18 W. PrasongsubJournal et alof. Associated Journal of AssociatedMedical Sciences Medical 2021; Sciences 54 (3): 2021; 18-26 54(3): 18-26
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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. Commercially concentration. available Sacha inchi oil is beneficial for health. Sacha inchi oil contains high amount of long-chain n-3 fatty acids and Total phenolic content (TPC) antioxidant compounds such as tocopherol and vitamin The TPC of Sacha inchi extracts were analyzed spec- A.11-13 In addition, dried Sacha inchi leaves and nutshell are trophotometrically using modified Folin-Ciocalteau colorimetric available as tea. Many customers who consumed Sacha inchi method.17 Sacha inchi extracts were dissolved to the concentration products experienced good controlled of blood glucose, of 1 mg/mL with deionized (DI) water. Samples (100 µL)
Total phenolic content (TPC) The TPC of Sacha inchi extracts were analyzed spectrophotometrically using modified Folin-Ciocalteau colorimetric method.17 Sacha inchi extracts were dissolved to the concentration of 1 mg/mL with deionized (DI) water. Samples (100 µL) were mixed with 100 µL of Folin-Ciocalteau’s phenol reagent (Sigma-Aldrich, MO). Then, 300 µL of 20% sodium carbonate solution were added into the reaction and incubated at room temperature for 15 min. After incubation, 100 µL of DI water were added into the reaction and centrifuged at 1,250 ×g at 25˚C for 5 min. Supernatant were collected and measured an absorbance at 765 nm by using UV-Visible spectrophotometer (SPECORD PLUS 250, Germany). Diluted extracts were used as sample blank. Gallic acid (0- 300 µg/ml) was used as a standard. Calibration curve was plot against concentration of gallic acid and absorbance at 765 nm. TPC of extracts were expressed as mg of gallic acid equivalents/gram dried weight (mg of GAE/g).
2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay This study aimed to evaluate the free radical scavenging capacity of each Sacha inchi extracts, to reduce the radical cation ABTS·+ to ABTS according to the method described by Kokina et al.18 Briefly, ABTS stock solution was prepared by mixing ammonium persulfate (4.9 mM) and ABTS (7 mM) in equal ratio and incubating at RT in dark for 12-16 hrs. The working ABTS·+ solution was prepared by dilution of the stock solution with 80% methanol to absorbance at 734 equals to 0.700±0.020. Different concentrations (1-20 mg/mL) of Sacha inchi extract (10 µL) were added to 990 µL of ABTS·+ working solution. An absorbance (734 nm) was measured at 1 min after incubation by using UV-Visible spectrophotometer. L-ascorbic acid was used as a standard. Calibration curve was plot against concentration of ascorbic acid and absorbance at 734 nm. The results were expressed as mg of ascorbic acid equivalent/gm dried weight.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay Free radical scavenging activity of Sacha inchi extract was investigated using DPPH assay as described by Hajlaoui et al.19 Sacha inchi extracts (40 µL) at various concentration (1-20 mg/mL) were mixed with 1.2 mL of DPPH reagent and incubated at room temperature for 15 min. Samples were centrifuged at 1,250 ×g at 25˚C for 5 min after incubation. The supernatants were collected and measured absorbance at 517 nm by using UV-Visible spectrophotometer. Diluted extracts were used as sample blank. Gallic acid was used as a standard. Calibration curve was plot against concentration of gallic acid and absorbance at 765 nm. Results were expressed as mg of gallic acid equivalents/gram dried weight (mg of GAE/gm).
HMG-CoA reductase activity assay This study aimed to investigate inhibitory effect of Sacha inchi extracts on HMG-CoA reductase enzyme activity. The HMG-CoA reductase catalyzed HMG-CoA into mevalonate using NADPH. The HMG-CoA reductase activity was measured by the reduction of NADPH at 340 nm. Briefly, 4 L of NADPH, 1 L of samples, 12 L of HMG-CoA substrate, and 181 L of assay buffer were mixed. Pravastatin at final concentration of 0.2 µg/mL was used as inhibition control. To initiate the reaction, HMG-CoA reductase catalytic domain was added into reaction 20 W. Prasongsub et mixal. tureJournal. The of absorbance Associated (340 Medical nm) was Sciences measured 2021; at 54(3): 37 °C every18-26 15 sec for 5 min by UV-Visible spectrophotometer. For blank, the assay buffer was added into reaction tube instead of HMG-CoA reductase. The enzyme activity were mixed with 100 µL of Folin-Ciocalteau’swas calculatedphenol reagent using the followingtube instead equation of HMG-CoA: reductase. The enzyme activity (Sigma-Aldrich, MO). Then, 300 µL of 20% sodium carbonate was calculated using the following equation: solution were added into the reaction and incubated at ( ΔA340 minsample - ΔA340 minblank) ×TV room temperature for 15 min. After incubation, 100 µL of Units mgP = 12.44 ×V ×0.6 ×LP DI water were added into the reaction and centrifuged at ⁄ ⁄ 1,250 ×g at 25˚ C for 5 min. Supernatant were collected and where ΔA:⁄ change of absorbance, TV: total volume of the where ΔA: change of absorbance, TV: total volume of the reaction in mL, 12.44: coefficient of NADPH, V: volume measured an absorbance at 765 nm by using UV-Visible reaction in mL, 12.44: coefficient of NADPH, V: volume of of enzyme used in the assay, 0.6: enzyme concentration in mg-protein, LP: light path in cm. spectrophotometer (SPECORD PLUS 250, Germany). Diluted enzyme used in the assay, 0.6: enzyme concentration in extracts were used as sample blank. Gallic acid (0-300 µg/ml) mg-protein, LP: light path in cm. was used as a standard. Calibration curveCholesterol was plot esterase against activity assay concentration of gallic acid and absorbance at 765Cholesterol nm. TPC esteraseCholesterol inhibitory esterase activity activity of Sacha assay inchi extracts was evaluated spectrophotometrically at 20 of extracts were expressed as mg of gallic acid25 °C equivalents/gram using the method of AsmaCholesterola and Ream esterase. The inhibitory cholesterol activity esterase of Sacha from inchi porcine pancreas (CEase) was dried weight (mg of GAE/g). solubilized in 1 mL of 0.1 extractsM sodium was phosphate evaluated spectrophotometricallybuffer pH 7.0 and stored at 25 at °C-80˚C using as a stock CEase. Prior to use, the method of Asmaa and Ream20. The cholesterol esterase working CEase was prepared by diluting a stock CEase to 5 µg/mL with the same buffer. The reaction mixture 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) from porcine pancreas (CEase) was solubilized in 1 mL of assay 0.1 M sodium phosphate buffer pH 7.0 and stored at -80˚ C This study aimed to evaluate the free radical scavenging as a stock CEase. Prior to use, working3 CEase was prepared capacity of each Sacha inchi extracts, to reduce the radical by diluting a stock CEase to 5 µg/mL with the same buffer. cation ABTS·+ to ABTS according to the method described by The reaction mixture consisted of 500 µL of Triton X-100 Kokina et al.18 Briefly, ABTS stock solution was prepared by (5% w/w), 20 µL of p-nitrophenyl butyrate (0.05 M in mixing ammonium persulfate (4.9 mM) and ABTS (7 mM) acetonitrile), 40 µL of 2% acetonitrile in 400 µL of assay in equal ratio and incubating at RT in dark for 12-16 hrs. buffer (100 mM sodium phosphate, 100 mM sodium chloride, The working ABTS·+ solution was nsist prepared 00 by dilution T it n of -100pH7.0) ( % and/ ) 20 µL samples. -nit n The mixture t t was (0.0 mixed in and t nit i ) 40 2% the stock solution with 80% methanol t nit i to absorbancein 400 at ss incubate (100 at 25 °Cs i for 5 s t min. The reaction 100 wass i initiated i by .0) n 20 734 equals to 0.700±0.020. Differents s concentrations. T i t s (1-20 i adding n in t of 20 µL t of2 working in CEase. T (5 ti n µg/mL). s After initi 15t min in 20 mg/mL) of Sacha inchi extract (10 µL) were added to 990 µL of incubation at 25oC, the absorbance was measured by in s ( / ). A t 1 in in ti n t 2 t s n s s of ABTS·+ working solution. An absorbance (734 nm) was spectrophotometer at 405 nm. NaF was used as inhibition s t t t t 40 n . s s s in i iti n nt . T nt in i iti n t measured at 1 min after incubation by using UV-Visible control. The percentage of inhibition were calculated using spectrophotometer. L-ascorbic acid sin was t used in as a standard. : the following formula: Calibration curve was plot against concentration of ascorbic acid AbsActivity-AbsSample and absorbance at 734 nm. The results were expressed as %Inhibition = ×100 mg of ascorbic acid equivalent/gram dried weight. AbsActivity
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay Statistical analysis Data were expressed as mean±standard deviation Free radical scavenging activityStatistical of Sacha analysis inchi extract was investigated using DPPH assay as described by Hajlaoui et al.19 (SD) of three independent experiments. The data were t ss s n st n i ti n ( p ) t in n nt i nts. T t Sacha inchi extracts (40 µL) at various concentration (1-20 analyzed by one-way ANOVA. <0.05 was considered to be n n A VA p 0 0 s nsi t st tisti si ni i nt mg/mL) were mixed with 1.2 mL of DPPH reagent- and statistically. . significant. . incubated at room temperature for 15 min. Samples were centrifuged at 1,250 ×g at 25˚ C Resultsfor 5 min after incubation. Results Total phenolic content and antioxidant activity of Sacha inchi extracts The supernatants were collected and measured absorbance Total phenolic content and antioxidant activity of Sacha s n ts n n t s s in i t t it t t . T t i nt at 517 nm by using UV-Visible spectrophotometer. Diluted inchi extracts t in i t ts t . t t s t i st i nt n ts extracts were used as sample blank. Gallic acid was used Leaves, baby nuts and nutshells of Sacha inchi were as a standard. Calibration curve n was plotn t againsts s concentration s ti (T extracted 1). T with t isti hot water. Total s yield percentagen ts s n of the Sacha n ts t t t ts of gallic acid and absorbance at 517 s n nm. inResults i were 1. inchi extracts were calculated. Leaf extract showed the expressed as mg of gallic acid equivalents/gram dried highest yield percentage followed by baby nuts and nutshells, weight (mg of GAE/g). Table 1. T t respectivelyi nt (Table t1). t The characteristic t ts of leaves, t nutshells in i. HMG-CoA reductase activity assay and baby nuts hot water extracts were shown in Figure 1. This study aimed to investigate inhibitory effect of TableSacha 1 Total inchi yield percentageYield of hot of water extract extracts (%) of each Sacha inchi extracts on HMG-CoA reductase enzyme activity. part of Sacha inchi. The HMG-CoA reductase catalyzed HMG-CoA into mevalonate s 1 . 3 using NADPH. The HMG-CoA reductase activity was measured Sacha inchi Yield of extract (%) t s s 12. 4 by the reduction of NADPH at 340 nm. Briefly, 4 µL of NADPH, Leaves 19.73 1 µL of samples, 12 µL of HMG-CoA substrate, and 181 µL Nutshells n ts 12.6414. of assay buffer were mixed. Pravastatin at final concentration of 0.2 µg/mL was used as inhibition control. To initiate the Baby nuts 14.57 reaction, HMG-CoA reductase catalytic domain was added into reaction mixture. The absorbance (340 nm) was measured at 37 °C every 15 sec for 5 min by UV-Visible spectrophoA - B C tometer. For blank, the assay buffer was added into reaction
D E F
Figure 1. t isti t st t i s. A: s : n ts s : n ts in i - : s n in i t t t ts ( - ).
T s t in sin in- i t i t i ss s. in i n ts n n t t ts s i T t t s i t 4. n 3. A /100 i i t
4
nsist 00 T it n -100 ( % / ) 20 -nit n t t (0.0 in t nit i ) 40 2% t nit i in 400 ss (100 s i s t 100 s i i .0) n 20 s s. T i t s i n in t t 2 in. T ti n s initi t in 20 in s ( / ). A t 1 in in ti n t 2 t s n s s s t t t t 40 n . s s s in i iti n nt . T nt in i iti n t sin t in :
AbsActivity-AbsSample %Inhibition = ×100 AbsActivity
Statistical analysis t ss s n st n i ti n ( ) t in n nt i nts. T t n n - A VA. p 0.0 s nsi t st tisti si ni i nt.
Results Total phenolic content and antioxidant activity of Sacha inchi extracts s n ts n n t s s in i t t it t t . T t i nt t in i t ts t . t t s t i st i nt n ts n n t s s s ti (T 1). T t isti s n ts s n n ts t t t ts s n in i 1.
Table 1. T t i nt t t t ts t in i.
Sacha inchi Yield of extract (%)
s 1 . 3
t s s 12. 4
n ts 14.
W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26 21
A B C
D E F espectively. Sacha inchi lea e t acts an oil sho e lo e hich e e 1 . an 10.0 m A /100 m ie ei ht ( i e ).
Figure 1. Characteristic of tested materials. A: leaves, B: nutshells, C: baby nuts of Sacha inchi, D-F: corresponding dried hot water extracts (D-F). 0 espectivelyFigure 1. t isti . Sacha inchi lea t st e t acts t i s an . A oil: s sho e : n ts lo e s : hich n t e es 1 . in i an 10- .:0 s n in m A /100 m ie i t t t ts ( - ). c TPC ei ht was determined( i e ). using 0 Folin-Ciocalteu calorimetric 74.8 mg and 63.6 GAE/100 g dried weight, respectively. assays. Sacha inchi nutshell and baby nut extracts showed Sacha inchi leaf extracts and oil showed lower TPC which T s t in sin in- i t i t i b ss s. in i n ts n n t t ts much higher TPC compared to 0 others, which were about were 18.2 and 10.0 mg GAE/100 g dried weight (Figure 2). s i T t t s i t 4. n 3. A /100 i i t 0 0 c 0 0 4 0 0 b 0 0 a 0 0 m m A 100 ie ei ht 0 10
0 0 a ea aby n t t shell il 0 m m A 100 ie ei ht 10 Figure 2. otal phenolic content o i e ent pa ts o Sacha inchi e t acts an Sacha inchi oil at concent ation o 1 m /m . he ata0 e e e p esse as the mean S o sample teste in in epen ent e pe iments. al es havin i e ent lette s i e ea si ni icantly (p 0 aby.0 ). n t Nutshell t shell il Figure 2. Total phenolic content of different parts of Sacha inchi extracts and Sacha inchi oil at concentration of 1 mg/mL. The data were expressed as the mean±SD of sample he testedantio i ant in 3 independent activity experiments. as ete mine Values havingby different an lettersA S a icaldiffer significantly scaven in (p<0.05). assay. es lts om otal phenolic content o i e ent pa ts o Sacha inchi e t acts an Sacha inchi oil at concent ation o Figureantio i ant 2. activity by both metho s e e simila to the es lts om . Sacha inchi n t shell e t acts The1 m antioxidantm he ata activity e e e p esse was determined as the mean S by DPPH o sample78 mg teste GAE/100 in g in epen ent dried weight e pe iments and 450 mg al es ascorbic acid p esente / . the hi hest an A S a ical scaven in activity hich e e m A /100 ie ei ht. an and ABTS radical scavenging assay. Results from antioxidantp equivalent/ 100 g dried weight, respectively. In contrast, havin 0 i e entm asco bic lette s aci i e e ivalent si ni icantly/ 100 ( ie 0.0 ) ei ht . espectively. In cont ast Sacha inchi oil ha the lo est activity by both methods were similar to the results from Sacha inchi oil had the lowest antioxidant activity (Figure 3 antio i ant activity ( i e an i e ). TPC. Sacha inchi henutshell antio i ant extracts activity presented as ete mine the highest by and an Figure A S 4). a ical scaven in assay. es lts om DPPH andantio i ant ABTS radical activity scavenging by both metho sactivity which e e simila were to the es lts om . Sacha inchi n t shell e t acts p esente the hi hest 0 an A S a ical scaven in activity hich e e m A /100 ie ei ht an 0 m asco bic aci e ivalent/ 100 ie ei ht espectively. In cont ast b Sacha inchi oil ha the lo est 0 antio i ant activity ( i e an i e ). b 0
0 0 b 0 0 b 0 0 a 0 0
m m A 100 e t act c 0 0
0 10 a 0 0
m m A 100 e t act ea aby n t Nutshell t shell c il 0 Figure 3. DPPH radical scavenging activity of different parts of Sacha inchi extracts and Sacha inchi oil at concentration of 1 mg/mL. The data were expressed as the mean±SD10 of sample tested in 3 independent experiments. Values having different letters differ significantly (p<0.05).
0 ea aby n t t shell il
Figure 3. a ical scaven in activity o i e ent pa ts o Sacha inchi e t acts an Sacha inchi oil at concent ation o 1 m /m . he ata e e e p esse as the mean S o sample teste in in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ). 22 W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26
00
b 00 b 00
00 a
00 100 m m asco bic aci e ivalent 100 e t act
0 ea aby n t tNutshell shell il
Figure 4. ABTS radical scavenging activity of different parts of Sacha inchi extracts and Sacha inchi oil at concentration of 1 mg/mL. The data were expressed as Figurethe mean±SD 4. A S of sample a ical tested scaven in in 3 independent activity experiments. o i e ent Values having pa ts different o Sacha letters inchi differ e t acts significantly an (p<0.05). Sacha inchi oil at concent ation o 1 m /m . he ata e e e p esse as the mean S o sample teste in in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ). HMG-CoA reductase inhibitory activity The rest of the Sacha inchi extracts and Sacha inchi oil The HMG-CoA reductase inhibitory effect of Sacha had no effect on HMG-CoA reductase activity. Inhibition of HMG-CoA reductase inhibitory activity inchi extracts and Sacha inchi oil were evaluated. Pravastatin HMG-CoA reductase activity by Sacha inchi nutshell extract was used as an inhibitory he control- oA which e ctase can inhibit inhibito y HMG-CoA e ect o wasSacha dose-dependent inchi e t acts manner. an Sacha It inhibited inchi oil HMG-CoA e e eval ate reductase. reductase activity avastatin for as 75%. se Only, as anSacha inhibito y inchi extractcont ol from hich canactivity inhibit up to- oA99% e ctase at the concentration activity o %of. nly 250 µg/mLSacha nutshells presentedinchi e t act HMG-CoA om n t reductase shells p esente inhibitory activity- oA e ctase(Figure inhibit 6). o y activity abo t % at the concent ation o about 65%1 at the/m concentration ( i e ). he estof 125 o theµg/mL Sacha (Figure inchi e t acts5). an Sacha inchi oil ha no e ect on - oA e ctase activity. Inhibition o - oA e ctase activity by Sacha inchi n tshell e t act as ose- epen ent manne . It 0 inhibite - oA e ctase activity p to % at the concent ation o 0 /m ( i e ). 0 b 0 c
0
0
0 inhibition 0
0
10 a a a 0 ont ol avastatin Nutshell t shell ea aby n t Figure 5. HMG-CoA reductase inhibitory activity of different parts of Sacha inchi extracts (125 µg/mL). Distilled water was used as a negative control and pravastatinFigure 5. (0.2 µg/mL)- oA e ctasewas used as a inhibito ypositive control. activity All data o i e entwere expressed pa ts as theo Smean±SDacha inchi of sample e t acts tested (in1 3 independent /m ). istille experiments. Values ate having as different se as letters a ne ative differ significantlycont ol an (p<0.05). p avastatin (0. µ /m ) as se as a positive cont ol. All ata e e e p esse as the mean S o sample teste in in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ).
1 0 100
0 b e 0 Inhibition 0 c
0
a a 0 ont ol avastatin 25 50 75 125 250 t shell e t act m
Figure 6. Inhibition o - oA e ctase activity by va io s concent ations o Sacha inchi n tshell e t acts. istille ate as se as a ne ative cont ol an p avastatin as se as a positive cont ol. All ata a e p esente as the mean S o samples teste in in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ).
Sacha inchi n tshell e t act as the analy e the type o en ymatic inhibition by ine eave - plot analysis. he ine eave - plot analysis sho e pa allel line patte n ( i e ) in icatin the ncompetitive inhibition.
0
0 b 0 c
0
0
0 inhibition 0
0
10 a a a 0 ont ol avastatin t shell ea aby n t
Figure 5. - oA e ctase inhibito y activity o i e ent pa ts o Sacha inchi e t acts (1 /m ). istille ate as se as a ne ative cont ol an p avastatin (0. µ /m ) as se as a positive cont ol. All ata e e e p esse as the mean S o sample teste in in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ).
W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26 23
1 0 100
0 b e 0 Inhibition 0 c
0
a a 0 ont ol avastatin 25 50 75 125 250 tNutshell shell e t act m Figure 6. Inhibition of HMG-CoA reductase activity by various concentrations of Sacha inchi nutshell extracts. Distilled water was used as a negative control andFigure pravastatin 6. Inhibition was used o as a positive- oA control. e ctase All data activity are presented by va io s as the concent ationsmean±SD of samples o tested Sacha in 3 independentinchi n tshell experiments. e t acts Values. having istille different ate letters as differ se significantly as a ne ative (p<0.05). cont ol an p avastatin as se as a positive cont ol. All ata a e
Sachap esente inchi nutshell as the mean S extract was o further samples analyzed teste thein in epen entThe Lineweaver-Burk e pe iments plot. analysis al es showedhavin i e entparallel line lette s pattern type of enzymatic i e si ni icantly inhibition (p 0by .Lineweaver-Burk0 ). plot analysis. (Figure 7), indicating the uncompetitive inhibition.
1 Sacha inchi n tshell e t act as the analy e 1 the type o en ymatic inhibition by ine eave - plot analysis. he ine eave - plot analysis sho e pa allel line patte n ( i e ) in icatin the ncompetitive inhibition. 1
o inhibito 0 mlmL o e t act 1 mlmL o e t act 0
0
0
0 0 0 1 0 0 0 0 0 1 0 1 S
Figure 7. Lineweaver-Burk plot analysis for HMG-CoA reductase in the presence of different concentrations of HMG-CoA (8, 16 and 32 mM) and two different Figureconcentrations 7. ine eave of Sacha- inchi plotnutshell analysis extract (75 o and 125 - oAµg/mL). e ctase Data represents in the mean+SD p esence of 3 independento i e ent experiments. concent ations o - oA ( 1 an m ) an t o i e ent concent ations o Sacha inchi n tshell e t act ( an 1 /m ). ata ep esents mean+S o in epen ent e pe iments.
Cholesterol esterase inhibitory activity Sacha inchi e t acts an Sacha inchi oil e e eval ate o thei choleste ol este ase inhibition activity. a se as a positive cont ol ma e ly inhibite the choleste ol este ase abo t .0%. Amon Sacha inchi e t acts only n tshell an baby n t e t acts eveale choleste ol este ase inhibito y activity by .1 an .1% espectively ( i e ). Sacha inchi oil ( 0 µ /m ) ha no e ect on choleste ol este ase inhibition ( ata not sho n).
c 0
0 b b
0 Inhibition 1
10
a a 0 ont ol Inhibito a t shell aby n t ea
Figure 8. ect o Sacha inchi e t acts (1 /m ) on choleste ol este ase activity. e centa e o en yme inhibition e e calc late . ont ol as ep esente the con ition itho t inhibito . ata ep esents mean + S o in epen ent e pe iments. al es havin i e ent lette s i e si ni icantly (p 0.0 ).
1 1
1
o inhibito 0 ml o e t act 1 ml o e t act 0
0
0
0 0 0 1 0 0 0 0 0 1 0 1 S
Figure 7. ine eave - plot analysis o - oA e ctase in the p esence o i e ent concent ations o - oA ( 1 an m ) an t o i e ent concent ations o Sacha inchi n tshell e t act ( an 1 /m ). ata ep esents mean+S o in epen ent e pe iments.
Cholesterol esterase inhibitory activity Sacha inchi e t acts an Sacha inchi oil e e eval ate o thei choleste ol este ase inhibition activity. 24 W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26 a se as a positive cont ol ma e ly inhibite the choleste ol este ase abo t .0%. Amon Sacha inchi Cholesterole t acts esterase only n tshell inhibitory an activity baby n t e t acts eveale choleste olbaby nut este ase extracts inhibito y revealed activity cholesterol by esterase.1 an inhibitory.1% espectivelySacha inchi extracts( i e and ). S Sachaacha inchiinchi oiloil were ( 0 evaluatedµ /m ) ha noactivity e ect onby choleste ol38.1 and 24.1%, este ase respectively inhibition (Figure( ata not 8). Sacha for their cholesterol esterase inhibition activity. NaF used inchi oil (750 µg/mL) had no effect on cholesterol esterase sho n). as a positive control markedly inhibited the cholesterol esterase inhibition (data not shown). about 26.0%. Among Sacha inchi extracts, only nutshell and
c 0
0 b b
0 Inhibition 1
10
a a 0 ont ol Inhibito a Nutshell t shell aby n t ea Figure 8. Effect of Sacha inchi extracts (125 µg/mL) on cholesterol esterase activity. Percentage of enzyme inhibition were calculated. Control was represented the condition without inhibitor. Data represents mean+SD of 3 independent experiments. Values having different letters differ significantly (p<0.05). Figure 8. ect o Sacha inchi e t acts (1 /m ) on choleste ol este ase activity. e centa e o en yme inhibition e e calc late . ont ol as ep esente the con ition itho t inhibito . ata ep esents mean + S Discussiono in epen ent e pe iments. al es havin i e ent lette sand i e Amaranthus si ni icantly viridis ( p 0leaf. 0 were). 84%, 74%, 70%, 62% and Various parts of Sacha inchi are edible. Products of 72%, respectively.25-27 Fraction 18 of methanolic Ficus virens Sacha inchi in the market include roasted nut, cold press Ait extract showed a large number of HMG-CoA reductase nut oil, and tea that made from leaves and nut shells.11 inhibition about 98%.28 Therefore, anti-HMG CoA reductase Plants which contain antioxidant activity have been reported compounds from plant origin was very interesting because to also exert lipid lowering property.21 As both oxidative they can be used to lower blood cholesterol level. Cholesterol stress and hyperlipidemia are involved in atherosclerosis, lowering effect of Sachi inchi via HMG-CoA reductase inhibition plant that exert both effects might be useful for CVDs prevention. was firstly investigated in this study. The mechanism of enzyme Among various parts of Sacha inchi and sacha inchi oil, inhibition of Sacha inchi nutshell extract was also studied. nutshell extract showed the highest antioxidant activity Unlike statin drug which competitively inhibit HMG-CoA as well as total phenolic content. This data suggested that reductase,29 Sacha inchi nutshell extract showed uncompetitive phenolic compound might be the active ingredient in Sacha inhibition pattern. This type of inhibitor is more specific and inchi nutshell extract. efficient compare to other types of inhibitors.30 Uncompetitive Sacha inchi products have been claimed to reduce inhibitors bind only to the enzyme-substrate complex, while cholesterol level. However, the scientific data and the exact competitive inhibitors have affinity for the enzyme and mechanism are still limited. HMG-CoA reductase, a rate compete for substrate binding. limiting step enzyme in cholesterol biosynthesis, is the target Besides inhibition of HMG-CoA reductase, inhibition of for treatment of hypercholesterolemia. Statins are synthetic enzymes that control cholesterol absorption and transportation drugs that competitively inhibit HMG-CoA reductase.22 is important target for lowering blood cholesterol. Cholesterol Statins are commonly used to treat hypercholesterolemia. esterase is one of those enzymes that plays an important However, the adverse side effects from long term use are role in the regulation of cholesterol metabolism by extending in a great concern.23 Thus, HMG-CoA reductase inhibitors cholesterol intestinal absorption and transportation to from natural origin are in a great interest.24 enterocytes.20 Sacha inchi nutshell extract showed the This is the first report on anti-HMG-CoA reductase strongest anti-cholesterol esterase activity. Inhibition activity from Sacha inchi nutshell. Hot water extract of Sacha of cholesterol esterase could block cholesterol absorption inchi nutshell inhibited HMG-CoA reductase activity ranging and finally lower the blood cholesterol level. In this study from 28-99% according to the concentrations of the extract Sacha inchi oil did not have either anti-HMG CoA reductase (50-250 µg/mL). Previous studies on HMG-CoA reductase (data not shown) or strong antioxidant activity. However, inhibitors from plant origin have been reported. HMG-CoA previous studies have reported lipid lowering properties reductase inhibitory activity of crude extract of Quercus of Sacha inchi oil in vivo.11,31 These data indicated that the infectoria, Basella alba, Rosa damascene, Myrtus communis, mechanism of cholesterol lowering activity of Sacha inchi W. Prasongsub et al. Journal of Associated Medical Sciences 2021; 54(3): 18-26 25 oil did not cause by direct inhibition of HMG-CoA reductase. [8] Krause BR, Sliskovic DR, Anderson M, Homan R. All data suggested that Sacha inchi nutshell extract Lipid-lowering effects of WAY-121,898, an inhibitor might be a potential lipid lowering agent which can be of pancreatic cholesteryl ester hydrolase. Lipids used as a supplement for treatment of hypercholesterolemia. 1998; 33: 489-98. [9] Bailey JM, Gallo LL, Gillespie J. Inhibition of dietary Conclusion cholesterol ester absorption by 3-BCP, a suicide In conclusion, Sacha inchi nutshell extract showed inhibitor of cholesterol-esterase. Biochem Soc Trans the highest antioxidant activity and total phenolic content. 1995; 23: 408S. It might be able to reduce blood cholesterol level through [10] Jeon SM, Kim HS, Lee TG, Ryu SH, Suh PG, Byun the uncompetitive HMG-CoA reductase inhibition, and SJ, et al. Lower absorption of cholesteryl oleate in cholesterol esterase inhibition. As oxidative stress and rats supplemented with Areca catechu L. extract. hyperlipidemia are contributed to CVDs development, Ann Nutr Metab 2000; 44: 170–6. consumption of Sacha inchi nutshell which contain high [11] Wang S, Zhu F, Kakuda Y. Sacha inchi (Plukenetia amount of antioxidant might prevent CVDs. However, more volubilis L.): Nutritional composition, biological investigation in an in vivo model could further confirm the activity, and uses. Food Chem 2018; 265: 316-28. potential of Sacha inchi nutshell extract in treating [12] Vicente J, de Carvalho MG, Garcia-Rojas EE. Fatty hypercholesterolemia. acids profile of Sacha Inchi oil and blends by 1H NMR and GC-FID. Food Chem 2015; 181: 215-21. Conflict of interest [13] Chirinos R, Zuloeta G, Pedreschi R, Mignolet E, There are no conflicts of interest associated with Larondelle Y, Campos D. 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