Antioxidant and 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitory activities of some plant samples

The antioxidant activity capacities of some plants, and their inhibitory effects on the HMG-CoA reductase enzyme, the rate-determining enzyme of cholesterol synthesis, were investigated in our study. Antioxidant activity capacity and inhibitory effect of the HMG-CoA reductase enzyme were detected in all plant extracts used in our research. From the results obtained, it was determined that both antioxidant activity and % inhibition values of HMG-CoA reductase enzyme increased as the plant extracts concentration increased. The strongest ABTS and DPPH radical scavenging activities were exhibited by pomegranate fruit extract (IC 50 = 1.07 ±0.04 mg/mL and IC 50 = 0.39 ±0.01 mg/mL, respectively). At the same time, lemon had the strongest DMPD radical scavenging activity (IC 50 = 9×10 -4 ±6×10 -5 mg/mL). The best HMG-CoA reductase inhibitory activity was observed in persimmon fruit extract (IC 50 = 0.71 ± 0 .18 µg/mL). The inhibitory power of this extract was much higher than that of the enzyme’s standard inhibitor, Atorvastatin (IC 50 = 1.76 ±0.12 µg/mL). The extracts' potent antioxidant and inhibitory properties can be attributed to the rich phytochemical composition of plant extracts. Thus, it may be a potential source of new bioactive compounds effective against oxidative stress


Introduction
Free radicals are metabolic by-products continuously produced via normal metabolic processes of the biological system.These molecules are highly reactive and unstable, thus detrimental to organisms' general well-being.In humans, the excessive production of these molecules, coupled with compromised antioxidant-oxidant balance, present severe health challenges such as cardiovascular dysfunction, premature ageing, neurological disorders and mutagenesis (Aruoma, 1994;Bagchi and Puri, 1998;Floyd, 1999;Njie-Mbye et al., 2013) these lead modification and destruction of biologically important macromolecules such as lipids, proteins, DNA and carbohydrates.The biological system is armed with an antioxidant defence mechanism that helps eliminate the toxic effect of free radicals.This antioxidant defence system involves the activity of antioxidant enzymes (i.e., superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) that works synergistically with the non-enzymatic antioxidant systems that include vitamins (vitamin A, C and E), antioxidant minerals (i.e.copper, zinc and selenium), appropriate biological levels of uric acid and bilirubin, plantbased antioxidants (such as polyphenols) etc.Some of these antioxidant molecules can unilaterally capture or detoxify free radicals, thus essential for healthy living.
Cholesterol is a lipid synthesised by all animal cells via the mevalonate pathway (Brown and Goldstein, 1997;Espenshade and Hughes, 2007).It is an indispensable component of the cell membrane and an important precursor of steroid hormones, bile and vitamin D (Yeagle, 1991;Hanukoglu, 1992;Haines, 2001;Payne and Hales, 2004).Despite the biological significance of cholesterol, its elevated level in human blood has health consequences such as the increased risk of atherosclerosis, heart attack, stroke, and peripheral artery disease (Lewington et al., 2007;Brunzell et al., 2008).Various studies have shown that lowering blood cholesterol levels can reduce the risk of coronary heart disease, regress existing atherosclerotic lesions in peripheral arteries, and reduce the risk of stroke risk and cardiovascular disease (Grundy et al., 2019).Thus, regulating both intake and biosynthesis of cholesterol is essential for normal well-being.The regulation of cholesterol levels in patients usually involves the use of statins.These group of compounds are known to be excellent inhibitors of hydroxy methylglutaryl-CoA (HMG-CoA) reductase (an important enzyme of the mevalonate pathway), thereby having the ability to slow down the biosynthesis of mevalonate and accumulation of cholesterol, and its accompanying defects (Nicholls, 2008;Kizer et al., 2010).Despite the proven effects of HMG-CoA reductase in reducing blood cholesterol levels and risk cardiovascular disease, they are suggested to cause liver damage, neuropathy, pancreatic dysfunction, muscle pain/damage, bleeding stroke, an increased risk of diabetes mellitus and sexual dysfunction (Golomb and Evans, 2008;Bellosta and Corsini, 2012;Naci et al., 2013;Collins et al., 2016;Lehrer and Rheinstein, 2020).Therefore, finding alternative food-based sources capable of HMG-CoA reductase inhibition is of medical significance.
Plants are outstanding sources of phytoactive compounds, thus, exhibit remarkable antioxidant activities (Magaji et al., 2022).Reports have shown that flavonoids and polyphenols can prevent the oxidation of haemoglobin to methaemoglobin through the scavenging of nitrites, thereby preventing anaemia (Frei et al., 1988;Choi et al., 1988;Kang et al., 1996).Moreover, plant-based chemicals such as carotenoids, flavonoids, glycosides, polyphenols, saponins, and terpenes act as inhibitors of enzymes while also exhibiting regenerative, wound healing, anti-inflammatory, and antitumor activity.A diet rich in these phytochemicals positively correlates with lower degenerative and pathological diseases and improved well-being (Willcox et al., 2004;Mahato and Sharma, 2019).In the present study, the antioxidant activity and HMG-CoA reductase inhibitory effects of 80% ethyl alcohol extract of some plant extracts were investigated.

Preparation of Ethyl Alcohol Extracts
Five grams of the dried plant sample was placed in a 250 mL Erlenmeyer flask containing 50 mL of 80% ethyl alcohol; the flask was closed with a parafilm.The mixture was stirred continuously in a dark cupboard at room temperature for 7 days.The solvent used for extraction was removed from the resulting filtrate under reduced pressure using a rotary evaporator.The amount of extract obtained was weighed and recorded.

ABTS Radical Scavenging Activity
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was performed according to the method of Arnao et al. (2001).ABTS was dissolved in distilled water to make a 7.4 mM, adding 1 mL of 2.6 mM potassium persulfate.The solution was kept away from light at room temperature for 12 to 16 hours.To 1 mL of the resulting solution, 60 mL of methyl alcohol was added to obtain the freshly prepared ABTS radical cation solution required for each experiment.Plant extracts (150 µL) were added to 2850 µL of ABTS radical cation, then kept in the dark for 2 hours before taking absorbance reading at a wavelength of 734 nm.Butylated hydroxyanisole (BHA) was used as a standard ABTS radical scavenger.% ABTS radical scavenging activity was calculated as follows: ABTS radical scavenging activity (%) = [(A0-A1) / A0] x 100 A0=Control absorbance value.
A1=Absorbance value of sample and standard.

DMPD Radical Scavenging Activity
The basis of DMPD radical scavenging activity is that in acidic pH, DMPD forms a stable-coloured radical cation in a suitable oxidant solution.The DMPD extreme shows a maximum absorbance value of 505 nm.Antioxidants that transfer H atoms to the DMPD extreme lead to decolourising the radical solution.
In the radical scavenging assay, 1 mL of the radical solution was added to 0.5 mL of extract solutions.After 10 minutes, absorbance was taken using a spectrophotometer at 505 nm against the buffer solution.Epicatechin was used as a standard.DMPD radical scavenging activity was calculated as follows: Percentage DMPD radical scavenging activity = [(A0-A1) / A0] × 100 A0: DMPD initial absorbance A1: Absorbance of the sample

DPPH Radical Scavenging Activity
1,1′-diphenyl-2-picrylhydrazil (DPPH) radical scavenging activity was determined according to the method developed by Brand-Williams et al. (1995).A 20 mg/L solution of DPPH in methyl alcohol was prepared daily.To 1.5 mL of the DPPH solution, 0.75 mL of various concentrations of plant extracts (0.001-5 mg/mL) were added.The absorbance value was read after 5 minutes against the blank at 517 nm using a spectrophotometer.A 0.75 mL methyl alcohol and 1.5 mL DPPH solution were used as control.Rutin (0.0001-1.0 mg/mL) was used as a standard percentage of DPPH radical scavenging activity and was calculated using the following formula: DPPH radical scavenging activity (%) = [(A0-A1) / A0] × 100 A0: Absorbance of control A1: Absorbance of sample and standard material

HMG-CoA Reductase Inhibition Assay
The inhibitory activity of the HMG-CoA reductase was assayed using the procedure outlined in the CS1090 kit of Sigma Aldrich.Briefly, 910 μL of buffer was placed into a test tube and added 5 μL of the inhibitors (0.01-10.00 µg/mL).The solutions were thoroughly mixed before adding 20 μL of NADPH, and 60 μL of HMG-CoA, then allowed to stand for 5 minutes at room temperature.After that, 5 μL of HMG-CoA reductase was added to the mixture, gently mixed, and allowed to stand for another 5 minutes.The absorbance of samples was read spectrophotometrically at 340 nm against corresponding reagent blanks.Atorvastatin was used as the standard inhibitor of HMG-CoA reductase.
The outcomes of the present finding agree with previous reports that indicated antioxidant and free radical scavenging activity of reported quince, apple, grapefruit, lemon, pomegranate, persimmon, radish and onion.Quince has been shown to contain high levels of phytochemicals.The fruit exhibit promising antioxidant activity such as reducing power, ferric reducing antioxidant power (FRAP) and DPPH radical scavenging activity in the range of 70.9-89.5%,1.40-1.68µM and 79.91-82.61%,respectively (Mir et al., 2016).Similarly, Muzykiewicz et al. (2018) (2021) reported that apple juice had DPPH radical scavenging activity and iron-reducing power of up to 86.20 ±2.28 µg/mL and 1.93 ± 0.66 mg/mL, respectively.The highest total antioxidant activity observed was 0.46 ±0.08 mg BHT equivalent/g.More so, a study by Siqueira et al. (2013) have shown that apple has both strong DPPH radical and ferric-reducing antioxidant power (FRAP), thus exhibiting excellent antiradical effects.
Comparative antioxidant analysis between astringent, nonastringent and wild types of persimmons by Aydin (2021) indicated that the wild type had higher antioxidant potential than the other types.The ABTS, CUPRAC, DPPH, and FRAP activities were 112.95 ± 1.48, 550.24 ± 1.30, 232.56 ± 3.08 and 542.69 ± 1.84 µmol Trolox/g, respectively.Research into the antioxidant potentials of radishes revealed that some varieties of the fruit could have superoxide radical scavenging activity of up to 68.87% at 1000 µg/mL and a DPPH activity of about 20.78% (Park et al., 2016).Onion is an essential source of bioactive and antioxidant compounds as a vegetable.The antioxidant activities of this vegetable are reported not to be drastically influenced by heating or cooking (Moreno-Ortega et al., 2020).FRAP and DPPH radical scavenging activities were 12.40 ±0.12 mg Trolox/g dry mass and 8.00 ± 0.23 mg Trolox/g dry mass before heating.After heating for 10 minutes at 80 ο C, the FRAP and DPPH radical scavenging activities were reduced to 10.57 ±0.34 and 6.50 ±0.37 mg Trolox/g dry mass (Ren et al., 2017).
In general, the antioxidant activities exhibited by fruits and the vegetable investigated in the present study can be attributed to the phytochemical component such as ascorbic acid, tocopherol, quercetin, and rutin, among other vitamins and polyphenols present in the plant extracts.(Collins et al., 2016).Plant-based statins (such as lovastatin, pravastatin, and simvastatin) derived from sterols during the fermentation process play a significant role in cholesterol biosynthesis via HMG-CoA reductase inhibition, thus reducing the risk of cardiovascular diseases and infarction (Furberg et al., 1994;Byington et al., 1995;Pedersen et al., 1998;Furberg, 1999).The HMG-CoA reductase inhibitory activity of fruits and vegetables observed in the present study is likely due to the rich phytochemical composition of the plant, which is in the form of polyphenols, sterols, vitamins, and fatty acids.

Conclusion
In conclusion, the fruits and vegetable extracts examined in the present study showed strong antioxidant activity.The plants also exhibited promising HMG-CoA reductase inhibition activity, especially persimmon and radish fruit.These potent antioxidant and inhibitory properties can be attributed to the rich phytochemical composition of plant extracts.Thus, it can be a potential source of new bioactive compounds effective against oxidative stress, hypercholesterolemia and cardiovascular complications.

Table 1 :
Antioxidant activities of ethyl alcohol extracts from various plants.

Table 2 :
HMG-CoA reductase inhibition activity of ethyl alcohol extracts prepared from various plants * Mean ± SD of triplicate values

Compliance with Ethical Standards Conflict of interests:
The author(s) declares that for this article, they have no actual, potential, or perceived conflict of interest.Authors declare that this study includes no experiments with human or animal subjects.The Scientific Research Project Coordination Unit of Istanbul University-Cerrahpaşa supported this work.Project No. 28070