Influence of Extracting Solvent on Pharmacological Activity and Cytotoxicity of Polygonum minus, a Commonly Consumed Herb in Southeast Asia.

OBJECTIVE: To investigate the antihyperlipidemic, antioxidant, and cytotoxic effect of aqueous and methanol extract of leaves of Polygonum minus.
MATERIALS AND METHODS: Acute antihyperlipidemic effect was studied on chemically induced hyperlipidemic rat model. Treated groups received aqueous and methanol extract of leaves of P. minus respectively (1000 mg/kg; oral) whereas standard treated group received atorvastatin (60 mg/kg; oral) for 3 consecutive days. Blood samples were collected at fixed intervals for lipid profile analysis. Antioxidant effects were studied using 1,1-diphenyl-2-picrylhydrazyl, 2,2-azinobis 3-ethylbenzothiazoline 6-sulfonate, and ferric reducing antioxidant power assays. The total flavonoids content and total phenolic contents were also estimated. Cytotoxicity of both extracts was studied on one normal and three cancer cell lines using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay method.
RESULTS: The methanol extract showed significant reduction in total cholesterol (P < 0.001), triglycerides (P < 0.01), LDL (P < 0.05), VLDL (P < 0.01), atherogenic index (P < 0.001), and elevation of HDL (P < 0.05) levels than the aqueous extract. Similarly, the antioxidant investigations also demonstrated that the methanol extract had higher antioxidant capacity than aqueous extract. Both extracts were not toxic to normal (EA.hy926) as well as to cancer (HCT116, HT29, and HeLa) cells. Significant correlation was demonstrated between total phenolic and total flavonoids contents with the antioxidant activity but not with the antihyperlipidemic effect, suggesting other groups of chemical constituents may be mainly responsible for the antihyperlipidemic effect of this plant.
CONCLUSION: The study demonstrated that the presence and extent of bioactivities are influenced by solvents used for extraction. This study confirmed the antihyperlipidemic effect of leaves of P. minus in acute hyperlipidemic rat model.
SUMMARY: Polygonum minus is an herbaceous flowering plant.This plant possess high amount of phenolics and flavonoidsThis study focused on the antioxidant, cytotoxicity and antihyperlipidemic effect of aqueous and methanol extracts of leaves of P. minusThe extracts possess significant antioxidant activity and antihyperlipidemic activity but they are not toxic to normal and cancer cells tested.The antioxidant activity is well correlated with phenolic and flavonoids contents but the antihyperlipidemic activity is not correlated with antioxidant effect. Abbreviations used: CVDs: Cardiovascular diseases, LDL: Low-density lipoprotein, DDPH: 1,1-Diphenyl-2-picrylhydrazyl radical, TPTZ: 2,4,6,-tris(1-pyridyl)-5-triazine, ABTS: 2,2'-Azino-di-[3-ethylbenzthiazoline Sulfonate], HDL: High-density lipoprotein, VLDL: Very low-density lipoprotein, TC: Total cholesterol, TG: Triglycerides, EC50: Half maximal effective concentration, LD50: Median lethal dose.

INTRODUCTION

Hyperlipidemia, the commonest form of dyslipidemia refers to an increase in the levels of blood lipids, mainly the cholesterol and triglycerides. It is the major risk factor for endothelial damage leading to cardiovascular diseases (CVDs) such as atherosclerosis.[1] CVDs are one of the major causes of health-related death across the globe and their contribution in disease burden has been increasing in the developing and under developed countries.[2] Hyperlipidemia and oxidative stress induced by free radicals contribute to the development of CVDs, in particular atherosclerosis and other chronic diseases.[3] Management of hyperlipidemia can be achieved by both nonpharmacological and pharmacological interventions. Dietary modification is a key factor in the nonpharmacological intervention.[4] Pharmacological way of managing hyperlipidemia relied on two major classes of drugs, such as the statins and nonstatins. Fibrates are the main nonstatin drugs, which helps in the reduction of blood triglycerides levels but not effective in reducing low-density lipoprotein (LDL). The use of statins are linked with adverse effects, in particular elevated liver enzymes and rhabdomyolysis,[56] whereas fibrates are linked to inflammation of liver and muscles and gall stone formation.[7] Thus, the search for effective and well-tolerated lipid-lowering drugs is still on-going.

Natural products play a vital role in the search of new treatment agents for various diseases. About 50% of the drugs introduced in the market during the last 20 years have been derived directly or indirectly from small molecules of natural origin.[89] The antihyperlipidemic activities of many natural products have been established using experimental animal models. Commonly available plants such as Camellia sinensis, Tamarindus indica, and Garcinia atroviridis showed potential antihyperlipidemic activity in animal models.[101112] In addition, medicinal plants and their derived products such as garlic, fenugreek, and curcumin were found to be effective in lowering cholesterol in human trials.[1314] The excellent antioxidant capacity of many natural products is of advantage, in particular to prevent lipids peroxidation.

In South East Asia, several types of herbs or vegetables are consumed raw as salad for improving general health and as preventive measures against diseases. In Malaysia, these include selom (Ooenanthe javanic), pegaga (Centella asiatica), and kesum (Polygonum minus).[15] Fresh leaves of P. minus are usually consumed raw along or it is used in the cooking for flavoring purpose. P. minus is traditionally used as for digestive problems and to remove dandruff.[16] The pharmacological activities of P. minus were reviewed and reported its anti-LDL oxidation, antimicrobial activity, digestive enhancing activity, antiulcer activity, and immunomodulatory activity. Clinical trial data showed that the combination of P. minus and Eurycoma longifolia is more effective than placebo in enhancing sexual performance in healthy male volunteers.[17] Despite this, many other potential pharmacological effects of this plant including antihyperlipidemic remain unexplored. This comparative study was carried out to evaluate and compare the influence of extracting solvents (aqueous and organic) on the pharmacological activities of leaves of P. minus such as antihyperlipidemic activity and antioxidant. In addition, this study also evaluated the cytotoxicity of those extracts.

MATERIALS AND METHODS

Chemicals, standards, and reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tris(1-pyridyl)-5-triazine (TPTZ), 2,2-azinobis 3-ethylbenzothiazoline 6-sulfonate (ABTS), poloxamer 407, quercetin, morin, and potassium persulfate were purchased from Sigma (St Louis, MO, USA). Folin–Ciocalteu reagent, methanol, gallic acid, and l-ascorbic acid were obtained from Merck (Darmstadt, Germany). Sodium carbonate, sodium acetate trihydrate, aluminum chloride, ferric chloride hexahydrate, carboxymethyl cellulose (CMC), and ferrous sulfate heptahydrate were purchased from R&M Chemicals (Essex, UK). Atorvastatin was obtained from Ranbaxy Malaysia Sdn. Bhd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide was purchased from Sigma (St Louis, MO, USA). Normal cell line – human umbilical vein cell line (EA.hy926) and cancer cell lines – human colon cancer cell line (HCT116), human colon cancer cell line (HT29), and human cervical cancer cell line (HeLa) are kind gift from the Oncology and Radiology Science Cluster, Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia.

Plant materials

The raw materials of P. minus (Polygonaceae) were obtained from the Balik Pulau, Penang Island, Malaysia. A voucher specimen (USM Herbarium. 11542) was deposited at the Herbarium, School of Biological Sciences, Universiti Sains Malaysia.

Preparation of extracts

Fresh leaves of P. minus were separated from stems and rinsed thoroughly with water to remove dirt. The leaves were dried in an oven below 50 °C for 3 days. The dried leaves were pulverized using a blender. The powdered leaves were extracted by maceration using distilled water or methanol (powder : solvent ratio of 1:10 [w/v]) for 5 days. The extracts were decanted and replenished with fresh solvent every day. The pooled extracts were filtered through a cotton plug followed by Whatman No. 1 filter paper. The filtrate was then concentrated in a rotary evaporator (Yamato Rotary Evaporator RE300; Yamato Scientific, Tokyo, Japan) and freeze-dried (Thermo Scientific, Massachusetts, USA) to obtain the dry extracts. The extract was kept in refrigerator until further use.

EVALUATION OF ANTIHYPERLIPIDEMIC EFFECT

Animals

Male Sprague-Dawley rats of 14–16 weeks old, weighing initially about 180–220 g were used in this experiment. The animals were kept at room temperature and maintained on a 12 h light/dark cycle. Animals were allowed free access to standard food pellets (Gold Coin, Penang, Malaysia) and drinking water. Animals handling was in accordance with institutional guidelines and approval to use the animals was obtained from the Animal Ethics Committee, Universiti Sains Malaysia (Ref. No. USM/Animal Ethics Approval/2014/(94) (479)) and Animal and Human Ethics Committee, AIMST University (Ref. No. AUHAEC 7/FOP/2013).

Evaluation of antihyperlipidemic in poloxamer 407-induced acute hyperlipidemic rats

The rats were fasted overnight before experimentation with free access to water. Poloxamer 407 solution for administration was prepared by mixing the agent with saline solution and then refrigerated overnight to facilitate its dissolution.[18] Rats were divided into five groups of six animals each; group 1 served as normal control group and given injection of normal saline intraperitoneally (i.p.), whereas animals from groups 2–5 were injected with 500 mg/kg poloxamer 407 i.p. After 6 h administration of poloxamer 407, groups 1 and 2 received 0.5% solution of CMC, group 3 received 60 mg/kg atorvastatin, whereas groups 4 and 5 received 1000 mg/kg aqueous and methanol extracts of leaves of P. minus, respectively. The extracts and atorvastatin were prepared in 0.5% CMC and administered by oral route, once daily for 3 days. Blood samples (250 μL) were collected from the retro orbital plexus in eye before and at 10 and 34 h after hyperlipidemia induction, for determination of total cholesterol (TC) and triglycerides, whereas terminal blood samples of 1 mL were collected at 58 h after hyperlipidemia induction for determination of TC, triglycerides, LDL, and high-density lipoprotein (HDL) levels. Blood samples were centrifuged at 5000 RPM for 10 min to obtain the serum, which were stored at −80 °C before analysis. Animals were euthanized and the carcase was disposed through AIMST university bio-waste management system. Serum lipids were determined using commercial kits following the manufacturer's protocol (Thermo Fisher Scientific, Massachusetts, USA). Very low-density lipoproptien (VLDL) concentration was calculated using the equation: (VLDL cholesterol = triglyceride/2.22), whereas the AI was calculated using the formula (TC-HDL)/HDL.[19]

ANTIOXIDANT ASSAY

The in vitro antioxidant assays were carried out on aqueous and methanol extracts of P. minus. All determinations were done in triplicate. Absorbance was measured using Lab-System Multiscan MS microplate reader (Model No. 354; Helsinki, Finland).

Total phenolic content

Total phenolic content was determined using Folin–Ciocalteu reagent solution following method described by Kumaran and Karunakaran[20] with gallic acid as a standard. In brief, Folin–Ciocalteu reagent, sodium carbonate, standard or test samples (10 mg/mL), and distilled water were mixed in a test tube in a ratio of 5:15:1:79 to the total volume of 200 μL. Absorbance of the reaction mixtures was read at 765 nm after incubation for 2 h at room temperature. The results are expressed as μg gallic acid equivalent/mg dry extract.

Total flavonoid content

Total flavonoid content was determined by the aluminum chloride method, as described by Chang et al.[21] using quercetin as reference standard. In brief, 100 μL of standard or extract (10 mg/mL) solutions were mixed with 20 μL of aluminum chloride (10%, w/v), 20 μL of 1 mol/L sodium acetate, 300 μL methanol, and 560 μL distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. The results are expressed as μg quercetin equivalent/mg dry extract.

1,1-Diphenyl-2-picrylhydrazyl radical scavenging

DPPH radical scavenging activity was determined by method described by Wang et al.[22] Briefly, 100 μL of the test samples (at six concentrations), dissolved in methanol:H2O (1:1) were added to 100 μL DPPH (200 μmol/L) prepared in methanol and incubated at room temperature for 30 min. Morin was used as reference standard. The amount of remaining DPPH was determined at 517 nm. The results are expressed as EC50, which is the concentration where the scavenging activity is 50%.

2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate] radical scavenging

ABTS radical scavenging activity was measured by the method as described by Re et al.[23] Briefly, ABTS radical cation (ABTS·+) solution was prepared by mixing 14 mmol/L ABTS and 4.9 mmol/L potassium persulfate solutions in equal volumes. The solution was allowed to react in the dark at room temperature for 16–20 h before use. Then, 1 mL of the solution was diluted with 40 mL deionized water to yield working ABTS solution with an absorbance equal to 0.70 ± 0.02 at 734 nm. To 180 μL ABTS working solution, 20 μL test samples (at six concentrations) were added. Absorbance of the samples was read at 734 nm after 6 min. Ascorbic acid was used as reference standard. The results are expressed as EC50, which is the concentration where the scavenging activity is 50%.

Ferric reducing antioxidant power

The FRAP assay was carried out by method of Benzie and Strain.[24] Briefly, 150 μL FRAP working solution (300 mmol/L acetate buffer, pH 3.6, 10 mmol/L TPTZ in 40 mmol/L HCl and 20 mmol/L FeCl3 in a ratio of 10:1:1) was added to 50 μL test samples (10 mg/L). The reaction mixtures were incubated for 8 min before absorbance measurement at 600 nm. Ferrous sulfate (FeSO4 7H2O) was used as reference standard and the results are expressed as nmol Fe2+ equivalent/mg dry extract.

CYTOTOXICITY EVALUATION

In vitro cell cytotoxicity of aqueous and methanol extract of P. minus was carried out by tetrazolium salt (MTT) assay as described by Denizot and Lang.[25] Four types of cell lines, such as HCT116, HT29, HeLa, and EA.hy926 were used for this study. The cells were cultured in minimum essential medium (MEM). The medium also contains 10% fetal calf serum, penicillin (100 U), and streptomycin (100 μg). Concentrations of extracts used were in the range of 200–12.5 μg/mL, prepared by serial dilution method. Extracts were initially dissolved in 1% dimethyl sulfoxide (DMSO) and further diluted with MEM medium. The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 × 105 cells/mL using medium. To each well of the 96-well microplate, 0.1 mL of the diluted cell suspension (approximately 10 000 cells) was added. After 24 h, the media was removed, cells were washed once, and 100 μL of plant extracts at different concentrations were added to the cells in microplates. The plates were then incubated at 37° C for 3 days in 5% CO2 atmosphere and microscopic examination was carried out every 24 h. After 72 h, the drug solutions in the wells were discarded and 50 μL of MTT in MEM was added to each well. The plates were gently shaken and incubated for 3 h at 37° C in 5% CO2 atmosphere. The supernatant was removed and 50 μL of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the formula

Statistical analysis

The results were presented as the mean ± SEM. The statistical significance of difference was evaluated by analysis of variance, followed by Tukey's post hoc test. Pearson correlation analysis was done for total phenolic and total flavonoids contents against antihyperlipidemic and antioxidant activities.[13] A P-value of less than 0.05 was considered significant.

RESULTS AND DISCUSSION

The leaves of P. minus are widely used in cooking as flavoring agent or consumed raw as a salad leaf in South East Asia, other than being used in traditional medicines. The plant is used to relieve stomach ache, body pain, and other aches. Different pharmacological activities of this plant were reported, in particular for aqueous or methanol extracts.[17] Despite the belief among locals that this plant may reduce blood lipids, the antihyperlipidemic potential of this plant was yet to be explored extensively. In this study, we report the influence of aqueous and methanol solvents on potential antihyperlipidemic effect, antioxidant activity, and cytotoxic effect of P. minus extracts.

Increased levels of lipids are one of the major risk factors of cardiovascular diseases.[26] In this comparative study, poloxamer 407 induced acute hyperlipidemic rats were used to evaluate the antihyperlipidemic potential of P. minus. Hyperlipidemia in experimental animals can be induced chemically using agents such as poloxamer 407 and triton WR1339. Acute hyperlipidemic rat model is preferred due to its convenience, reproducibility, and lack of undesirable underlying pathological conditions.[1927] However, poloxamer-induced hyperlipidemic rat shows higher total cholesterol and triglyceride levels than that of diet-induced hyperlipidemia. Therefore, this method is a more suitable method for agents that have strong antihyperlipidemic activity.[28] The initial serum TC and triglycerides levels between all groups were not significantly different in this study. Upon induction with poloxamer 407, the hyperlipidemic control animals showed marked increases in TC and triglycerides levels, which were significantly higher than the normal control throughout the study. The antihyperlipidemic effect of aqueous and methanol extracts of P. minus leaves in hyperlipidemic rats is summarized in Table 1. The TC of the aqueous and methanol extract treated group was significantly lowered at 34 and 58 h whereas the triglycerides level showed no significant changes at 34 h but showed significant reduction at 58 h. However, the methanol extract treated group showed significant reduction of both TC and triglycerides levels at 34 and 58 h, and this effect was similar to atorvastatin-treated group.

Table 1

Effect of aqueous and methanol extracts of Polygonum minus on lipid parameters of Poloxamer 407-induced hyperlipidemic rats

The effects of aqueous and methanol extracts of P. minus leaves on other lipid parameters (LDL, HDL, VLDL, and AI) are shown in Table 1. The LDL, VLDL, and AI levels of hyperlipidemic control animals are significantly higher but the HDL level is significantly lower compared to the normal controls. Hyperlipidemic animals treated with methanol extract has significant reductions in LDL, VLDL, and AI, whereas aqueous extract treated group showed significant reductions of VLDL levels and AI but not LDL. In general, the findings showed that the methanol extract had better antihyperlipidemic activity than the aqueous extract. AI is used as one of investigational tool to evaluate the risk for coronary heart disease. Elevation and oxidation of LDL were related to the formation of plaque in blood vessels, leading eventually to atherosclerosis.[19] In this study, both methanol and aqueous extracts reduced the AI and the results suggest their beneficial role in atherosclerosis and coronary heart disease.

Further study was carried out to evaluate the antioxidant capacity of the aqueous and methanol extracts. The results are summarized in Table 2. A single antioxidant assay is not sufficient to clearly demonstrate the antioxidant capacity of an agent in a biological system.[29] Therefore, in this study, few antioxidant assays were used. The results showed that the antioxidant capacity of methanol extract of P. minus leaves were better than aqueous extract, whereby methanol extract had higher phenolic and flavonoids contents, ferric reducing power, and DPPH and ABTS scavenging activities (as indicated by lower EC50 values). The antioxidant and free radical scavenging capacity of plant extracts is mainly contributed by the presence of phenolic compounds, in particular the flavonoids and tannins. The redox property of the compounds contributes to the reducing, hydrogen donating and free radical scavenging abilities; other than acting as singlet oxygen quenchers and metal chelators.[30] Previous phytochemical investigations on P. minus reported the presence of flavonoids such as myricetin, quercetin, rutin, methyl flavonol (6, 7-4’,5’-dimethylenedioxy-3,5,3’-trimethoxyflavone), and flavone (6, 7-methylenedioxy-5, 3’,4’,5’-tetramethoxyflavone). These compounds possessed antioxidant activity and could contribute at least in part to antioxidant activity of P. minus extracts.[313233] The findings on the antioxidant activity of P. minus in this study is in agreement with previous reports, which found that aqueous, ethanol, and methanol extracts of P. minus possessed high antioxidant activities. The high polyphenol content, presence of vitamin C, and β carotene was suggested to be responsible for the antioxidant activity of P. minus.[34] Similarly, our study also found that the aqueous and methanol extract of P. minus had high amount of polyphenolic compounds.

Table 2

Antioxidant activities of aqueous and methanol extracts of Polygonum minus

Pearson correlation analysis was used to analyze the correlative relationships between total phenolic and flavonoids contents of the aqueous and methanol extract with their antioxidant and antihyperlipidemic effects. The results are tabulated in Table 3. The analysis showed that total phenolic and flavonoids contents had a significant negative correlation with DPPH and ABTS scavenging activities, and a significant positive correlation with FRAP for both the aqueous and methanol extracts of P. minus leaves. In contrast, both total phenolic and flavonoids contents showed no correlation with the antihyperlipidemic effects (any of the serum lipid parameters or AI). Therefore, the findings suggest that the phenolics and/or flavonoids may contribute to the antioxidant activities but not the antihyperlipidemic effect of the P. minus aqueous and methanol extracts. The antihyperlipidemic effect of P. minus extract may be due to the some other chemical constituents or due to the combination of antioxidant principles with other chemical constituents such as myricetin and tetradecanal,[3536] which are found to be effective in lipid-level modulation.

Table 3

Correlation between total phenolic content (TPC) and total flavonoids content (TFC) with antioxidant and antihyperlipidemic activity

The cytotoxicity potential of P. minus extracts is shown in Table 4. The results showed that both aqueous and methanol extracts are not cytotoxic against all the three cancer cell lines, with LD50 values of over 200 μg/mL. At 200 μg/mL, both extracts had maximum cell death of lesser than 42% against HCT116, HT29, and HeLa cells. In the case of normal cell (EA.hy926), the LD50 was found to be 92.2 and 214 μg/mL for aqueous and methanol extracts, respectively. Previously, cytotoxicity profile of P. minus on normal cell and cancer cell was reported on lung fibroblast cell line (Hs888Lu) and HeLa cells, respectively. The results showed that the extract was not cytotoxic on Hs888Lu cells but cytotoxic on HeLa cells at 0.1 mg/mL.[34] This study demonstrates that both the plant extracts are relatively safe toward living cells even at higher concentrations.

Table 4

In vitro cytotoxicity study of aqueous and methanol extracts of Polygonum minus against selected cell lines

CONCLUSIONS

This study evaluated the influence of different extracting solvent, namely water and methanol on the pharmacological activities and cytotoxicity of P. minus leaves extracts. Both aqueous and methanol extracts showed antihyperlipidemic effect but the methanol extract showed better effect and significant reductions in all lipid parameters (except an increase in HDL) and AI. Similarly, the antioxidant investigations also demonstrated that the methanol extract had higher antioxidant capacity than aqueous extract. Both extracts were not cytotoxic on normal as well as cancer cells tested. Significant correlation was demonstrated between total phenolic and total flavonoids contents with the antioxidant activity but not with the antihyperlipidemic effect of P. minus leaves extracts, suggesting other groups of chemical constituents may be mainly responsible for the antihyperlipidemic effect of this plant. In conclusion, the study demonstrated that the presence and extent of bioactivities of the extracts are influenced greatly by solvents used for extraction. The antihyperlipidemic effect of P. minus in acute model warrants further evaluation of its antihyperlipidemic effect in diet-induced chronic models and elucidation of the possible mechanism of action.

Financial support and sponsorship

This work was supported in part by AIMST University Internal research Grant (AURGC/18/FOP/2013).

Conflicts of interest

There are no conflicts of interest.

Vikneswaran Murugaiyah

ABOUT AUTHOR

Dr. Vikneswaran Murugaiyah, is an Associate Professor at Universiti Sains Malaysia, Penang, Malaysia. His research interest is on neurohormetic phytochemicals and cholinesterase inhibitors for neurodegenerative and lipid disorders. He is currently supervising Ph.D and M.Sc students in the field of pharmacology and phytochemistry. He has published a number of research and review articles in reputed impact factor journals.