In Vitro Antioxidant, Hypolipidemic and Anti-Lipase Potential of Joha Rice of Assam, India

R. Chakraborty; P. Kalita; P. Manoj Kumar; S. Sen

doi:10.36468/pharmaceutical-sciences.1121

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Short Communication

In Vitro Antioxidant, Hypolipidemic and Anti-Lipase Potential of Joha Rice of Assam, India

R. Chakraborty¹, P. Kalita², P. Manoj Kumar³ and S. Sen^*

Faculty of Pharmaceutical Science, Assam down town University, Guwahati, Assam 781026, ¹Assam Don Bosco University Institute of Pharmacy, Assam Don Bosco University, Sonapur, Assam 782402 ²Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam 781026 ³The Dale View College of Pharmacy and Research Centre, Thiruvananthapuram, Kerala 695575, India

*Corresponding Author:: S. Sen
Faculty of Pharmaceutical Science, Assam down town University, Guwahati, Assam 781026, India
E-mail: saikat.pharm@rediffmail.com

Date of Received	12 January 2023
Date of Revision	12 January 2023
Date of Acceptance	20 April 2023
Indian J Pharm Sci 2023;85(2):539-543

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DOI: 10.36468/pharmaceutical-sciences.1121

Abstract

Present research accounts for in vitro antioxidant, hypolipidemic and pancreatic lipase inhibitory potential of two scented rice of Assam, India, namely ketoki joha and kola joha. Our result showed that ethanol extract of ketoki joha and kola joha (whole rice), particularly ketoki joha, exhibits a potent antioxidant potential when tested through DPPH•, NO•, H₂O₂ scavenging and lipid peroxidation inhibition assay. Among different whole rice extracts, ethanol extract of ketoki joha and kola joha at 100 μg/ml produced 76.67 % and 62.47 % inhibition of β-Hydroxy β-methylglutaryl-CoA reductase activity respectively. Ethanol and petroleum ether extracts potently inhibit cholesterol esterase. Ethyl acetate extract of ketoki joha showed the highest pancreatic lipase half maximal inhibitory concentration (IC₅₀)= 254.28 μg/ml). In conclusion, the results showed that both rice varieties, mainly ketoki joha, possess potent antioxidant, β-Hydroxy β-methylglutaryl- CoA reductase, pancreatic lipase inhibitory effect, and cholesterol esterase inhibitory effect. Observations might suggest the role of whole joha rice in combating oxidative stress and obesity-related cardiovascular diseases, including atherosclerosis.

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Keywords

Whole Rice, antioxidant, hypolipidemic, antiobesity, Joha rice

Rice (Oryza sativa) is a staple food of most of the global population that is a rich source of carbohydrates and nutritive substances[1]. Assam, a northeastern state of India, produces different rice varieties that are categorized under sali (winter), boro (summer), ahu (autumn) and bao (deep water) rice. Peoples of Assam are cultivating different Joha rice cultivars (categorized under sali rice), which are known for their aroma, superfine kernel and excellent palatability[2]. Recently, joha rice has received the geographical indications tag from Govt. of India. Research from different parts of the world demonstrated that different rice germplasms exhibit antioxidant, antidiabetic, anti-inflammatory, anti-hyperlipidemic, immunomodulatory and anticancer activities and contain bioactive phytochemicals like polyphenol, phytosterol, carotenoids and flavonoids[1,3]. It was also evident that intake of 90 g of whole grains daily reduces the risk of cardiovascular disease, cancer, and mortality[4]. Although much research has been undertaken to investigate the pharmacological activity of different rice cultivars, quality research on joha rice is still limited. Our present study, aimed to evaluate in vitro hypolipidemic, antiobesity and free radical scavenging activity of two joha rice cultivars i.e., Ketoki Joha (KTJ) and Kola Joha (KJ). Seeds of two joha rice cultivars viz., KTJ and KJ samples were collected from the Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, India. The whole rice was processed for de-husking using a traditional instrument called "URAL" to get unpolished rice (rice with bran). Powdered whole rice sample was extracted by petroleum ether, ethyl acetate, ethanol and water using Soxhlet apparatus. Extracts were concentrated in reduced pressure using a rotary vacuum evaporator and stored in a cool place. In vitro antioxidant activity of extracts was completed using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), nitric oxide radical (NO•), hydrogen peroxide (H₂O₂) scavenging assay and Lipid Peroxidation (LPO) inhibitory assay method. Estimation of DPPH• scavenging effect of extracts was determined as DPPH solution (violet color) is reduced to diphenylpicryl hydrazine (yellow colored product), which was measured by taking absorption at 517 nm[5]. In NO• scavenging assay, Griess Illosvoy reaction is used to quantify nitrite generated from Sodium Nitroprusside (SNP). Different concentrations of extracts were mixed with SNP solution and incubated at 37° for 2 h followed by the addition of Griess reagent to generate a chromophore measured at 570nm[5]. In H₂O₂ scavenging activity, different concentrations of extracts were mixed with H₂O₂ solution (43 mM) in phosphate buffer and the absorbance was measured at 230 nm[6]. Prior approval from Institutional Animal Ethics Committee (IAEC) of Assam down town University, Guwahati has been taken to collect experimental animal blood and rat liver required for LPO assay and 3-hydroxy-3- methylglutaryl-CoA (HMG CoA) reductase assay. Malondialdehyde (MDA) is a cytotoxic product produced during lipid peroxidation. Rat liver homogenate was used as a source of polyunsaturated fatty acid in LPO assay. Chromophore produced by MDA and thiobarbituric acid reaction was measured at 532 nm against blank[5]. Ascorbic acid was used as a standard for in vitro antioxidant activities. The percentage of inhibition was estimated against blank using the formula

Percentage inhibition=(A₀-A₁)/A₀×100

Where, A₀= Absorbance of control and A₁ = Absorbance of test/standard. Antihyperlipidemic activity of extracts was investigated using the in vitro HMG-CoA reductase assay and Cholesterol Esterase (CEase) inhibition assay method. Rat liver homogenate was used to prepare microsomes using a standard protocol that acts as an HMG CoA reductase enzyme source. Different concentrations of whole joha rice extracts (25, 50, 75 and 100 μg/ ml) were mixed with liver microsomes and HMGCoA reductase activity of the extract was measured considering the relative oxidation of NADPH and release of NADP+[7]. Atorvastatin was used as standard. CEase inhibitory activity was performed in the presence of sodium taurocholate with p-Nitrophenyl Butyrate (p-NPB) as substrate. About 430 μl of sodium phosphate buffer (pH 7.0), 500 μl of taurocholate solution, 40 μl of acetonitrile, 10 μl of p-NPB solution, and 10 μl of extract were mixed and incubated for 2 min at 25° followed by the addition of 10 μl of enzyme solution. Absorbance was measured at 405 nm to measure in vitro CEase inhibitory potency of extracts[8]. In vitro pancreatic lipase inhibition assay, the ability of joha rice extracts was determined considering the hydrolysis of p-nitrophenyl butyrate (p-NPB) to p-nitrophenol using a standard method. Porcine pancreatic lipase was mixed with different extract concentrations and the volume was made up to 1 ml by adding Tri-HCl solution. The reaction mixture was incubated at 25° for 15 min, followed by adding p-NPB solution in acetonitrile. The reaction mixture was incubated for 30 min at 37°, and optical activity was measured at 405 nm. Orlistat was used as a standard[9]. IC₅₀ value indicating the concentration at which an extract/ standard would inhibit free radicals/reactive oxygen species/enzyme activity by 50 % was also calculated. Results obtained from in vitro antioxidant activity are presented in Table 1. Whole rice extracts of KTJ and KJ exhibited profound antioxidant activity. In the DPPH• scavenging assay, the IC₅₀ value of the ethanol extract of KTJ, ethanol extract of KJ and ascorbic acid was found to be 102.77, 112.01 and 32.40 μg/ ml respectively. DPPH• scavenging activity resulted in the reactivity of test substances with constant free radicals. In visible spectroscopy, DPPH• exerted prevailing absorption bands at 517 nm due to the odd electron quantity[10]. DPPH• scavenging activity of KTJ and KJ extracts indicated that extracts, particularly ethanol extract, contain potent phytochemicals that can effectively scavenge free radicals. Among all the extracts, ethanol extract of KTJ had the lowest IC₅₀ value (752.13 μg/ml), followed by ethyl acetate extract of KTJ (938.90 μg/ml) and ethanol extract of KJ (962.61 μg/ml) when tested through NO• scavenging assay. NO• is an important pleiotropic mediator that regulates several physiological activities. Nevertheless, increased generation of NO• may induce oxidative damage and cause disorders like atherosclerosis, inflammation, cancer etc[11]. Nitric oxide synthase and reaction initiated by superoxide radicals can induce the generation of peroxynitrite excessively, which may involve the pathogenesis of different degenerative diseases and oxidative damage[10]. Results showed the potent antioxidant activity of KTJ and KJ, suggesting a possible role of these extracts against free radical induced diseases. The concentration of the ethanol and water extract of KTJ required to scavenge H₂O₂ by 50 % was 80.53 and 106.87 μg/ml, respectively, while the IC₅₀ value of ethanol extract of KJ was found to be 111.87 μg/ml. A lesser IC₅₀ value indicates higher antioxidant activity. H₂O₂ can be highly reactive in nature and in the presence of Fe²⁺ or Cu²⁺, and H₂O₂ induces the most reactive hydroxyl radical through the Fenton reaction[12]. Extracts effectively scavenge H₂O₂ in a concentration dependent manner. KTJ and KJ also effectively inhibit lipid peroxidation. The IC₅₀ of ethyl acetate extract of KJ and KTJ was 109.78 and 132.61 μg/ml respectively, in lipid peroxidation inhibition assay, while IC₅₀ of ascorbic acid was found to be 76.6 mg/ml. Lipid peroxidation causes degradation of the lipid bilayer of the cells membrane, thus inducing disruption of normal cell function and cellular homeostasis. End-products of lipid peroxidation can further induce mutagenesis or protein oxidation involved in the pathogenesis of different disease condition[5,13]. Extracts effectively inhibit lipid peroxidation and the generation of malondialdehyde. Hence, increasing interest in finding the level of protection can confer by an extract that may help avert cardiovascular disease. The result of the present study reports that whole joha rice extract exerts in vitro antioxidant and lipid peroxidation inhibitory activity in a dosedependent manner. Previous studies suggested that rice contains diverse polyphenolic compounds[1,3]. These results indicated that whole rice could be helpful in the prevention of oxidative stress and its associated diseases. Endogenous cholesterol biosynthesis in the liver is mainly controlled by rate limiting HMG-CoA reductase, a rate limiting enzyme primarily responsible for controlling the biosynthesis of endogenous cholesterol in the liver. HMG-CoA reductase involves the Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-dependent conversion of HMG-CoA to mevalonate[14]. The inhibition of HMG-CoA reductase efficiently decreases cholesterol levels and is considered a key target for developing a hypolipidemic drug. Data presented in fig. 1 shows that KTJ and KJ extracts significantly inhibited HMG-CoA reductase activity in a concentrationdependent manner. At 100 μg/ml concentration, KTJ ethanol extract, KJ ethanol extract, and KTJ petroleum ether extract inhibit HMG-CoA reductase activity by 76.67 %, 62.47 %, and 61.43 %, respectively, while atorvastatin (50 μg/ml) inhibit enzyme activity by 61.5 % when compared with control. Thus, whole rice extract of KTJ and KJ can be considered potent inhibitors of this HMG-CoA reductase and can be considered lead in drug development targeting hyperlipidemia. Recent research supports that effective inhibition of digestion and absorption of dietary fat is a new approach to preventing hyperlipidemia. CEase induce hydrolysis of cholesterol ester in the small intestine to unesterified cholesterol and free fatty acids. Further, it also regulates the incorporation of cholesterol into mixed micelle[15,16]. CEase inhibitory activity of rice extracts is given in Table 2. The findings supported the strong CEase inhibitory activity of ethanol extract of KTJ (IC₅₀=87.11 μg/ml), pet ether extract of KTJ (IC₅₀=111.96 μg/ml), petroleum ether extract of KJ (IC₅₀=136.53 μg/ml). Several phenolic and flavonoids compounds, such as gallic acid, catechin, and epicatechin exhibited CEase inhibitory activity[16]. CEase inhibitors may confer as a solution to control the absorption of free cholesterol and reduce the bioavailability of dietary cholesterol resulting from cholesterol esters. Therefore, inhibition of CEase by whole rice extract of KTJ and KJ could play an essential role in managing hyperlipidemia and obesity by reducing dietary cholesterol absorption. Obesity results from an imbalance between energy intake and expenditure that increases the risk of cardiovascular, metabolic and endocrine disorders and has become a major global health concern[17]. Obesity is responsible for increasing triglycerides, low-density lipoprotein cholesterol, blood glucose and blood pressure, and reduction of high-density lipoprotein cholesterol[17]. Pancreatic lipase is synthesized and secreted by the pancreas and exerts a vital role in lipid digestion; almost 50 %-70 % of total dietary lipids are digested by lipase enzyme[9]. Therefore, the anti-lipase activity of phytoextract/phytochemicals is one of the most important mechanisms for finding an anti-obesity agent. Results obtained from our study showed that ethyl acetate extracts of KTJ and KJ have high porcine pancreatic lipase inhibition potential with IC₅₀ values of 254.28 and 259.05 μg/ml respectively, in comparison with the orlistat, which has an IC₅₀ of 151.20 μg/ml. Many previous researches concluded that flavonoids and other phenolic compounds have porcine pancreatic lipase inhibitory activity by binding to the enzymesubstrate complex and causing a reduction in lipid absorption[9]. In our study, rice extracts showed porcine pancreatic lipase inhibition assay, which may be due to flavonoids and other phenolic compounds present in the extract. Recent studies investigated that joha rice contains diverse antioxidant phytochemicals, including apigenin, tricin, avenasterol, coumarin, coumaric acid, phenyl alanine, caffeic acid, and α-tocophenol[3,18]. An imbalance of linoleic acid (ω-6-fatty-acid) and linolenic acid (ω-3-fatty-acid) has been associated with the pathogenesis of metabolic and cardiovascular diseases. Presence of ω-6 and ω-3- fatty-acid are also reported in different joha rice cultivars[3]. Further, rice bran is an important source of dietary antioxidants that have been found effective against obesity and cardiovascular disease[1], though research on joha rice is limited. In our study, we have taken unpolished rice, and thus antioxidant molecules present in bran might be responsible for investigated pharmacological activities. The present study results concluded that both KTJ and KJ, particularly KTJ, produced strong antioxidant activity, hindering the enzymatic activity of HMG-CoA and CEase, and exhibiting an anti-lipase effect. Further studies are needed to isolate the phytochemicals and find the role of whole joha rice in managing or preventing hyperlipidemia, hypercholesterolemia, and cardiovascular diseases.

Extract	IC₅₀ value (µg/ml)
Extract	DPPH• scavenging assay	NO• scavenging assay	Lipid peroxidation inhibition assay	H₂O₂ scavenging assay
KTJ water extract	253.06±3.68	1009.42±24.58	224.95±6.19	106.87±1.59
KTJ ethanol extract	102.77±1.76	752.13±8.10	194.34±5.28	80.53±0.63
KTJ ethyl acetate extract	255.41±5.90	938.9±15.25	132.61±5.37	118.03±1.85
KTJ petroleum ether extract	320.09±4.12	1109.67±16.64	205.44±4.90	146.5±2.82
KJ water extract	226.4±1.80	1102.93±16.90	215.67±3.41	112.42±1.24
KJ ethanol extract	112.01±2.34	962.61±13.38	151.02±5.99	111.87±1.42
KJ ethyl acetate extract	290.17±9.15	1122.83±13.64	109.78±3.33	181.78±2.56
KJ petroleum ether extract	338.85±11.42	1293.36±6.79	178.21±0.15	166.4±4.76
Ascorbic acid	32.40±0.44	623.20±9.86	76.6±4.02	26.80±2.98

Note: Values are expressed as mean±standard error of mean (n=3); KTJ: Ketoki Joha; KJ: Kola Joha

Table 1: In Vitro Antioxidant Activity of Whole Rice Extracts of Ktj and Kj

Fig. 1: Effect of whole rice extract of KTJ and KJ. Values were expressed as mean±standard error of mean (n=3); KTJ: Ketoki Joha; KJ: Kola Joha; Data were analyzed by one way analysis of variance, statistical significance was expressed as *p<0.05, **p<0.01 and ***p<0.001 in comparison to control group; (): 25 µg/ml; (): 50 µg/ml; (): 75 µg/ml and (): 100 µg/ml

Extract	IC₅₀ value (µg/ml)
Extract	Cholesterol esterase inhibitory activity	Pancreatic lipase inhibition assay
KTJ water extract	241.55±5.77	409.24±19.83
KTJ ethanol extract	87.11±3.36	354.55±2.90
KTJ ethyl acetate extract	240.73±7.65	254.28±2.11
KTJ petroleum ether extract	111.96±4.30	428.08±3.84
KJ water extract	260.44±7.04	450.92±5.72
KJ ethanol extract	136.87±2.33	323.39±1.19
KJ ethyl acetate extract	278.51±7.86	259.05±1.14
KJ petroleum ether extract	136.53±9.38	331.96±17.37
Standard drug	60.95±1.06 (simvastatin)	151.20±3.88 (orlistat)

Note: Values are expressed as mean±standard error of mean; n=3; KTJ: Ketoki Joha; KJ: Kola joha

Table 2: Inhibitory Effect of Whole Rice Extract of Whole Rice Extracts of Ketoki Joha and Kola Joha on Cholesterol Esterase and Pancreatic Lipase

Acknowledgments:

The present research work was funded by the Department of Biotechnology (NER-BPMC), Ministry of Science and Technology, Govt. of India, under the network project on ‘DBT’s Scented Rice Program of the NE’ (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 to carry out the study. All the experiments reported in this research were performed in Assam down town University, Guwahati.

Conflict of interests:

The authors declared no conflict of interests