibrosis in rodents. Gomes *et al*. conducted a study in adult male Swiss mice [12]. Twenty-four hours after administration of CCl4 animals were anesthetized by sodium phenobarbital. Blood was collected and assessed for markers of hepatic damage. Liver sections were then numerically graded to assess histological features for degree of acute hepatic injury. Enzyme activities and bilirubin levels in plasma were measured. Caspase 3 and Caspase 9 activities were measured using a Caspase Glo- assay kit. Bradford method was employed to measure protein concentration using Bovine serum albumin as a standard. The data of experimental and control groups were compared.

Liver tissue from mice exposed to CCl4 compared to the control group revealed extensive injury, vascular congestion and hepatic fibrosis. *γ*- oryzanol supplementation reduced hepatic fibrosis and reduced degree of liver damage (injuries). No histological changes were noted. Results revealed that CCl4 exposure increased liver peroxidation. Two-way ANOVA of caspase 3 and caspase 9 revealed a significant interaction with the treatment of *γ* - oryzanol and silymarin.

Hepatic fibrosis induced by means of CCl4 is brought about by oxidative, inflammatory and apoptopoic alterations. *γ*- oryzanol supplementation led to intermittent suppression of pathological alterations in the model of hepatic fibrosis. Damage caused in the liver by CCl4 was principally shown as hepatic fibrosis brought about by single administration. The findings indicated that *γ*- oryzanol prevented hepatotoxicity and consequently protected against CCl4 induced hepatic fibrosis. Lipid peroxidation is one of the primary causes of CCl4 induced hepatic fibrosis. The findings reinforced antioxidant potential of *γ*- oryzanol

supplementation. It is implied that one of the mechanisms involved in the hepatoprotective effect of *γ*- oryzanol is the regulation of oxidative stress. Antiinflammatory compounds have the potential to serve as therapeutic agents for various diseases, including, but not limited to, hepatic fibrosis. In the study conducted, it was observed that hepatic inflammatory reactions induced by CCl4 could be suppressed by *γ*- oryzanol. The study demonstrated that CCl4 administration produced a worsening effect by caspases 3 and 9 in the liver of mice, indicating that the apoptopic process had intensified. Several studies have shown that hepatic fibrosis induced by CCl4 can lead to apoptopic pathway *in-vitro* and *in-vivo* [26, 27].

The author suggested that *γ* - oryzanol supplementation prevents CCl4 induced hepatic fibrosis by modulating activity of caspases and that antioxidant action of *γ*oryzanol may be at least partially associated with normalization of apoptopic process. Study demonstrates that *γ*- oryzanol supplementation was able to prevent CCl4 induced hepatic fibrosis in mice by preventing oxidative, inflammatory and apoptopoic modifications.

The hepatoprotective role of *γ*- oryzanol in various models of liver injury are shown in **Figure 3**.

#### **3.6 Effect on hypoadiponectinemia**

The decrease in plasma adiponectin level is integrally involved in the development of insulin resistance and the resulting type 2 diabetes. In adipocyte and myotube model via nuclear factor kB (NF-kB) transcription factor pathways, palmitate, a major fatty acid observed in meat fat such as beef tallow, was known to cause insulin resistance [10].

In a study conducted by Nagasaka *et al.* (2011), serum adiponectin levels in mice were reduced from 48 to 96 h by oral administration of Beef Tallow and palmitate to approximately half the initial levels. Then γ-oryzanol, a major bioactive ingredient in rice bran, was administered and its effects on hypoadiponectinemia were examined [13].

Single dose of 0.5 ml beef tallow (beef tallow group, n = 5), 0.5 ml maize oil (control group, n = 5) or palmitate (0.17 mg/ml maize oil, n = 5) were administered orally to C57BL6j male mice aged 7 weeks and weighing between 18 and 21 g. Separately, 0.5 ml of beef tallow and maize oil containing 0.025 mmol oryzanol were also given. Blood samples were obtained from the caudal vein from 0 to 120 h per 24 h after administration. In order to estimate the total amount of adiponectin monomer secreted from Adipocyte, serum adiponectin levels were estimated. The

#### **Figure 3.** *Hepatoprotective role of γ- oryzanol in various models of liver injury.*

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

immunoreactivity was observed and digitally acquired using an Odyssey Infrared imaging system. Signal intensity for adiponectin was evaluated by Image-J.

In the beef tallow group, the level of adiponectin was significantly suppressed from 48 to 72 h similarly palmitate significantly suppressed serum adiponectin levels from 48 to 96 h after administration. The administration of oryzanol dissolved in corn oil increased adiponectin level from 48 to 72 h gradually and then got the level back almost to the initial level at 120 h. Oryzanol supplementation to beef tallow increased significantly adiponectin levels at 96 h compared to the control group and successfully recovered the hypoadiponectinemia induced by the beef tallow administration. Adiponectin levels at 120 h reverted almost to the initial level, suggesting that effects of the single doses of beef tallow and oryzanol should disappear within 120 h probably due to metabolism or secretion. The rise in the secretion of adiponectin is considered a priority for the development of drugs and the treatment of metabolic diseases associated with obesity.

#### **3.7 Impact on immune system**

Fujimoto *et al.* conducted a study on cycloartenyl ferulate (CAF), a major component of rice bran derived γ -oryzanol as an anti-allergic agent in passive cutaneous anaphylaxis reaction and mast cell degranulation and its effect on IgE [14]. Gamma oryzanol was extracted from domestic Japanese rice using flash chromatography on silica gel. Passive cutaneous anaphylaxis (PCA) reaction carried out as the allergic model in Sprague Dawley rats was used in the study. Mast cell degranulation was estimated by the release of β- hexosaminidase. Light and heavy chains of anti-DNP or anti-TNP were identified by using SDS polyacrylamide gel electrophoresis. The concentrations of the compounds are given in **Table 2**.

The major component of *γ*- oryzanol was discovered to be CAF, having >90% compounds affiliated to CAF. Anti-allergic reaction of *γ*- oryzanol and CAF were found to be alike. The effect of CAF on RBL-2H3 mast cell degranulation was studied to verify that the effect of CAF on PCA reaction. Following DNP-HSA stimulation, anti-DNP IgE sensitized RBL-2H3 cells generated *β*- hexosaminidase. Anti-DNP IgE incubated with CAF was added to RBL-2H3 cells. The degranulation triggered by successive stimulation of DNP-HSA was inhibited in a concentrationdependent manner by CAF. The effect of 24-methylene cycloartanyl ferulate, cyclobranyl ferulate, and *β*- sitosteryl ferulate on mast cell degranulation on affiliation to CAF were studied by purifying the compounds. The results showed that, Cyclobranyl ferulate was more potent that cycloartanyl ferulate in inhibiting degranulation while 24 – methylene cycloartanyl ferulate and *β*-sitosteryl ferulate were found to be less effective.

It was found that CAF significantly inhibited mast cell degranulation. Binding of IgE to mast cells led to the failure of CAF to inhibit the degranulation. The researchers also found that CAF failed to inhibit degranulation once IgE binds


#### **Table 2.**

*The predominant ferulates present in γ- oryzanol.*

to mast cells. This is suggestive of some effect of CAF on the ability of IgE bind with Fc*ϵ*RI.

The concentration of IgE were measured by ELISA. Anti- TNP IgE on incubation for an hour with *γ*- oryzanol or CAF decreased IgE concentration in a dose dependent manner. The effect was also found to be dependent on incubation time. It was also observed that ELISA failed to detect IgE when incubated with CAF.

To confirm whether CAF acted by sequestration of IgE from anti-IgE antibody or IgE configuration change SDS-PAGE analysis was performed. The amount of IgE in the supernatants decreased when IgE was incubated with CAF suggesting CAF's sequestering role on IgE which makes it undetectable in ELISA. The study demonstrated that CAF found in *γ* - oryzanol encapsulates IgE and prevents it from binding to Fc*ε*RI. Thereby, attenuating allergic reaction. The report of Nagasaka et al. supports the immune response of CAF as they found this molecule inhibits NFκb activity preventing the late delayed phase of allergic inflammation [22].

#### **3.8 Anti-inflammatory activity**

Serum lipid levels and pro-inflammatory mediators which are prime factors for cardiovascular diseases are greatly influenced by dietary oils. The study of Rao *et al.* investigated the effect of minor constituent of rice bran oil (RBO), *γ*- oryzanol on secretion of pro-inflammatory mediators by peritoneal macrophages of male Wistar rats [28]. 2 mL of fresh medium Roswell Park Memorial Institute (RPMI)-1640 was added to the macrophages and incubated with LPS. ELISA was used to study the cytokines.

The macrophages from the rats that were fed a diet with rice bran oil with unsaponifiable fraction (RBO-N) gave rise to lower levels of superoxide anion (51%) and nitric oxide (45%) compared to groundnut oil containing unsaponifiable fraction (GNO-N). However, the macrophages from rats fed rice bran oil with minor constituents removed (RBO –MCR) exhibited lower levels of superoxide anion (16%) and nitric oxide (8%) compared to GNO-N fed rats. This suggested that the extraction of unsaponifiable fraction from RBO had compromised potential to reduce the production of reactive oxygen species (ROS) by macrophages.

Lower levels of Prostaglandin E2 (PGE2), Thromboxane B2 (TXB2), Leukotriene B4 (LTB4) and Leukotriene C4 (LTC4), were secreted by macrophages from rats that were fed RBO-N diet compared to GNO-N diet fed rats. RBO-MCR diet fed group secreted lower levels of PGE2 compared to macrophages from rats fed GNO-N. The secretion of TXB2, LTB4 and LTC4 in RBO-MCR and GNO-N or GNO-MCR diet fed rats showed no remarkable differences. This deduced that the removal of unsaponifiables from RBO-N impacted its potential to effect the eicosanoid secretion by macrophages. On the contrary, rats fed RBO-N diet showed an enhanced secretion of 6-keto PGF1a by 36% compared to rats given GNO-N diet.

A decrease in levels of pro-inflammatory cytokines like TNF-*α* (by 65%) and IL-6 (by 40%) was observed in the macrophages of rats fed with RBO diet in contrast to rats fed with GNO diet. TNF- *α* and IL-6 were secreted in lower levels by macrophages from rats that were fed an unsaponifiable removed RBO diet compared to rats fed with GNO diet. Pro-inflammatory response in hosts was also influenced by lysozyme enzymes secreted by macrophages. Lower levels of collagenase, elastase and hyaluronidase by 42%, 43% and 55% respectively were secreted by macrophages of rats fed with RBO diet compared to rats fed with GNO diet. GNO-MCR diet fed rats secreted collagenase, elastase and hyaluronidase in similar levels compared to GNO diet fed rats.

Pro-inflammatory compounds were secreted in lower levels by macrophages of rats fed RBO diet as compared to that observed from rats fed GNO. Reactive oxygen *Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

species, lysosomal enzymes, eicosanoids, cytokines and matrix metalloproteases are over produced by macrophages when activation of NF-kB induces the proinflammatory signaling pathway [29]. The authors demonstrated how macrophages in rats fed RBO containing *γ*- oryzanol secrete less IL-1*β* in contrast to rats fed hydrogenated fat [30]. Expression of adiponectins was up regulated by RBO and down regulated expression of Toll like receptors (TLR-2 and TLR-4). The secretion of inflammatory compounds was lowered suggesting that the removal of unsaponifiables from RBO led to a decrease in the potential of RBO.

#### **3.9 Neuroprotective role**

The various neuropharmacological actions of *γ***-**oryzanol are represented in **Figure 4.**

#### *3.9.1 Anti-parkinsonian activity*

Gamma oryzanol has shown potential in reduction of tumor growth and plasma cholesterol levels [31]. Ferulic acid a constituent of *γ*-oryzanol has protective function towards Alzheimer's, Parkinson's disease and stroke [32]. Parkinson's disease can be induced in Drosophila and rodents models using a chemical, rotenone [33]. Rotenoids consists of a toxic agent called rotenone [34]. Rotenone acts as an inhibitor with high affinity towards mitochondrial NADH dehydrogenase (complex I) [35]. It is suggested that dopaminergic cell death is caused by rotenone. It also causes increase in free radicals and oxidative stress in mitochondria [36].

*Drosophila melanogaster* is used as a genetic tool for studying biological problems because of its similarities with mammals. They have similar biological, physical and neurological properties and 75% of human disease-causing genes [37]. Several studies on neurodegenerative diseases used Drosophila as a model as it shares genetic similarity with humans in related to Parkinson's disease [38].

Araujo *et al*. performed a study using both genders of *Drosophila melanogaster* of age 1 to 5 days by dividing them into four groups of 50 flies each [16]. Control, oryzanol 25 *μ*M, rotenone 500 *μ*M, and oryzanol 25 *μ*M + rotenone 500 *μ*M. The groups were administered a diet containing rotenone and oryzanol for 7 days. Ethanol and sucrose were used as diluents with rotenone (500 *μ*M) and oryzanol (25 *μ*M) respectively. The dose of oryzanol was decided after conducting an experiment using different doses of oryzanol. Doses of 25 *μ*M, 50 *μ*M and 75 *μ*M were

**Figure 4.** *Neuroprotective action of γ- oryzanol.*

checked. After studying mortality and behavioral test negative geotaxia of flies, 25 *μ*M concentration was deemed best for the experiment.

The flies were administered rotenone (500 *μ*M) and oryzanol (25 *μ*M). Two controls were taken one with 1% ethanol and 1% sucrose. Results presented control group with ethanol and sucrose because there was no statistical difference observed in all groups. The diet contained 1% agar w/v, 1% w/w milk powder, 0.08% w/v nipagin, 2% w/v sucrose and 1% yeast w/v beer. During the experimental period, the survival rate of the flies was estimated daily. The time taken to achieve 8 cm height from the base of the glass tube was measured to determine negative geotaxis [39]. Negative geotaxis behavior assay was used to evaluate locomotor activity of the flies [40]. Ice was used to induce anesthesia to 10 flies of both genders. The flies that were unable to climb above the mark were noted. The activity and movement of the fly were estimated by dividing 15 flies with one square cm distance in a covered petri dish [41].

A homogenate of flies was prepared which was later subjected to HPLC to analyze dopamine concentration. The cell viability was estimated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay and the resazurin reduction assay. ELISA (enzyme linked immunosorbent assay) was used for incubation of supernatant and fluorescence was noted. The flies were centrifuged to obtain mitochondria. Spectrophotometry was used with Ellmann reagent to evaluate Thiol protein and non-protein content [42]. Followed by evaluation of reactive oxygen species (ROS) and lipid peroxidation. Catalase activity was determined using method of Aebi [43]. Inhibition of quercetin auto oxidation was used to find the superoxide dismutase activity [44]. 1-chloro-2-4- dinitrobenzene (CDNB) was used according to Habig et al. procedure for the estimation of glutathione-s-transferase activity (GST) [45]. Bradford method was used to estimate protein concentration [46].

The mortality was lower in the group that was administered oryzanol, in contrast to the rotenone group. Hence, suggesting its potential to prevent rotenone induced mortality. An adverse effect was observed on the locomotor behavior on flies given rotenone, which was a decrease in climbing rate compared to the control. This was overcome by the treatment of oryzanol. In the open field test, there was a decrease in exploratory activity compared to control group, preventing a locomotor deficit. Dopamine levels in the head dropped by 42% in flies in the rotenone group compared to control group. Oryzanol prevented the drop in dopamine levels induced by rotenone in flies. Group exposed to rotenone showed MTT reduction and in cell viability observed in the fly homogenates, verifying the reduction in cell viability. Rotenone also led to a decrease in MTT reduction in mitochondria. Oryzanol reversed both the effects.

Resazurin reduction test was used to estimate cell viability. Toxicity of rotenone cell level was verified by decrease in cell. A marker of lipid peroxidation, Malondialdehyde (MDA) was estimated. MDA levels and rotenone induced DCFDA (2<sup>0</sup> ,70 -dichlorodihydrofluorescein diacetate) oxidation was reduced by simultaneous exposure to oryzanol and rotenone. Oryzanol inhibited decrease Superoxide dismutase activity (SOD), catalase (CAT) and glutathione S-transferase (GST) caused by rotenone. The amount of protein thiols and non-protein thiols did not change and remained the same in all groups.

Motor function, dopamine levels and activity of enzyme acetylcholinesterase improved with the treatment of oryzanol. In addition, oryzanol strengthened antioxidant defenses, oxidative stress, mitochondrial dysfunction protecting from rotenone toxicity. The constituent of oryzanol, ferulic acid esters could be responsible for the neuroprotective role and its anti-oxidant ability. Ferulic acid demonstrated anti-oxidant activity in neuronal cell culture and arrested apoptosis in focal cerebral

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

ischemic injury showing neuroprotective action. Ferulic acid on long term administration in mice ameliorated memory deficits induced by centrally administered *β*amyloid [47]. Decrease in expression of active caspase-3 in the rat striatum, increase in interlukin-1 an immunoreactive component, levels of endothelial nitric oxide synthase and 3- nitrotyrosine of mouse hippocampus can be mediated by feruilic acid. It also provides neuroprotection against striatal neuronal cells exposed to oxidized low-density lipoproteins [48]. Neuromotor deficits, geotaxia negative tests (climbing) and open field test (rating exploratory capacity) were successfully reversed by oryzanol.

Rotenone is used to induce symptoms of Parkinson's disease. As Parkinson's disease is related to mitochondrial dysfunction because of anomaly in complex I of electron transport chain, similar symptoms can be induced using rotenone [49]. A decrease in anti-oxidant and an increase in iron levels along with oxidative stress on dopaminergic neurons could be carried out by the inhibition of complex I. Dopamine metabolism involves synthesis, storages, release, reuptake and degradation of neurotransmitter [50]. The flies that were treated with rotenone showed a decrease in dopamine levels. Earlier studies verified that dopamine levels in flies reduce upon treatment with rotenone including loss of dopaminergic neurons in brain and reduction in vesicular monoamine transporter (VMAT) [51, 52]. Oryzanol displayed a neuroprotective effect by preventing the dopamine loss. Further, ferulic acid has shown neuroprotective effect via inhibition COX-2 enzyme, which in turn prevents the oxidation of dopamine and prevents accumulation of *α*- synuclein [53].

Neurodegeneration in Parkinson's disease is also induced by oxidative stress [54]. The increase in MDA, ROS levels and decrease in CAT, SOD and GST during the study confirms that exposure to rotenone causes oxidative stress. Oryzanol was able to reduce oxidative markers thus, confirming its anti-oxidant ability. Biological membranes can be protected from lipid peroxidation, peroxyl and alkoxyl radicals by ferulic acid [55].

Cell viability was reduced when exposed to rotenone and led to increase in mortality of flies. However, oryzanol treatment increased cell viability and reduced mortality. This suggests the presence of bioactive compounds in it that suppress free radicals and contribute to the anti-oxidant defense system. Finally, the study concluded that oryzanol prevented the toxicities caused by rotenone in *Drosophila melanogaster*. Thereby, confirming its neuroprotective role in Parkinson Disease.

#### *3.9.2 Anti-anxiety*

It is believed that chronic stress is correlated to structural degeneration and compromised brain function which could be the reason for increased risk of advancement of neuropsychiatric disorders like anxiety, depression and dementia. In an experiment conducted by Akter *et al*. five-week-old ICR male mice were put through restraint stress by a wire mesh bag (3x6x12) [17]. The mice were subjected to 1st phase chronic restraint treatment for 14 days followed by a recovery phase and second phase for another 5 days. Since, most studies employed 0.5% dose of *γ* oryzanol and found it to be effective, 0.5% *γ* - oryzanol was administered to the mice.

Open field and elevated plus maze tests were used as behavioral tests. Mice were sacrificed at ZT 5.5 to 7.5 post 3 h CRST exposure and EPM test. Samples were drawn and serum was separated after centrifugation for 15 min at 3000 rpm and stored at 80°*C*. Brain matrix was used for excision of hippocampus, cerebral cortex and amygdala. Neuroanatomical landmarks from the brain atlas was used to dissect the brain. ELISA was used to determine levels of serum corticosterone.

The neurochemical mechanisms involved in anxiolytic like effects were studied by estimating the levels of centrally acting monoamine neurotransmitters such as noradrenaline and serotonin and their metabolites 5-hydroxyindole acetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG), in the various dissected parts of the brain by HPLC-ECD following the behavioral tests.

GORZ treatment did not show any significant changes in the behavioral parameters in both the tests. Daily food intake sharply decreased in the stressed mice group during the early days of CRST. However, there was substantial recovery in the later period. As a result of CRST negative control group significantly lost body weight when compared to the normal control and test group. The treatment of 0.5% *γ*- oryzanol reduced such effects indicating that the *γ*- oryzanol treatment prevented the stress induced weight loss.

A mild reversal of CRST induced decrease in time spent in the central zone was observed by the treatment of *γ*- oryzanol. 0.5% oryzanol treatment significantly increased number of entries in the central zone in CRST while slightly increasing the total distance traveled in both the conditions. These are suggestive of the decreasing effect of *γ*- oryzanol on anxiety like behavior. In elevated plus maze test, *γ*- oryzanol significantly increased time spent in open arm and distance traveled in open arm under cold restrained stress. At the same time oryzanol treatment reduced the distance traveled in the closed arm. These observations indicate the anxiolytic effect of *γ*- oryzanol. Serum corticosterone levels rose significantly in control and *γ*oryzanol treated animals in contrast with the unstressed control animals in CRST.

A mild reduction in the levels of neurotransmitters and their metabolites following CRST was observed in the hippocampus and cerebral cortex. CRST led to a decrease in serotonin and 5-HIAA levels with no significant change in amygdala. The slight decrease in serotonin and nor-adrenaline and their metabolites was restored by oryzanol in the hippocampus and cerebral cortex. Under CRST an increase in 5-HIAA, a metabolite of 5-HT was seen in amygdala of mice that received 0.5% of oryzanol. Similarly, an increase in a noradrenaline metabolite (MHPG) was noticed in oryzanol treated group without stress. These facts are implicative of anti-anxiety potential of *γ*- oryzanol in chronic stress conditions. CRST induces morphological alterations in BBB. It also remarkably decreased body weight (b.w.) by inhibiting food intake through the reduction of mRNA expression of food intake related genes like ghrelin, pro-opiomelanocortin in hypothalamus. Gamma oryzanol is indicated to be a weak modulator of stress response in hippocampus. Its anxiolytic effect could be related to the up regulation of centrally acting monoamines in amygdala. The anti-stress and anti-anxiety effects are unrelated to corticosterone activity. In gist, the observations of elevated plus maze test evidence the anti-anxiety effect of oryzanol. The locomotor activity was found to be unaffected.

#### *3.9.3 Anti-Alzheimer*

The neuroprotective and cognitive enhancement effect of *γ*- oryzanol in Alzheimer's disease was investigated by Jha and Panchal [18]. In the study, the researchers performed *in-vitro* DPPH assay, AchE enzyme activity inhibition assay, cell viability assay on SH-SY 5Y cell line and alamar blue assay. The nootropic activity was assessed using Y and radial arm maze. The brain corticular homogenate was subjected to estimation of biochemical markers such as catalase, glutathione, malondialdehyde, brain mitochondrial ATPase, brain acetylcholinesterase activity and C-reactive protein. The slices of brain were finally subjected to amyloid-βplaque staining, immunohistochemistry and histopathology. The IC50 of *γ*- oryzanol was found to be 227.03 17.24 μM in DPPH assay and 34.04 3.20 μM in AchE

inhibitory assay. *γ*- oryzanol has shown a dose-dependent enzyme inhibition by preventing hydrolysis of ATCI from AchE *in-vitro*. *γ*- oryzanol treatment increased cell survival by 1.07-1.104 folds in the cell viability assay at concentrations 100 nM, 1 μM and 100 μM. In both Y and radial arm maze tests, *γ*- oryzanol raised entry in Y maze, exhibited significant increase in total arm entry and correct arm entry, reduced reference as well as working memory errors. Memory score improved with the treatment of Donepezil and *γ*- oryzanol treated animals. *γ*- oryzanol increased the level of brain mitochondrial ATPase (BMA), catalase (CAT) and glutathione (GSH) while significantly decreasing the malondialdehyde (MDA), acetylcholinesterase activity and C reactive proteins (CRP) thereby exhibiting free radical scavenging action in a dose dependent manner and was found to be more potent than ferulic acid.

The histopathological examination revealed regular morphological and cytological characteristics in normal control group and disorganized cellular and morphological architecture, presence of dead cells, loss of neuronal cells in CA1, CA2, CA3 and DG regions, altered thickness (CA1 and CA2), aberrantly scattered CA3 pyramidal cells, cell arrangement in granular cell layer (GCL), dentate gyrus ectal limb (DGEC), dentate gyrus endal limb (DGEN) and loss of neuronal cells in entorhinal cortex (ERC) in disease control group. There were no such observations in Donepezil and *γ*- oryzanol treated brains. Immunohistochemical analysis showed non-significant but marked reduction in Mean GFAP count, decrease in active astroglial cells and inflammation. *γ*- oryzanol group brain sections improved synaptic connectivity, indicated by increase in the mean synaptophysin count in CA1, CA2, CA3, DG and ERC regions. This highlighted protective effect of *γ*- oryzanol in streptozotocin induce cerebral damage.

The qualitative and quantitative analysis of cortical area of brains observed as bright red fluorescence revealed higher mean amyloid-β count in disease control group which was lowered by both Donepezil and *γ*- oryzanol as it decreased plaque formation. The immunohistochemical parameters (GFAP and synaptophysin) expression and amyloid β-12 was inhibited by *γ*- oryzanol. The overall effects of *γ*oryzanol were suppression of neuroinflammation and plaque formation (β-amyloid aggregation) while improving synaptic connectivity and neuronal energy catastrophe in cerebral region preventing neuronal loss. *γ* - oryzanol proved to be beneficial and therapeutic candidate in the experimental model of sporadic Alzheimer disease and demonstrated potent anti-oxidative, anti-inflammatory, cognitive enhancing and amyloidogenesis terminating effects. The researchers recommended further studies and exploration of *γ*- oryzanol use in the neurodegenerative disorder.

#### **3.10 Anti-obesity potential**

As *γ*- oryzanol proved to have the ability to treat hyperlipidemia, hyperglycemia, hypoadiponectinemia, etc., the nutraceutical was considered to be a good candidate for screening in obesity induced kidney injury by Francisqueti *et al*. [19].

Male Wistar were divided into 2 groups, control group and high sugar-fat diet (HSF) group after 20 weeks, the rats were treated with *γ*- oryzanol. Post treatment, the rats caloric intake, body weight and adiposity index were used to estimate their nutritional profile. Glucose concentration, triglycerides and adiponectin were estimated using glucometer, automatic enzyme analyzer system and enzyme-linked immunosorbent assay (ELISA) respectively. Plasma and urine were used to estimate the renal function. Amount of urea and creatinine in plasma was recorded along with the glomerular filtration rate (GFR). The renal tissue was homogenized and centrifuged. ELISA was used to measure tumor necrosis factor - alpha (TNF – *α*),

interlukin - 6 (IL-6) and monocyte chemoattractant protein - 1 (MCP–1) levels. Protein amount was used to verify the results.

The caloric intake showed no change and HSF showed higher values for all parameters. The renal function of the group that was given HSF and *γ*- oryzanol presented lower proteinuria and high GFR. Renal tissue of the group that was administered *γ*- oryzanol showed decrease in inflammatory response unlike the control group. During the study, the group given HSF diet developed obesity, insulin resistance, hypertension, chronic inflammation, dyslipidemia and oxidative stress.

Antioxidant defense is impaired in subjects suffering from renal insufficiency. *γ*-oryzanol treatment exhibited a rise in antioxidant capacity - superoxide dismutase (SOD) and catalase activity. Abnormal levels of adiponectin are seen in obesity, diabetes, chronic kidney disease, etc. The secretion of adiponectin by adipose tissue has a great impact on kidney disease. In contrast to reduced levels of adiponectin when it comes to obesity, chronic kidney disease shows rise in the same. Therefore, the higher levels of adiponectin related to GFR in the HSF fed group helped to deduce kidney disease. On the contrary, the HSF diet group treated with *γ*- oryzanol showed reduced adiponectin levels. The expression of PPAR – *α* increases in tissue with high mitochondrial and *β*oxidation activity. Increased PPAR – *α* expression suggests metabolic control in an organ, and it controls several factors involved in renal damage [56, 57]. Thus, supporting the study to conclude that HSF diet with oryzanol showed no inflammation in kidney, as well as low levels of TNF-*α*, IL-6, and MCP-1.

#### **3.11 Wound healing activity**

*γ* – Oryzanol has both anti-inflammatory and antioxidant properties, which makes it a prospect for wound healing. However, due to the large molecular weight and water insolubility, it is incompatible for topical application. Penetration enhancers containing vesicles were formulated using used transcutol and labrosol in a study conducted by Aldalaen *et al.* [20]. Alpha – bisabolol (BISA) is a derivative of essential oil. BISA is an unsaturated sesquiterpene alcohol. Reports indicate that BISA is employed as a nutraceutical compound in treating wounds. Owing to its anti-inflammatory property, BISA was included as co-penetrator to boost the permeation ability of *γ* - oryzanol. Thus, increasing its antioxidant and antiinflammatory properties.

The formulation was developed using the thin film hydration technique. *γ*-oryzanol (25 mg) and the phospholipid Epikuron (200 mg) were dissolved in a mixture of chloroform: methanol (2:1) v/v. This mixture was then subjected to vacuum evaporation at 40°*C* and 150 rpm. The lipidic film was then hydrated with 10 ml phosphate buffer (pH 7.4) comprising of BISA oil (10 *μ*L) and penetration enhancers (transcutol and labrosol). The vesicular dispersion was treated with rotation for 30 mins at 40°*C* and sonication for 1 hr. Finally, storing it at 4°*C*.

The experiment was conducted in Wistar rats. Intraperitoneal (i.p) ketamine HCL (50 mg/kg) and xylazine HCL (20 mg/kg) were used as anesthetics. The animals were subjected to excision wound model. The posterior of the rats were shaved and cleaned with 70% ethanol. Two circular wounds were induced on the dorsal skin of the rats. A biopsy punch of 20 mm in diameter was utilized for the removal of the skin. The Wistar rats were divided into three groups. The wound on the upper side of the dorsal back was treated while the lower side was left untreated which was used as control. The PEV formulation with *γ*- oryzanol was given to group 1, the PEV formulation with *γ* -oryzanol and BISA was given to group 2, and group 3 was treated with commercial wound healing product (Healosol). Fifty *μ* L of each formulation was applied on the upper side of the wounds every day. The rats were photographed, and *Functional and Therapeutic Potential of* γ*-Oryzanol DOI: http://dx.doi.org/10.5772/intechopen.97666*

the diameter of wounds were measured on the 3,5,7,10,14 and 21 days. The rats were subjected to euthanasia and the tissue formed was cut out leaving 5 mm of skin. The skin samples were then examined histopathological changes.

During the experiment it was observed that the wound treated with PEV formulation including *γ* -oryzanol and BISA exhibited a better anti-inflammatory response and wound healing capacity. This formulation could enhance the wound healing due to the pharmacological effects of both *γ* -oryzanol and BISA, and their antioxidant properties. Gamma oryzanol induced morphological abnormalities in the testes of ram indicating some form of reproductive toxicity. But this necessitates careful scrutiny of the effects of *γ* – oryzanol on testes and ovaries.
