18 W. PrasongsubJournal et alof. Associated Journal of AssociatedMedical Sciences Medical 2021; Sciences 54 (3): 2021; 18-26 54(3): 18-26 Thai-Journal Citation Index Centre (TCI) & ASEAN Citation Index (ACI) Journal of Associated Medical Sciences Journal of Associated Medical Sciences Journal homepage: https://www.tci-thaijo.org/index.php/bulletinAMS/index Anti-HMG-CoA reductase and antioxidant activities of Sacha inchiPlukenetia ( volubilis L.) nutshell extract Wattanapong Prasongsub1 Nattaporn Pimsan1 Chantanatda Buranapattarachote1 Khanittha Punturee1,2* 1Division of Clinical Chemistry, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai Province, Thailand 2Cancer Research Unit of Associated Medical Sciences (AMS-CRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai Province, Thailand ARTICLE INFO ABSTRACT Article history: Background: Hypercholesterolemia is one of the major risks of cardiovascular Received April 2021 diseases (CVDs). Hypercholesterolemia and oxidative stress are involved in the Received in revised form May 2021 pathogenesis of atherosclerosis. Thailand has high percentage of unawareness of Accepted as revised June 2021 hypercholesterolemia, with low percentage of treatment and control. HMG-CoA Available online June 2021 reductase is a rate limiting step enzyme in cholesterol biosynthesis. Synthetic drugs such as statin are normally used to lower cholesterol level, however it causes Keywords: adverse effects on the liver and muscle. Thus, HMG-CoA reductase inhibitors of Sacha inchi, Plukenetia volubilis, plant origin are needed. Plukenetia volubilis Linneo, commonly known as Sacha HMG-CoA reductase inhibitor, inchi or inca peanut is a potential oilseed crop. The seeds of this plant are rich in antioxidant, hypercholesterolemia omega-3 fatty acid. Some studies have shown that consuming Sacha inchi could reduce blood cholesterol. However, the exact mechanism of their lipid lowering activity is still unknown. Objectives: This study aimed to investigate the anti-HMG-CoA reductase, anti-cholesterol esterase and antioxidant activities of different parts of Sacha inchi extracts. Materials and methods: Hot water extracts of 3 different parts of Sacha inchi (nutshell, baby nut and leaf) and Sacha inchi nut oil were evaluated the anti-HMG-CoA reductase and antioxidant activities. HMG-CoA reductase inhibitory activity was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as a substrate. HMG-CoA reductase inhibitory mechanism was also analyzed by using Lineweaver-Burk plot. Antioxidant activity was determined by ABTS and DPPH radical scavenging assay. Total phenolic content was also measured by Folin-Ciocalteu reagent. Moreover, cholesterol esterase inhibition assay was also performed. Results: Sacha inchi nutshell extract revealed the highest anti-HMG-CoA reductase activities at about 99% at concentration of 250 µg/mL, with uncompetitive inhibition in Lineweaver-Burk plot analysis. It also showed the highest cholesterol esterase inhibitory activity around 38% at concentration of 125 µg/mL. Moreover, Sacha inchi nutshell extract also showed the highest antioxidant activity by both ABTS and DPPH assay. Antioxidant activity was correlated to total phenolic content. Conclusion: The experimental data suggested that Sacha inchi nutshell extract is a source of antioxidant compound and may lower cholesterol level by inhibiting HMG-CoA reductase and cholesterol esterase enzymes. Investigation in an in vivo model could further confirm the potential use of Sacha inchi nutshell extract as a supplement for treatment of hypercholesterolemia. * Corresponding author. Author’s Address: Division of Clinical Chemistry, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai Province, Thailand. ** E-mail address: [email protected] doi: E-ISSN: 2539-6056 W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26 19 Introduction decreased cholesterol level especially LDL-cholesterol and Cardiovascular diseases (CVDs) are becoming a leading increased high-density lipoprotein cholesterol (HDL-cho- cause of death in developing countries due to demographic lesterol).14 Sacha inchi seeds showed antioxidant activity15 transition and lifestyle changes.1 The prevalence of CVDs and immunomodulatory activity,16 however other parts of has increased rapidly. According to WHO fact sheet in plant have never been studied before. This study aimed 2016, there are 17.9 million people died from CVDs around to investigate anti-HMG-CoA reductase, anti-cholesterol the world. In Thailand, the prevalence and mortality rate esterase and antioxidant effects of Sacha inchi extracts have also increased and most of CVDs patients are adults.2 (dried leaves, nutshells, and baby nuts) as well as Sacha A lack of income, reduced productivity, and increased inchi oil health care costs lead to overall economic losses. Thus, the prevention of CVDs can save not only billions for the economy Materials and methods but also many lives. Major risk factors of CVDs include dyslipidemia, diabetes, smoking, high blood pressure, and Chemicals family history of atherosclerotic cardiovascular diseases The 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic (ASCVD). Moreover, additional risks of CVDs have also acid (ABTS), gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), been reported such as alcohol consumption, unhealthy 4-nitrophenyl butyrate (pNPB), cholesterol esterase from diet, sedentary lifestyle, obesity, stress, and air pollution.3 porcine pancreas and HMG-CoA reductase assay kit were Pathogenesis of CVDs begins with the development of purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol atherosclerosis causing by an accumulation of cholesterol and isopropanol were purchased from MERCK (Darmstadt, in the intima layer of blood vessel. Low density lipoprotein Germany). L-ascorbic acid (vitamin C), and sodium hydrogen (LDL) contains the highest amount of cholesterol compare phosphate were obtained from Ajax Finechem Pty Limited to other lipoprotein particles. Accumulation of LDL in intima (Taren Point, New South Wales, Australia). Acetonitrile and layer of blood vessel and modification of LDL particle by sodium chloride were purchased from RCI Labscan Limited free radical and carbohydrate trigger the development of (Bangkok, Thailand). Sodium fluoride (NF) was acquired atherosclerosis. Thus, hypercholesterolemia and oxidative from BDH Laboratory Supplied (Poole, England). Triton X-100 stress status directly involved in atherosclerosis. Moreover, was purchased from Amresco (Solon, Ohio, USA) All reagents excess adipose tissue in obesity can cause inflammation, were analytical or HPLC grade. Distilled water and ultrapure induce oxidative stress by releasing of pro-inflammatory water were used for all experiments. cytokines and induce insulin resistant which leads to type Plant materials and extracts 2 diabetes mellitus (T2DM).4,5 Lowering cholesterol level Sacha inchi leaves, nutshells, and baby nuts were and improving oxidative stress status can prevent CVDs provided by Oil Star Tak Limited Partnership, Tak province, and T2DM. Thailand. Each part of plant was dried and ground to powder. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG One hundred grams of Sacha inchi leaf, nutshell, and baby CoA) reductase is a rate limiting step enzyme in cholesterol nut powder was extracted by boiling with 1 liter of distilled biosynthesis pathway. Inhibition of this enzyme can lower water for 15 minutes and further incubated for 4 hours. cholesterol level in blood. Statin, HMG-CoA reductase Solution was filtered through straining cloth and centrifuged inhibitor, is effectively used for treatment of hypercho- at 3,500 rpm for 5 minutes. After centrifugation, supernatant lesterolemia. However, statins increased risk of liver and was filtered through Whatman paper filter no.1. The filtrate muscle-related adverse effects in long-term use such as was completely dried in a freeze-dryer and stored at -20°C myalgia, myopathy and rhabdomyolysis.6 Thus, HMG-CoA until further use. The total yield percentage of the Sacha reductase inhibitors of plant origin are needed. Medicinal inchi extract was calculated by the formula, yield percentage plant that contains flavonoids showed anti-atherosclerotic (%) = (weight of extract obtained)/ (total weight of sample effect.7 Another way to reduce cholesterol level is inhibition loaded)×100. of cholesterol esterase (CE). The hydrolysis of dietary cholesterol Sacha inchi nuts were mainly used to produced ester into cholesterol by CE in the intestinal lumen is an sacha inchi oil which contain high amount of polyunsaturated essential process for absorption. Therefore, inhibition of fatty acid and antioxidant compounds such as tocopherol cholesterol esterase enzyme activity could inhibit absorption and vitamin A. Cold pressed Sacha inchi oil was received of dietary cholesterol esters.8-10 from Oil Star Tak limited partnership, Tak province, Thailand. Sacha inchi (Plukenetia volubilis L.) belongs to the The Sacha inchi oil was stored at room temperature. The family Euphorbiaceae. It is also known as inca peanut. It oil was dissolved in isopropanol into the desired concentration is a tropical rain forest Amazonian plant. Sacha inchi becomes before performing any experiments or added directly into an economic crop in Southeast Asia especially in Thailand. the reaction mix in the volume that meet the desired Sacha inchi seeds contain mainly oil and protein.