Phenolic Profile, Antioxidant, Antihyperlipidemic and Cardiac Risk Preventive Effect of Pigmented Black Rice Variety Chakhao poireiton in High-Fat High-Sugar Induced Rats

Raja CHAKRABORTY, Pratap KALITA, Saikat SEN

(1Faculty of Pharmaceutical Science, Assam down town University, Assam 781026, India; 2Institute of Pharmacy, Assam Don Bosco University, Assam 782402, India; 3Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam 781026, India)

Abstract: The present study aimed to investigate the hypolipidemic, antioxidant and cardiac risk-suppressing effects of Chakhao poireiton (CP), a GI-tagged pigmented black rice from India. In vitro and ex vivo studies confirmed that whole rice extracts of CP have potent antioxidant, 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol esterase inhibitory, and antilipase effects. An in vivo study was conducted to evaluate the effects of the ethanol extracts of CP on high-fat high-sugar induced hyperlipidemic rats. The ethanol extract significantly ameliorated lipid parameters and liver enzymes to normal. Levels of lactate dehydrogenase, creatine kinase-N-acetyl cysteine, C-reactive protein, and lipoprotein a were significantly lower in the extract-treated groups than those in the disease control group. A marked reduction of ApoB/ApoA1 and other atherogenic indices were observed in extract-treated groups. Twelve phenolic compounds, i.e. rosamarinic acid, gallic acid, protocatechuic acid etc., were quantified in CP. This study provided the first evidence of the antihyperlipidemic and cardiac risk inhibitory effects of CP, which would be beneficial in preventing and managing hyperlipidemia, associated oxidative stress, and cardiac complications.

Key words: antioxidant; cardiac risk; high fat; high sugar; hypolipidemic; phenolic compound;pigmented rice; whole rice

Dyslipidemia is characterized by increased levels of total cholesterol (TC), triglycerides (TG) or both, and is considered a fundamental risk factor for cardiovascular diseases (CVD) (Thongtang et al, 2022). Recently,enhanced low density lipoprotein cholesterol (LDL-C),TG and/or reduced high-density lipoprotein cholesterol(HDL-C) levels have become a major health concern.Furthermore, a positive association between increased levels of TC, mainly LDL-C, with a greater risk of cardiovascular events and an inverse association between the risk of CVDs and HDL-C has been well established (Soppert et al, 2020; Thongtang et al, 2022).Therefore, effectively addressing the dyslipidemic condition in the early stage is a crucial approach to preventing cardiovascular events and deaths related to CVDs (Thongtang et al, 2022).

Rice is not only known for its nutritive value but also screened for its potential therapeutic utilities.Various pigmented rice varieties (i.e. black, red,purple, brown, red-brown) have been investigated for their nutritional and health-promoting effects (Samyor et al, 2017). Pigmented rice is a rich source of anthocyanin, proanthocyanidin, phenolic acids, flavanols,isoflavones, carotenoids, phytosterol, γ-oryzanol, and vitamin E, etc. Such pigmented rice has also been successfully investigated for its antioxidant, antiinflammatory, antidiabetic, hypolipidemic, anti-obesity,anticancer, antitumor, anti-allergic, anti-influenza, and anti-inflammatory activities (Samyor et al, 2017; Sen et al, 2020; Ed Nignpense et al, 2022a). Black, red,and purple rice varieties, cultivated in China, Korea,Indonesia, Japan, etc., have been primarily explored for their therapeutic potential (Samyor et al, 2017; Sen et al, 2020). Some of the Indian pigmented rice varieties cultivated in Kashmir (Zag, Kaw quder, Shel kew, Samarkand, Kaw kareed, Gull zag, and Teli zag rice cultivar), Arunachal Pradesh (Lingkang taker ame,Umling ame rice cultivar), and Kerala (Njavara rice cultivar) have been successfully investigated for potent antioxidant activity and profiled few antioxidant molecules (Parvathy et al, 2014; Samyor et al, 2016; Bhat and Riar, 2017). Although the morphological characteristics, bioactivity, bioactive components, and nutritive value of differ among rice cultivars. Chakhao [Chakhao poireiton (CP), Chakhao amubi, Chakhao angouba, etc.], scented, sticky (gluten free), and indigenous black rice from Manipur, India,is less known and considered important in the socio-cultural rituals of the local Meitei community(Borah et al, 2018). With a geographical indication(GI) tag, Chakhao is getting popular owing to its possible health-promoting effect. Several studies on Chakhao, including investigation of antioxidant activities and isolation of few antioxidant molecules,were reported (Asem et al, 2015; Bhuvaneswari et al,2020; Moirangthem et al, 2021; Devi and Badwaik,2022; Singh et al, 2022). However, no investigation has been conducted on the effect of Chakhao on metabolic disorders and their associated complications.Henceforth, to substantiate the knowledge and facilitate the consumption of Chakhao for better health, the present investigation was undertaken to investigate the antihyperlipidemic, antioxidant, and cardioprotective effects of CP (Oryza sativaL. subsp.indica) throughin vitroandex vivomodels and to quantify certain phenolics compounds present in CP.

RESULTS

In vitro and ex vivo antioxidant activity

Whole rice extracts of CP, i.e. petroleum ether extract(PetE-CP), ethyl acetate extract (EtOAc-CP), ethanol extract (EtOH-CP) and water extract (H2O-CP)showed potent antioxidant activity (Fig. 1). EtOH-CP exhibited strong 2,2-diphenyl-1-picryl-hydrazy radical(DPPH·), hydrogen peroxide (H2O2), and nitric oxide radical (NO·) scavenging activities with IC50values of 41.41, 27.88, and 668.92 µg/mL, respectively. In the lipid peroxidation inhibition assay, EtOAc-CP and EtOH-CP exhibited strong scavenging effects with low IC50values(86.78 and 99.60 µg/mL). Additionally, ascorbic acid exhibited superiorin vitroantioxidant activity.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)reductase, cholesterol esterase and pancreatic lipase inhibitory assay

Data showed that CP extracts inhibited HMG-CoA reductase in a concentration-dependent manner (Fig.2-A). At 100 µg/mL concentration, H2O-CP, EtOH-CP,EtOAc-CP, and PetE-CP inhibited HMG-CoA reductase activity by 49.8%, 78.8%, 55.9%, and 64.6%, respectively.Cholesterol esterase (CEase) inhibitory activity of extracts was also analyzed. EtOH-CP (IC50= 72.9µg/mL) and PetE-CP (IC50= 74.9 µg/mL) inhibited CEase potently comparable to simvastatin (IC50= 60.9µg/mL) (Fig. 2-B). The extracts demonstrated moderate to strong pancreatic lipase inhibitory effect. The results showed that EtOAc-CP was a strong inhibitor of porcine pancreatic lipase with an IC50value of 177.9 µg/mL (Fig. 2-B).

Fig. 1. Free radical scavenging activities of Chakhao poireiton rice extracts.

Fig. 2. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitory activity (A), and cholesterol esterase and pancreatic lipase inhibitory activities(B) of Chakhao poireiton rice extracts.

Acute toxicity study

Acute oral toxicity of EtOH-CP was investigated,finding that EtOH-CP at 2 000 mg/kg did not produce any toxic effect. Behaviour and mortality of experimental animals were observed closely, and no sign of behavioural change, morbidity or mortality were observed. Therefore, 2 000 mg/kg dose of EtOH-CP was categorized under Globally Harmonized Classification System category 5 (safe dose) following Organisation for Economic Cooperation and Development guideline 423 (Annexure 2 d).

Effect of CP on lipid profile, liver enzymes and body weight

After being fed a high-fat high-sugar (HF-HS) diet for 45 d, rats in the disease control group had significant increases in TC, TG, LDL-C, and very low density lipoprotein cholesterol (VLDL-C), and a decrease in HDL-C level. EtOH-CP (200 and 400 mg/kg)significantly ameliorated the levels of lipid parameters to near normal (Table 1). The effect of the higher dose of EtOH-CP on reducing the levels of TC, TG, LDL-C,and VLDL-C was comparable to standard atorvastatin.The level of HDL-C in the animals treated with a higher dose of EtOH-CP and atorvastatin was 38.63 and 42.23 mg/dL, respectively. In our study, animals supplemented with EtOH-CP for 30 d were found to prevent the rise in TC, TG, LDL-C, and VLDL-C and depletion of HDL-C, which indicated that the extract has the potency to avert hypercholesterolemic and hypertriglyceridemic conditions and cardiovascular complications. The body weight of rats in the healthy control, disease control, standard, EtOH-CP (200 and 400 mg/kg) groups increased by 13.7%, 22.2%, 18.1%,17.7%, and 16.8%, respectively. The levels of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkalinephosphatase (ALP) were increased in the disease control group compared with the healthy control group, indicating impairment in a hepatic cell under hyperlipidemic conditions. Oral administration of EtOH-CP and atorvastatin significantly improved the above parameters (Table 1).

Table 1. Effect of Chakhao poireiton rice extract on lipid profile and kidney function on high fat high sugar induced hyperlipidemia.

Table 2. Effects of ethanol extract of Chakhao poireiton rice on LDH, CK-NAC, endogenous antioxidants and lipid peroxide.

In vivo antioxidant activity

Substantial reduction in the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase(GPx) and reduced glutathione (GSH) was observed in the disease control group compared with the healthy control group. These levels were significantly enhanced significantly (P＜ 0.001) in the EtOH-CP(400 mg/kg) treated group. The level of GSH in animal treated with a higher dose of extract was 197.61 µmoL/L, which is comparable with the health control group (199.98 µmoL/L). The level of plasma lipid peroxide (PLP) was the highest in the disease control group (8.77 nmoL/mL), which was highly reduced in rats treated with a higher dose of EtOH-CP(3.52 nmoL/mL) (Table 2).

Effect on lactate dehydrogenase (LDH) and creatine kinase-N-acetyl cysteine (CK-NAC)

The disease control group recorded higher levels of LDH and CK-NAC compared to the healthy control group.The beneficial effect of EtOH-CP on the level of LDH and CK-NAC is depicted in Table 2. In the EtOH-CP(400 mg/kg) group, the LDH and CK-NAC levels were 637.20 and 330.13 U/L, which was significantly(P＜ 0.001) less than the disease control group.

Effect on C-reactive protein (CRP), lipoprotein a[Lp(a)], apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), and atherogenic indices

The effects of the extract on CRP, Lp(a), ApoA1 and ApoB were given in Fig. 3-A. The results showed that the CRP and LP(a) levels were significantly (P＜0.001) reduced in the EtOH-CP treated group. The level of ApoA1 in the EtOH-CP (400 mg/kg) treated group was found to be 91 mg/L, which was significantly high compared with the disease control group, while the level of ApoB was reduced. In the present study, the ratio of ApoB to ApoA1 increased in the disease control group (2.078) compared to the healthy control (0.915), which was reduced to a normal level in rats treated with the standard (0.910)and a higher dose of EtOH-CP (0.952) treated group(Fig. 3-A).

Fig. 3. Effects of Chakhao poireiton on CRP, Lp(a), ApoA1, ApoB, and ApoB/ApoA1 (A), and on atherogenic indices (B).

The atherogenic index, atherogenic coefficient and cardiac risk ratio increased while the cardioprotective index reduced in the disease control group compared to the healthy control group. EtOH-CP (400 mg/kg)administration positively ameliorated atherogenic indices. The cardiac risk ratio reduced to 2.77, while the cardioprotective index increased to 1.15 in rats treated with a higher dose of EtOH-CP, indicating the beneficial effect of CP (Fig. 3-B).

Profiling of phenolic compounds

The quantification of certain free and bound phenolic compounds was completed using high performance liquid chromatography (HPLC) analysis by the standard comparison method. Twelve phenolic compounds i.e.,p-coumaric acid,o-coumaric acid,t-coumaric acid,caffeic acid, syringic acid, gallic acid, rosamarinic acid, protocatechuic acid, sinapic acid, vanillic acid,phytic acid and chlorogenic acid, were quantified in rice variety CP by reversed phase-HPLC (RP-HPLC)method (Fig. 4). The levels ofp-coumaric acid,t-coumaric acid and rosamarinic acid were found as 4 055.9, 2 753.4 and 2 969.2 mg/kg, respectively.

DISCUSSION

Fig. 4. Profiling of phenolic compounds in Chakhao poireiton.

Chakhao poireiton (in the local language ‘Chakhao’indicates delicious rice) is a lesser known aromatic black rice variety from Manipur, India. In recent years,research on pigmented rice, including Chakhao, has intensified owing to its potential nutraceutical,antioxidant and other health promoting effects (Borah et al, 2018). Oxidative stress is implicated in the pathogenesis of different CVDs. Preventing oxidative stress is an important approach to treating or preventing many health conditions including hyperlipidemia and other CVDs (Sen et al, 2020).DPPH·, NO·, and H2O2scavenging assays are widely used to find the extent of scavenging activity of antioxidant substances. In this study, ethanol extract potently scavenges free radicals like DPPH·, NO·, and non-radical species like H2O2, which is also effective in averting lipid peroxidation, thiol oxidation, and oxidation and nitration of protein tyrosine. Earlier investigations on CP successfully reported strongin vitroantioxidant potential of seed, bran and straw of the rice variety through DPPH· scavenging and other assay models (Asem et al, 2015; Bhuvaneswari et al,2020; Devi and Badwaik, 2022; Singh et al, 2022).This study also found that extracts of CP, particularly ethanol extract, exhibited promisingin vitroantioxidant activity. Stability of the membrane is disrupted through lipid peroxidation by altering the membrane’s permeability, causing damage to membrane receptors,membrane transport protein distraction, and reduction of enzyme activity bound to the membrane (Sen et al,2013; Bhuvaneswari et al, 2020). EtOAc-CP and EtOH-CP exhibited very strong lipid peroxidation inhibition effects that infer the role of CP in controlling oxidative stress and associated complications. Endogenous antioxidants like SOD, CAT and GPx are considered the first line of defense in maintaining redox balance by averting ROS/RNS-induced stress. A deficit of SOD may weaken the immune response and promote the progression of diseases, including CVDs and vascular problems (Ighodaro and Akinloye, 2018).Gene mutation, polymorphism, and deficit of CAT and GPx are linked with the pathogenesis of various pathological conditions like diabetes mellitus, cancer,neurological disorders, endothelial dysfunction, and CVDs (Ighodaro and Akinloye, 2018). GSH is a crucial non-enzymatic antioxidant. Maintenance of GSH homeostasis is essential to avert the progression of many diseases (Aquilano et al, 2014). By producing malonaldehyde, lipid peroxidation is directly linked to the pathophysiology of numerous diseases, including CVD (Sen et al, 2013; Kumari et al, 2018). Extract prevented the downregulation of SOD, CAT, GPx, and GSH, and halted the increase of PLP. Thus, the free radical scavenging effect, enhancement of endogenous antioxidants, and prevention of lipid peroxidation can be considered a key mechanism of CP associated with its antihyperlipidemic potential.

HMG-CoA reductase catalyzes the rate-limiting stage in cholesterol biosynthesis, promoting HMG-CoA conversion to mevalonate (Baskaran et al, 2015). In this study, the inhibition of HMG-CoA reductase by extracts of CP may reflect the potential of extracts in cholesterol reduction, which is a crucial therapeutic strategy for hyperlipidemia. In the lumen of the small intestine, CEase is responsible for the hydrolysis of esterified cholesterol to produce free fatty acids and it regulates the incorporation of cholesterol into mixed micelles (Heidrich et al, 2004). In the intestine,pancreatic cholesterol esterase also plays a key role in controlling the conversion of cholesterol from dietary cholesterol esters and in assisting in the transfer of free cholesterol to the enterocyte. Therefore, CEase could be an important target of therapeutics for limiting cholesterol absorption (Heidrich et al, 2004).Therefore, CEase inhibitory activity of CP extracts could be important in the effective management and prevention of hyperlipidemia and obesity by declining dietary cholesterol absorption.

Pancreatic lipase is a key lipolytic enzyme, playing an important role in TG digestion. It eliminates fatty acids from dietary TG to form β-monoglycerides and fatty acids (saturated and polyunsaturated). Most of the total dietary fat is hydrolyzed through pancreatic lipase (Birari and Bhutani, 2007). Obesity is a key risk factor for hyperlipidaemia, and pancreatic lipase inhibitors from natural sources are considered a key therapeutic approach to controlling weight gain by regulating lipid metabolism (Liu et al, 2020). Weight gain was restricted in rats that received EtOH-CP compared with the disease control group. This study highlighted the pancreatic lipase inhibitory effect of CP, which extrapolates the role of CP in controlling obesity and dyslipidaemia.

Hypercholesterolemia, atherosclerosis, CVDs, and metabolic disorders like obesity and diabetes are positively linked with enhanced plasma TC, TG, and LDL levels (Munshi et al, 2014). Reducing LDL-C level is a main tactic to reduce the risk of CVDs(Soppert et al, 2020). VLDL-C is produced in the liver and involved in transporting TGs (Packard et al, 2020).HDL-C is termed as ‘good cholesterol’ that plays an important role in reverse cholesterol transport, exhibits antioxidative and anti-inflammatory effects, reduces the production of oxidized LDL, displays a pro-survival effect, inhibits platelet activation by removing an excess quantity of plasma membrane cholesterol, and exhibits vasoprotective efficacy (Soppert et al, 2020).In our study, the rats supplemented with EtOH-CP for 30 d were found to prevent the rise in TC, TG, LDL-C,and VLDL-C and depletion of HDL-C, which indicated that the extract has the potency to avert hypercholesterolemic and hypertriglyceridemic conditions and cardiovascular complications.

Sangkitikomol et al (2010) reported that anthocyaninrich the extract from Thai black sticky rice exhibits antihyperlipidemic and antioxidant effects on HepG2 cells. Bae et al (2014) found that supplementation with Superjami (a Korean pigmented rice cultivar) in high fat fed animals exhibits hypolipidemic potential through the regulation of antioxidant and other regulatory enzymes, hepatic lipogenesis, and production of adipokine.Another pigmented rice from Korea (Heugjinjubyeo cultivar) was found to attenuate hepatic steatosis in experimental rodents via fatty acid oxidation (Jang et al,2012). Our present study reported the antioxidant, and antihyperlipidemic potential of India’s non-pigmented rice cultivar (Maniki Madhuri) (Sen et al, 2022). Rice bran or its constituents, including rice bran oil, were found to increase LDL receptor, cholesterol 7α-hydroxylase,sterol regulatory element-binding protein-2, HDL-C,and the excretion of bile acids and cholesterol through feces while inhibiting the absorption of cholesterol,HMG-CoA reductase, and fatty acid synthase (Sen et al,2020). The present study provides the first evidence of the antihyperlipidemic effect of CP through regulating HMG-CoA reductase, cholesterol esterase, pancreatic lipase, and antioxidant potency. The potency of the extract in averting dyslipidaemia is clearly observed from the results obtained.

A high-fat diet may induce hepatic cell lysis that, in turn, releases enzymes into circulation (Arora et al,2022). SGPT, SGOT, and ALP increased in the disease control group, indicating impairment in a hepatic cell in hyperlipidemic conditions. Conversely, EtOH-CP administration causes a decrease in the levels of SGOT, SGPT, and ALP, indicating that the extract may assist to maintain the structural integrity of hepatocytes by preventing dyslipidaemia.

High LDH and CK-NAC levels in hyperlipidemic animals indicate the impairment of plasma membrane integrity (Hassan et al, 2011). EtOH-CP was effective as the levels of LDH and CK-NAC were lower in the extracttreated group. Results suggest that CP supplementation averted myocardial cell damage associated with hyperlipidaemia by positively influencing lipid profile and reducing oxidative stress. CRP is recognized as an important and subtle inflammatory marker used to monitor the stage of CVDs and diabetes (Aramwit et al, 2013). High CRP activity is linked with an increased risk of acute coronary events; thus, the effect of EtOH-CP is useful in averting the risk of CVDs. Lp(a) is an LDL particle that is attached to a polypeptide, and ApoA is considered an independent risk factor for CVD linked with atherosclerosis,particularly in those with an increased level of LDL-C and a reduced level of HDL-C. Therapies that can reduce Lp(a) and LDL-C could offer better results in minimizing the risk of CVDs (Soppert et al, 2020;Forbes et al, 2016; Saeedi and Frohlich, 2016).Therefore, reducing Lp(a) and LDL-C by EtOH-CP may be useful in preventing atherosclerosis and subsequent CVD events.

ApoA1 is an integral protein of HDL that exerts antioxidative properties. ApoA1 halts the linking of oxidized lipids by confiscating these molecules (Saeedi and Frohlich, 2016). ApoA1 also plays a crucial role in the ‘reverse cholesterol transport’ process and has been found to possess anti-inflammatory and antioxidant potential (Lu et al, 2011). ApoB is the major apolipoprotein in LDL, VLDL and IDL, and to some extent, the plasma ApoB level indicates the level of cholesterol, and TGs field particles (Lu et al, 2011).The ratio of ApoB to ApoA1 indicates the risk involved in CVDs (Walldius and Jungner, 2006). The therapeutic benefit of EtOH-CP is confirmed by the results obtained as EtOH-CP efficiently increases in ApoA1, decreases in ApoB and reduces the ratio of ApoB to ApoA1. Enhancing HDL-C and ApoA1 and reducing LDL-C are important to manage hyperlipidemia,possibly through regulating cholesterol synthesis and augmenting reverse cholesterol transport through the HDL (ApoA1) pathway.

Results showed that the atherogenic index, atherogenic coefficient, and cardiac risk ratio increased while the cardioprotective index decreased in the disease control group compared with the healthy control group. These observations were consistent with the results discussed till now. These parameters are considered a scale of potential progression of atherosclerosis and are important in understanding the risk of CVDs development (Baskaran et al, 2015; Oršolić et al, 2019). Thus, this study provided pre-clinical confirmation of the beneficial effect of EtOH-CP in preventing hyperlipidemia and the progression of atherosclerosis.

Plant-based secondary metabolites called polyphenols have demonstrated potential for preventing and treating diseases based on clinical and preclinical studies. These polyphenols found in pigmented cereals are thought to improve health outcomes by means of their antioxidant properties (Ed Nignpense et al, 2022b). Phenolic compounds in food grains are an essential part of the human diet, and the antioxidant properties of these compounds have potential health benefits (Shahidi and Ambigaipalan, 2015). Dietary phenolic compounds may confer their beneficial effect in preventing CVDs through several mechanisms like reducing cholesterol absorption, upregulating LDL receptor, altering ApoB secretion, reducing microsomal transfer protein and acyl-CoA cholesterol acyltransferase activities, and decreasing TG, lipoprotein lipase, antioxidant, and anti-inflammatory effects (Zern and Fernandez, 2005).Bran of rice is a key source of phytochemicals particularly phenolic compounds with antioxidant properties (Sen et al, 2020). A few groups of researchers estimated total phenolic content, total flavonoid content, and total anthocyanin content, and identified a few of anthocyanins,i.e., peonidin-3-O-glucoside, delphinidin 3-arabinoside,delphinidin 3-galactoside, cyanidin 3-glucoside, and cyanidin 3-galactoside in CP (Asem et al, 2015;Bhuvaneswari et al, 2020; Singh et al, 2022). In our study, we did not profile anthocyanins but reported a few phenolic compounds for the first time in CP.Phenolic compounds are well known antioxidants and have hypolipidemic potency (Sen et al, 2020).Therefore, the phenolic compounds reported in the present study and anthocyanins reported in earlier investigations may be linked with the beneficial activity exerted by CP.

Fig. 5. Antioxidant, antihyperlipidemic and cardiac risk preventing effect of Chakhao poireiton and possible mechanism of action.

This is the first study to quantify certain phenolic components and describe the hypolipidemic, antioxidant,and hypolipidemic potential of CP. The present study confirmed that unpolished CP is helpful in averting or decelerating the progression of hyperlipidemia and in reducing the cardiac risk associated with dyslipidaemia by exerting antioxidant activity, inhibiting cholesterol synthesis, augmenting cholesterol elimination, reducing LDL-C and VLDL-C, enhancing HDL-C and ApoA1,regulating reverse cholesterol transport, and decreasing ApoB, CRP, LDL, Lp(a), and CK-NAC levels (Fig. 5).However, further studies and clinical evaluation are required to establish its antihyperlipidemic effects.

In summary, CP was successfully evaluated for its hypolipidemic, antioxidant, and cardioprotective effects.The presence of twelve phenolic compounds was also quantified in the rice sample. The presence of phenolic compounds may be linked to the biological activity of the studied rice sample. The beneficial effect of CP may be linked with the regulation of cholesterol synthesis and elimination, regulation of LDL-C synthesis, upregulation of HDL-C, prevention of myocardial damage, antioxidant activity, reduction of ApoA1, and enhancement of ApoB. Our findings supported the hypothesis that CP is a key source of phenolic compounds and was useful in preventing lifestyle-related disorders. The results also offered novel insights into the prospect of CP as a supplement or inclusion in the diet to prevent hyperlipidemia and atherosclerosis-related coronary events.

METHODS

Collection and extraction of rice sample

Seeds of CP were collected from the College of Agriculture,Central Agricultural University, Imphal, Manipur, India. They were manually de-husked using ‘ural’ (a traditional tool) to get unpolished (rice with bran) CP. Solvent (petroleum ether, ethyl acetate, ethanol and water) extracts of sample were carried out using a Soxhlet apparatus, and then evaporated to dryness using a rotary evaporator at reduced pressure to get solvent free PetE-CP, EtOAc-CP, EtOH-CP, and H2O-CP, and finally stored at 4 °C.

In vitro and ex vivo antioxidant activit y

In vitroandex vivoantioxidant activities were screened using DPPH·, NO·, H2O2scavenging assay, and lipid peroxidation inhibition assay model. DPPH· scavenging capacity of extracts was measured in terms of hydrogen donating or radical scavenging capability using stable DPPH·, a previously defined method (Sen et al 2013). NO· was produced from sodium nitroprusside and estimated by the Griess Illosvoy reaction. The absorbance was measured at 570 nm, and the percentage scavenging activity was calculated (Sen et al, 2013). H2O2scavenging ability of extracts was measured using the protocol described by Kumari et al (2018).

The ability of whole rice extracts of CP to inhibit lipid peroxidation was determined by using liver tissue from the rats according to a standardex vivoexperimental protocol (Sen et al,2013). Generation of malonaldehyde, a cytotoxic product of lipid peroxidation, was measured at 532 nm.

The percentage scavenging activity of extracts/standard was calculated using the following equation,

Where,Acmeans absorbance control andAsmeans absorbance extract/standard. The control experiment was also carried out in a similar manner, using solvent in the place of extract or standard.

HMG-CoA reductase, cholesterol esterase, and pancreatic lipase inhibition assay

Liver microsomes were prepared from rat liver using a standard procedure and used as an enzyme source in HMG-CoA reductase inhibition assay. The microsomes were treated with various concentrations (25, 50, 75, and 100 µg/mL) of PetE-CP,EtOAc-CP, EtOH-CP, and H2O-CP. The HMG-CoA reductase ability of extracts was measured based on the relative nicotinamide adenine dinucleotide phosphate (NADPH) oxidation and NADP+release (Mishra, 2014). CEase inhibitory assay was carried out in sodium taurocholate withp-nitrophenyl butyrate as a chromogenic substrate. Hydrolysis is executed with a high enzyme concentration and estimated spectrophotometrically.The ability of whole rice extracts to inhibit pancreatic lipase was analyzed by estimating the hydrolysis ofp-nitrophenyl butyrate top-nitrophenol, adopting a standard procedure(Jaradat et al, 2017). Orlistat was used as a standard, and the relative lipase inhibition activity (%) was calculated.

Animals and research protocol approval

Wistar albino rats (150-200 g, either sex) were procured and acclimatized in standard environmental conditions, and examinations were conducted to confirm the normality.Animals were fed with a commercially available pellet diet and water ad libitum. The Institutional Animal Ethics Committee has reviewed and approved all the experimental procedures(approval No. AdtU/IAEC/2016/005). Guidelines from the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Govt. of India were followed during the experiment.

Acute oral toxicity study

Acute oral toxicity test of EtOH-CP was carried out, following the protocol of Organisation for Economic Co-operation and Development (OECD guidelines 423, annexure 2 d).

Experimental protocol: drug intervention

A standard HF-HS diet-induced hyperlipidemic model described by Munshi et al (2014) was adopted in this study.Rats were divided into five groups, each containing six rats,with the healthy control group received a normal diet for the entire duration. Experimental animals in Group II to Group V were fed with the HF-HS diet for 45 d to induce hyperlipidemia.The high-fat diet contained Indian vanaspati ghee and coconut oil in a ratio of 3:1 and was administered to rats at a dose of 3 mL/kg per day, while 25% fructose (high sugar) was given orally. Atorvastatin and EtOH-CP were prepared in 0.5%carboxy methyl cellulose suspension as vehicle and administered orally once daily using oral gavage needle. Rats of the healthy control and the disease control received vehicle, while animals of standard drug and extract treated groups received atorvastatin (2 mg/kg) and EtOH-CP (200 and 400 mg/kg)orally from the 15th to the 45th day.

Biochemical analysis

Blood samples were collected from rats under light ether anaesthesia on the 46th day to analyze biochemical parameters.Finally, the animals were sacrificed using ketamine overdose.Serum lipid profile (TC, TG, LDL-C, HDL-C, very low density lipoprotein), SGOT, SGPT, ALP, LDH, Lp(a), ApoA1, ApoB,CK-NAC, and CRP were measured using commercial kits of Agappe Diagnostics Ltd, Kerala, India, on the Mispa ace biochemical autoanalyzer.

VLDL-C was calculated as per the Friedewald formula (Devi and Singh, 2017).

Change body weight and atherogenic indices

Atherogenic indices, i.e. atherogenic index of plasma, atherogenic coefficient, cardiac risk ratio and cardioprotective index were calculated using the formula following by Orsolic et al (2019).

In vivo antioxidant activity

Blood samples collected from experimental animals were centrifuged at 3 000 ×gfor 10 min, and packed red blood cells were washed with an isotonic solution of NaCl to remove the buffy coat. Hemolysis was assessed by mixing RBC suspension in ice-cold distilled water, and the sample was then centrifuged at 12 000 ×gfor 40 min. The cell content was carefully separated and taken to estimate antioxidant activity. Estimation of SOD, CAT, GPx, and GSH in blood and blood lipid peroxides was carried out using a previously reported method(Chandra et al, 2007).

Profiling of phenolic compounds through RP-HPLC analysis

Polyphenolic compounds of rice samples were profiled using the specified extraction procedure described by Shao et al(2014a, b) with minor modifications. Alcoholic extraction was carried out after defatting rice sample withn-hexane. Extracted residue was digested with 4 mol/L NaOH, and concentrated HCl was added to maintain pH of 1.5-2.0. The slurry was extracted with ethyl acetate and dried using a rotary vacuum evaporator. The concentrated residue was mixed with HPLC grade methanol and stored in a deep freezer until analysis.

Phenolic compounds were analyzed by binary HPLC system(Agilent, USA) with UV/Vis detector. C18 column (250 mm ×4.6 mm) with 5 µm particles was used for analysis. The solvent system used was: A (0.1% acetic acid in water) and B (0.1%acetic acid in methanol). Linear gradient analysis procedure was followed, and detection was carried out at 280 nm.Pre-established HPLC mobile phase running method was set as described by Shao et al (2014a, b) with a flow rate of 1 mL/min.

Statistical analysis

Results were expressed as Mean ± SE (n= 3 forin vitroexperiments andn= 6 forin vivoanalysis). Statistical analysis was performed with ANOVA followed by the Tukey test using GraphPad Prism software.

ACKNOWLEDGEMENTS

The authors acknowledge the Department of Biotechnology,Ministry of Science and Technology, Govt. of India, for funding (Grant No. DBT-NER/AGRI/29/2015). Authors are thankful to Assam down town University, the host institute of the project for providing the necessary infrastructure and facilities for the study. All experiments reported in this research were conducted in Assam down town University, Guwahati.The authors express gratitude to Dr. J. M. LAISHRAM, College of Agriculture, Central Agricultural University, Imphal,Manipur, for providing the experimental rice samples.