Anti-proliferative, in vitro antioxidant, and cellular antioxidant activities of the leaf extracts from Polygonum minus Huds: Effects of solvent polarity

ABSTRACT

The present study reports the antioxidant and anti-proliferative activities of Polygonum minus leaf extracts obtained through sequential extraction using four solvents of varying polarities (i.e. hexane (HX), ethyl acetate (EA), methanol, and water). The antioxidant potential was evaluated by measuring the total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical-scavenging, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical-scavenging, superoxide anion and nitric oxide scavenging, ferrous ion-chelating (FIC), and cellular antioxidant activity (CAA) assays. The highest antioxidant potential was generally shown by the methanol extract (PM-MeOH). PM-MeOH exhibited the highest values for TPC (174.00 ± 0.18 mg GAE/g), TFC (53.19 ± 0.71 mg GAE/g), FRAP (1728.33 ± 0.96 µmol Fe²⁺/g), ABTS (226.25 ± 4.25 µmol TE/g), DPPH (1276.81 ± 7.08 µmol TE/g), and nitric oxide scavenging assays (IC₅₀, 675 ± 32.33 µg/mL). In the CAA assay, PM-MeOH dose-dependently inhibited the peroxyl radical-induced oxidation of 2ʹ,7ʹ-dichlorodihydrofluorescein (DCFH₂) to 2ʹ,7ʹ-dichlorofluorescein (DCF) in HCT116 cells, with an EC₅₀ value of 263.92 ± 21.60 µg/mL. Liquid chromatography and mass spectrometry analyses of PM-MeOH suggested the presence of tannins and flavonoids including apigetrin, hyperoside, isoquercetin, astragalin, miquelianin, quercetin, and quercitrin. P. minus hexane (PM-HX) and ethyl acetate (PM-EA) extracts showed selective cytotoxicity towards HCT116 with IC₅₀ values of 40.00 ± 0.83 µg/mL and 43.18 ± 0.67 µg/mL, respectively. Taken together, these results highlight the potential of P. minus as a source of bioactive phytochemicals that may be useful in cancer therapeutics and nutraceutical industry.

KEYWORDS:

• Polygonaceae
• Polygonum minus
• antioxidant
• antiproliferative
• sequential extraction
• kesum
• HCT116
• CCD841

Introduction

Chronic diseases, which include cardiovascular diseases, cancers, respiratory diseases, and diabetes, are the leading cause of disability and mortality globally. According to the World Health Organization (WHO), chronic diseases were responsible for 38 million (68%) of the world’s 56 million deaths in 2012.^[1] These diseases may develop from prolonged cellular redox imbalances in the body. Metabolic reactions in the living cells can produce reactive oxygen species and reactive nitrogen species (ROS/RNS), which when present in excessive amounts can oxidize and cause damage to various biological molecules such as lipid, DNA and proteins.^[2,3] This particularly happens during oxidative stress, arising from the disruption of cellular redox signalling and control, which causes increase in the pro-oxidant:antioxidant ratio of the body.^[4,5]

Among the chronic diseases, cancer, which caused 21.7% of the deaths worldwide in 2012, is of global challenge. Severe oxidative stress condition is a known factor that can contribute to the development of cancer.^[1] The presence of excessive ROS/RNS can cause DNA oxidation, leading to chromosomal defects, oncogene activation, abnormal gene transcription alteration, and ultimately malignant transformation.^[6,7] Cancers are generally treated by radiation therapy or radical surgery, which is often combined with adjuvant chemotherapy. However, these treatments present toxic side effects and could also exacerbate the disease condition by inducing an increase in the ROS/RNS levels, leading to poor antioxidant status in the body. This can further lead to severe oxidative stress, which could promote apoptosis resistance in cancer cells.^[8]

The uses of medicinal plants in complementary and alternative medicine have gained considerable attention lately, particularly for the treatment of chronic diseases. Plant-sourced foods are of particular significance, as they are rich in antioxidant molecules that could help prevent oxidative stress or redox imbalances and reduce the risk of developing chronic diseases.^[9,10] Antioxidants are defined as “any substance that can prevent, reduce, or repair the ROS/RNS-induced damage to a target biomolecule”.^[11] Dietary plant antioxidants have the potential to exert anticancer effects through several mechanisms, which include their ability to interfere with the various stages of carcinogenesis.^[9]

Polygonum minus Huds. (syn. Persicaria minor [Huds.] Opiz) is a tropical herb from the family Polygonaceae, locally known as “Kesum”, “Cenohom”, or “Daun Laksa” in Malaysia.^[12,13] It is frequently used as vegetable salad and as flavouring enhancer in many dishes. Traditionally, the leaves have been used to treat skin fungal infection, indigestion, dandruff, postnatal tonic, sprains, and alleviating body aches.^[12–14] The fragrant nature of P. minus is due to its high contents (72.54%) of volatile aromatic oils, with at least 48 different essential oils identified.^[15] Approximately 77 metabolites have been identified in P. minus by Gas Chromatography Mass Spectrometry (GC-MS) and these were mainly sesquiterpenes and aliphatic aldehyde compounds.^[16] The major essential oils were decanal (16.26%), 1-decanol (12.68%), and dodecanal (43.47%).^[15–17] The variation of dodecanal content among four different accessions of P. minus was reported to be about 10% (37.83–48.28%).^[18] The phytochemicals identified from P. minus include myricetin, quercetin, flavone, and methyl flavonol.^[19] P. minus methanolic extract was non-toxic to Sprague Dawley rats at a dosage of 2 g/kg^[20] and only caused moderate DNA strand breaks (16–31.4%) in lymphocytes at 250–2000 µg/mL.^[21] The n-hexane extract of P. minus leaf showed antimicrobial activity against methicillin-resistant Staphylococcus aureus.^[16] The water and methanol extracts of P. minus leaf showed antioxidant and acetylcholinesterase enzyme inhibition activities.^[16,22] The ethanolic extract of P. minus demonstrated the complete inhibition of 5-LOX and COX-1 activities at 100 µg/mL. The aqueous extract also demonstrated anti-inflammatory activity in a rat animal model through the inhibition of carangeenan-induced paw oedema.^[23] The ethyl acetate (EA) fraction of P. minus was cytotoxic and capable of inducing apoptosis and S-phase cell-cycle arrest in HepG2 cells.^[24] P. minus extracts have also been reported to show the following activities: antioxidant,^[25,26] anti-HSV1, anti-vesicular stomatitis virus,^[27] anti-fungal, anti-ulcer,^[28] anti-hyperlipidemic,^[29] anti-acetylcholinesterase,^[30] antimicrobial, and cytotoxicity towards HeLa cells.^[27,31] Other members of Polygonaceae have also been reported to show antimicrobial, antioxidant, anticancer, cytotoxic, anti-angiogenic, and anticholinesterase activities.^[32–34] The ethno-medicinal uses of P. minus and published data on the bioactivities of Polygonaceae family members provide justifications for the undertaking of the present study, to further characterise the antioxidant and anti-proliferative activities of P. minus. This may provide scientific insights of its benefits and justification for its ethno-medicinal uses.

Materials and methods

Reagents, solvents, and chemicals

The main reagents and chemicals used in this study were of analytical grade and mostly purchased from Sigma-Aldrich. Solvents used for the extraction of plant materials were obtained from Fisher Scientific and Merck Millipore. The following are some of the reagents used: gallic acid (Sigma, G7384); quercetin (Sigma, Q4951); trolox (Merck, 648471); DMSO (Sigma, D4540); DPPH reagent (Sigma, D9132); Ferrozine (Sigma, 82950); MTT reagent (Thermofisher Scientific, M6494); 2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ) (Sigma,T1253); Folin–Ciocalteu’s phenol reagent (Merck, Cat.# 1.09001); ABTS (Merck, 194430); and p-nitroblue tetrazolium chloride (NBT) (Merck, 484235).

Samples preparation

Fresh and healthy P. minus leaves were purchased from the local wet market and processed on the same day. The identity of the sample was confirmed by Dr Sugumaran Manickam of Rimba Ilmu, Institute of Biological Sciences, University of Malaya. The leaves were cleaned with water, sliced into smaller pieces, and dried in a 40 °C oven until no weight reduction was observed. The dried leaves were powdered using a table blender and stored at –20°C until further analysis.

P. minus extracts preparation

The dried leaves were extracted sequentially, using the following solvents in the order indicated: HX > EA > methanol > water.^[35] Dried leaves of 30 g weight were extracted using 300 mL of HX in an incubator shaker at 37°C for 8 h and centrifuged at 200G for 5 min. The supernatant was filtered through Whatman filter paper (No. 4). The extraction was repeated twice, and the residues were dried. The 900 mL of HX solvent that was collected after the extraction was evaporated at 40°C using a rotary evaporator (BUCHI Rotavapor R-215). The extraction was continued using the same method for each of the remaining solvents (EA, methanol, and water) following the method as indicated above. The filtrate of the water extract was dried using a freeze-dryer. The plant extracts were dissolved in 10% dimethyl sulfoxide (DMSO) at 2 mg/mL and kept at –20°C until use.

Antioxidant potential assays

Total phenolic content

Total phenolic content (TPC) was determined using Folin–Ciocalteu reagent (FCR).^[36] A total of 50 μL of aqueous FCR (10% v/v) was mixed with 100 μL of the plant extract and incubated for 5 min. Next, 350 μL of 1M Na₂CO₃ was added to the mixture and incubated at room temperature for 2 h, followed by absorbance measurement at 765 nm, using a polystyrene cuvette (BRAND®, Sigma-Aldrich, product #Z330418) and a VARIAN Cary®50 UV-VIS spectrophotometer. A standard calibration curve was constructed using gallic acid, using concentrations up to 0.2 mg/mL. Results were expressed as mg gallic acid equivalents (GAE) per gram of dried extract (DE).

Total flavonoid content

Total flavonoid content (TFC) was determined according to Chang et al.^[37] with minor modifications. Aluminium trichloride (10% w/v) and potassium acetate (1M), both at 5 μL volumes, were mixed with 25 μL of plant extract in a 96-well microplate. A total of 75 μL of ethanol (95% v/v) and 140 μL of distilled water were added to the mixture successively and mixed. The mixture was incubated for 30 min at room temperature followed by absorbance reading at 415 nm using Tecan Infinite® M1000 Pro Multimode reader (Tecan Trading AG, Switzerland) (Tecan-I). Quercetin was used as a standard and flavonoid values were expressed as milligram quercetin per gram of DE.

Ferric reducing antioxidant power

Ferric reducing antioxidant power (FRAP) was determined according to Benzie and Strain^[38] with minor modifications. FRAP reagent was freshly made by mixing the following solutions in a 10:1:1 volume ratio (v/v/v): acetate buffer (300 mM, pH 3.6), 10 mM TPTZ in 40 mM HCl, and FeCl₃.6H₂O (20 mM). Ten microliters of the plant extract was mixed with 300 μL of FRAP reagent in a 96-well microplate and absorbance reading at 593 nm was taken 30 min thereafter using a Tecan-I microplate reader. A calibration curve was plotted using FeSO₄ solution (0–1 mM). The results were expressed as μmol Fe²⁺ per gram of DE. Quercetin, gallic acid, ascorbic acid, and trolox were used as positive controls.

ABTS^●+ radical-scavenging activity

ABTS^●+ (2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid) radical-scavenging activity was determined according to Re et al.^[39] with minor modifications. ABTS^●+ radical cations were made by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate and left in the dark for 12–16 h at room temperature. The resulting solution absorbance was measured at 734 nm and adjusted to 0.7 using ethanol. Around 20 μL of plant extract was mixed with 180 μL of ABTS^●+ reagent in a 96-well microplate, and the absorbance at 734 nm was taken after 30 min using a Tecan-I microplate reader. Quercetin, gallic acid, ascorbic acid, and trolox were used as positive controls. The TEAC values were calculated for the extracts using the trolox calibration curve (0–1.6 mM). Results were expressed as μmol trolox equivalents (TE)/g DE.

DPPH^• radical-scavenging activity

The 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical-scavenging activity was determined according to Brand-Williams et al.^[40] with minor modifications. One hundred micromolar DPPH solution was prepared in methanol and left in the dark for at least 30 min. Next, 20 μL of plant extracts, at different concentrations (up to 1000 μg/mL), was mixed with 150 μL of 100 μM DPPH solution in a 96-well microplate. After 30 min, the absorbance of the reaction mixture was taken at 515 nm using a Tecan-I microplate reader. Quercetin, gallic acid, ascorbic acid, and trolox at concentrations up to 1000 μg/mL were used as positive controls. A calibration curve was constructed using trolox at concentrations up to 400 mM. DPPH radical-scavenging activity of the extracts was expressed as μmol TE per gram of DE. The DPPH radical-scavenging activity was calculated using the formula [(A₀—A_x) /A₀] x 100%, where A_x and A₀ are the absorbances in the presence and absence of test compounds, respectively. When significant scavenging activity was detected, the EC₅₀ value was also estimated.

Superoxide anion radical (O₂^•‒)-scavenging activity

Superoxide anion radical-scavenging activity was evaluated according to Robak and Gryglewski^[41] with slight modifications. Reagents consisted of 150 μM NBT, 468 μM nicotinamide adenine dinucleotide (NADH), and 60 μM phenazine methosulphate (PMS) in 0.1M phosphate buffered saline. Around 50 μL of extract, at different concentrations (0–1000 μg/mL), was mixed with 50 μL of NBT, 50 μL of NADH, and 50 μL of PMS in a 96-well microplate. After incubation in the dark for 10 min at room temperature, the absorbance was read at 570 nm using a Tecan-I microplate reader. Gallic acid was used as a positive control. The results are expressed as percentage inhibition of O₂^{• ‒} radicals. EC₅₀ was estimated when significant O₂^{• ‒} scavenging activity was observed.

Nitric oxide (^●NO) radical-scavenging activity

Nitric oxide scavenging activity was measured according to Awah and Verla^[42] with minor modifications. This assay uses sodium nitroprusside (SNP) to generate ^●NO. Griess reagent was used to measure nitrite (NO₂⁻) that was formed from the oxidation of ^●NO. Now, 25 μL of the extract, at different concentrations (0–1000 μg/mL), was mixed with 25 μL of freshly prepared 5 mM SNP solution in phosphate buffered saline (pH 7.3) in a 96-well microplate. The mixture was then incubated for 60 min under a visible polychromatic light (150 W). Griess reagent (50 μL), containing equal volumes of 1% sulfanilamide in 5% phosphoric acid (H₃PO₄) and 0.1% of naphthylethylenediamine dihydrochloride, was added to the mixture and incubated for 5 min before absorbance was read at 550 nm using Tecan-I microplate reader. Gallic acid was used as a positive control. Results were expressed as a percentage of NO radical-scavenging activity. EC₅₀ was estimated when a significant scavenging activity was observed.

Ferrous (Fe²⁺) ion-chelating ability

The ferrous (Fe²⁺) ion-chelating FIC ability was determined according to the method of Decker and Welch^[43] with minor modifications. For each determination, the following solutions were added and mixed sequentially in a 96-well microplate: 50 μL of each of the extracts (0–2000 μg/mL), 20 μL of 0.5 mM FeCl₂, 160 μL of distilled water, and 20 μL of 2.5 mM ferrozine. The mixture was allowed to stand for 10 min at room temperature. The ferrous iron–ferrozine complex formation was then monitored by taking the absorbance at 562 nm against a blank reaction mixture, which substituted the tested extract with 50 μL of distilled water. Ethylenediaminetetraacetic acid (EDTA) was used as a positive control. The EC₅₀ value is the concentration (mg/mL) at which the chelating activity of the tested compound was at 50%.

Cellular antioxidant activity assay

The cellular antioxidant activity (CAA) assay was carried out according to Wolfe and Liu^[44] with minor modifications. American Type Culture Collection (ATCC) human colon cancer cells HCT116 were seeded at a density of 5 x 10⁴ cells/well on a 96-well black microplate in 100 µL of growth medium. The cells were then incubated for 24 h, after which the growth medium was removed and the wells were washed with Phosphate Buffered Saline (PBS). Triplicate wells were treated with 100 µL of various concentrations of P. minus methanol extract (0–500 µg/mL) containing 25 µM DCFH-DA dissolved in the treatment medium. After 1 h of incubation, the media was removed and the cells were once again washed with 100 µL of PBS. Then, 600 µM of ABAP was applied to the cells in 100 µL of Hank's Balanced Salt Solution (HBSS) and the fluorescence intensity was measured with the Tecan-I microplate reader. Emission at 538 nm was measured with excitation at 485 nm every 5 min for 1 h. Each plate included a triplicate control and blank. The control wells consisted of cells treated with DCFH-DA and 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), while blank wells contained cells treated with the dye and HBSS only. After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value according to the following formula:

where ʃ SA is the integrated area under the sample fluorescence versus time curve and ʃ CA is the integrated area from the control curve. The median effective dose (EC₅₀) was determined from the median effect plot of log (ƒ_a/ƒ_u) versus log (dose), where ƒ_a is the fraction affected and ƒ_u is the fraction unaffected by the treatment.

Mass spectrometry analyses: Liquid chromatography ion-trap time-of-flight mass spectrometry (LCMS-IT-TOF)

Two mg of the DE was dissolved in 1 mL of HPLC-grade methanol and filtered using a 0.25 μm nylon filter membrane. The extract was analysed on a Shimadzu liquid chromatography ion-trap time-of-flight

(LCMS-IT-TOF) system, equipped with a Photodiode Array (PDA) UV detector (220, 254, and 350 nm) and an ion trap TOF mass spectrometer. The analysis was performed in positive and negative ion modes, using the Waters XBridge BEH C₁₈ column (PN 186003085, 2.1 × 50 mm, 2.5 μm) with the following parameters: solvent A: 0.1% formic acid; solvent B: acetonitrile with 0.1% formic acid; flow rate: 0.25 mL/min; column temperature: 40 °C; injection volume: 25 µL; gradient: 0–100% over 14 min. The compounds were identified by comparison of the MS data to those reported in the literature.

Anti-proliferative activity assay

The anti-proliferative activity of the extracts was evaluated through cell viability (MTT) (3,(4,5-dimethythiazol-2-yl)-2,3-diphenyl tetrazolium bromide) assay, according to Twentyman and Luscombe^[45] with minor modifications. HCT116 (passage #30) and CCD841 (passage #10) cell lines were cultured and maintained according to the protocol specified by ATCC. The cells (~5.0 × 10³) were incubated overnight in a 100 μL culture media/96-well plate and thereafter treated with 20 μL of plant extracts at different concentrations. After 48 h of incubation, 20 μL of MTT solution was added into each well and incubated for another 4 h. The culture solution was then discarded and 150 μL DMSO was added to solubilize the formazan crystals. Absorbance was then taken at 595 nm using a Tecan-I microplate reader. Each determination was performed in six replicates. When significant anti-proliferative activity was observed, the IC₅₀ value was determined.

Statistical analyses

Unless otherwise specified, each determination was performed in triplicate. Results were presented as mean ± standard error of mean (SEM) and analysed using SPSS statistical software (version 20.0, SPSS Inc.). One-way analysis of variance (ANOVA) and Tukey test were used to compare means among groups (three and more groups). Independent t-test was calculated to compare the mean between two groups. Pearson correlation was used to investigate the correlation between antioxidant components and the antioxidant activities. An observation is considered statistically significant if the p value was less than 0.05 (p < 0.05).

Results and discussion

Extraction yield

The types of solvents used for the extraction of phytochemicals from herbs are known to affect the yield and bioactivity of the resulting extract.^[35,46] The use of solvents with varying polarities can provide a better mass transfer medium, with a wider coverage of extraction efficiency. The present study is the first to report the sequential extraction of P. minus leaves using HX, EA, methanol (MeOH) and water (W), to characterize its anti-proliferative and antioxidant activities.

Extraction yields obtained for the various solvents are shown in Table 1. PM-MeOH extract demonstrated the most significant yield of 15.41%, while PM-W showed the second highest yield, which was 7.09%. When combined, the yield using polar solvents (i.e. MeOH and W) was 22.05%, while the yield from nonpolar solvents (i.e. HX and EA) was only 3.41%, suggesting that P. minus leaf extracts had more polar components. The distinctive structures and functional groups of plant phytochemicals are known to affect their polarity and solubility in the different extraction solvents used, and thus the yields would be dependent on such characteristics. Methanol is frequently reported to generate the highest yields when used as an extracting solvent.^[35]

Table 1. Extraction yields and antioxidant components of P. minus leaf extracts.

Extraction solvent	Total Phenolic Content, TPC (mg GAE/g extract)	Total Flavonoid Content, TFC (mg QE/g extract)	Extraction Yield (%)
Hexane	2.45 ± 0.05^a	10.18 ± 0.20^a	1.56
Ethyl acetate	37.18 ± 0.13^b	47.22 ± 0.71^b	1.85
Methanol	174.00 ± 0.18^c	53.19 ± 0.71^c	15.41
Water	73.90 ± 0.13^d	43.65 ± 0.36^d	7.09

Values are mean ± SEM (n = 3). For each column, means with different letters (^a–d) are significantly different (p < 0.05). GAE: gallic acid equivalent; QE: quercetin equivalent.

Total phenolic content

Phenolics are a major group of phytochemicals responsible for the nutritional and antioxidants activities in plants. They are able to become electron/hydrogen donor, reducing agent or metal chelator, which underlies the health benefits associated with the dietary consumption of plants. Thus, profiling of phenolic content is frequently carried out to justify the potential health benefits and bioactivity of plant-sourced foods.^[47,48] In the present study, the range of TPC values obtained among the extracts was from 2.45 ± 0.05 to 174.00 ± 0.18 mg GAE/g DE (Table 1). The TPC value for PM-MeOH in the present study was apparently higher than that reported by Huang et al.,^[49] which used freeze dried leaf/stem or whole plant materials from four different Polygonum species. The TPC values for PM-MeOH and PM-W extracts in the present study were also higher compared to those previously reported: 55.1 ± 0.9 mg GAE/g (water extract)^[21] and 12.9 ± 2.8 mg GAE/g extract (acetone extract),^[50] although they were lower compared with the ethanol extract (207 ± 0.011 mg GAE/g extract) that was reported by Qader et al.^[25]

Total flavonoid content

Flavonoids are phenolic compounds with diverse beneficial health-promoting activities, including reducing the risk of dementia, preventing LDL-oxidation and atherosclerosis, lowering cholesterol, and antioxidant and antihypertensive activities. The hydroxyl groups of flavonoids contribute to the radical-scavenging ability.^[9] In the present study, flavonoids were detected in all the extracts tested (Table 1), and their values ranged from 10.18 ± 0.20 to 53.19 ± 0.71 mg QE/g DE. PM-MeOH again showed the highest TFC value (53.19 ± 0.71 mg QE/g extract), followed by EA, W, and HX extracts, in descending order.

FRAP assay

This assay uses ferric tripyridyltriazine (Fe³⁺–TPTZ) complex that reacts with reducing agents or antioxidant molecules to form an intense blue Fe²⁺–TPTZ complex with a λ_max at 593 nm, and the colour intensity generated is proportional to the amount of antioxidant molecules present in the reaction mixture. In the present study, the FRAP values of all the extracts ranged from 26.11 ± 0.28 to 1728.33 ± 0.96 µmol Fe²⁺/g extract (Table 2). PM-MeOH showed the highest FRAP value (1728.33 ± 0.96 µmol Fe²⁺/g extract), followed by (in descending order) PM-W, PM-EA, and PM-HX. Borneo et al.^[51] categorized the strength of ferric-reducing capacities of antioxidants into four groups: low antioxidant power (<100 μmol Fe²⁺/g), medium (100–250 μmol Fe²⁺/g), high (250–625 μmol Fe²⁺/g), and extremely high (>625 μmol Fe²⁺/g). Based on such a classification, PM-MeOH and PM-W are considered to have extremely high antioxidant power. The respective FRAP values for PM-MeOH and PM-W extracts were about two and thirteen times higher than the values that were previously reported: 781.32 ± 4.2 µmol Fe²⁺/g MeOH extract^[24] and 68.3 ± 3.3 µmol Fe²⁺/g water extract.^[21]

Table 2. In vitro antioxidant activity assays of P. minus leaf extracts.

Extraction solvent	FRAP (µmol Fe^Citation2+/g extract)	ABTS^●+ (µmol TE/g extract)	DPPH^• (µmol TE/g extract)	Superoxide anion, IC50 (µg/mL extract)	Nitric oxide, IC50 (µg/mL extract)	FIC, EC50 (µg/mL extract)
Hexane	26.11 ± 0.28^a	25.43 ± 1.02^a	7.57 ± 0.67^a	NA	NA	NA
Ethyl acetate	351.14 ± 3.24^b	133.23 ± 0.92^b	255.59 ± 4.81^b	NA	NA	NA
Methanol	1728.33 ± 0.96^c	226.25 ± 4.25^c	1276.81 ± 7.08^c	195.64 ± 10.57^a	675 ± 32.33^a	NA
Water	898.33 ± 2.40^d	190.60 ± 2.48^d	699.83 ± 7.24^d	103.77 ± 3.86^b	1239 ± 42.15^b	341.54 ± 11.15^a
Positive controls
Gallic acid	23186.24 ± 549.37^e	42107.87 ± 45.21^e	21653.16 ± 295.67^e	25.60 ± 0.00^c	49.45 ± 0.95^c	–
Quercetin	12265.60 ± 376.32^f	12977.3 ± 69.36^f	9201.22 ± 335.19^f	–	–	–
Ascorbic acid	19399.65 ± 273.18^g	1567.27 ± 1.87^g	6464.54 ± 28.97^g	–	–	–
EDTA	–	–	–	–	–	38.62 ± 0.84^b

Values are mean ± SEM (n = 3). The mean values in each column with different letters (^a–g) are significantly different (p < 0.05). TE: trolox equivalent; IC₅₀: the concentration of the extracts that inhibited 50% of radicals; EC₅₀: the effective concentration of the extracts that chelated 50% of ferrous ions; EDTA: ethylenediaminetetraacetic acid.

ABTS^●+ radical-scavenging activity

ABTS^●+ reagent is a stable radical monocation that is directly produced from the oxidation of ABTS by potassium persulfate.^[39] The ABTS^●+ blue/green chromophore decolorizes when it is scavenged by antioxidant molecules and this can be monitored at 734 nm. The decrease in ABTS^●+ absorbance in the presence of the tested extract reflects the percentage of ABTS^●+ inhibition. The ABTS^●+ scavenging activity of P. minus has never been reported in the literature. The scavenging activities of PM leaf extracts are shown in Fig. 1A. PM-MeOH and PM-W extracts showed very prominent ABTS^●+ scavenging activity with IC₅₀ values of 159.62 ± 14.51 and 198.36 ± 15.27 µg/mL, respectively. When the ABTS^●+ scavenging was expressed as TEAC values (Table 2), they ranged from 25.43 ± 1.02 to 226.25 ± 4.25 µmol TE/g extract, with PM-MeOH and PM-W extracts showing the most prominent TEAC values.

Figure 1. In vitro antioxidant activities of P. minus extracts. (A) ABTS^●+radical-scavenging. (B) DPPH radical-scavenging. (C) Superoxide anion (O₂^•-) radical-scavenging. (D) Nitric oxide (^●NO) radical-scavenging. (E) Ferrous ion-chelating activity. The positive controls were: quercetin (Qn), gallic acid (GA) and ascorbic acid (AA). Values are means ± SD (n = 3). PM, P. minus; HX, hexane; EA, ethyl acetate; MeOH, methanol, W, water.

DPPH^• radical-scavenging activity

DPPH^•-scavenging activity is frequently used to evaluate the in vitro antioxidant activity of plant extracts. The scavenging of DPPH^• by antioxidants causes the bleaching of the purple-coloured solution. This assay is compatible for the evaluation of hydrophilic and lipophilic antioxidants activities.^[30] The DPPH^•-scavenging activity of the extracts is shown in Fig. 1B. With the exception of the HX extract, all other extracts showed concentration-dependent DPPH^•-scavenging activity. PM-MeOH and PM-W scavenging activities appeared to increase rapidly and reached their maximum values at ~500 μg/mL. The IC₅₀ values for PM-MeOH and PM-W extracts were 125.51 ± 4.70 and 210.16 ± 1.35 μg/mL, respectively, and these values imply a higher scavenging activity compared with that reported by Ghazali et al.,^[24] which were 540.0 ± 10.8 μg/mL for PM-MeOH and 472.5 ± 15.2 μg/mL for PM-W.

Superoxide (O₂^•‒) anion-radical scavenging activity

Superoxide anions can be produced in the human body through normal cellular metabolism. However, excessive amounts can cause cellular damage.^[11] The O₂^•‒ scavenging activity was assayed using the PMS-NADH-NBT system, where O₂^•‒ was generated by the oxidation of NADH in the PMS/NADH coupling reaction, followed by the reduction of NBT. The presence of antioxidants that can scavenge O₂^•‒ is indicated by the decrease in absorbance at 570 nm. The O₂^•‒ scavenging activity for PM leaf extracts is shown in Fig. 1C. The present study is the first to report the O₂^•‒ scavenging activity of P. minus. PM-W and PM-MeOH extracts showed concentration-dependent inhibition of O₂^•‒. In this case, PM-W showed the strongest activity, reaching 90% O₂^•‒ inhibition at ~500 μg/mL, followed by PM-MeOH, reaching 80% O₂^•‒ inhibition at ~1000 μg/mL. The IC₅₀ values for PM-W and PM-MeOH were 103.77 ± 3.86 and 195.64 ± 10.57 µg/mL, respectively (Table 2).

Nitric Oxide (^●NO) Radical-scavenging Activity

Nitric oxide is an important signalling molecule in various biological functions, which include vasodilation, neurotransmission, regulating hormone secretion, destruction of pathogenic microbes and killing of tumour cells.^[3] However, ^●NO can also pose undesired biological effects. It is pro-oxidant at high concentrations and could generate extremely reactive peroxynitrites, which damages biomolecules.^[3] Thus, the evaluation of ^●NO-scavenging activity of plant extracts is frequently carried out to evaluate their antioxidant potential. The present study is the first to assess the ^•NO scavenging activity of P. minus, which is shown in Fig. 1D. Only two of the extracts showed robust ^●NO-scavenging activity and they were PM-MeOH and PM-W, with IC₅₀ values of 675 ± 32.33 and 1239 ± 42.15 µg/mL, respectively. The IC₅₀ value for gallic acid was 49.45 ± 0.95 µg/mL. PM-MeOH and PM-W extracts were earlier observed to have among the highest TPC values (Table 1). Substantial amounts of tannins (natural plant polyphenols), the active components underlying the ^●NO-scavenging activity in traditional crude drugs,^[52] have been previously reported in the MeOH extracts of Polygonum species.^[49] The presence of galloyl groups in polyphenols, especially tannins, plays an important role in ^●NO-scavenging activities.^[53] This may provide an explanation on the prominent ^●NO-scavenging activity of PM-MeOH extract that was observed in the present study.

FIC activity

Ferrous ions (Fe²⁺), which are involved in processes such as enzyme activity and oxygen transportation, are essential in biological systems. However, Fe²⁺ produces highly reactive hydroxyl radicals when reacting with hydrogen peroxide, and such reactive species can induce oxidative damage.^[11] Thus, the evaluation of the metal-chelating ability of antioxidant candidates is of significance to gauge their antioxidant potential. In this assay, the ferrozine reagent reacted with Fe²⁺ to form a stable Fe²⁺–ferrozine complex. The magenta-coloured complex can be disrupted in the presence of antioxidants that can act as a metal chelator. The bleaching of Fe²⁺–ferrozine complex can be spectrophotometrically monitored at 562 nm to deduce the coexisting metal chelator(s).^[43] In the present study, all extracts of P. minus showed concentration-dependent metal chelating activity, although their FIC activities differed (Fig. 1E). PM-W extract showed robust FIC activity with an EC₅₀ value of 341.54 ± 11.15 µg/mL extract (Table 2) and reached > 70% chelation activity at less than 500 µg/mL. The EC₅₀ of PM-W extract was recently reported to be 5400 µg/mL,^[26] which was ~15 times higher compared with the value obtained in the present study, suggesting that the sequential extraction employed in this study produced PM-W extract with a much better FIC activity.

Correlation analysis

When analysed together, TPC and TFC values of the extracts imply strong (r > 0.8) and positive correlations (r = 0.60–0.79) with FRAP, ABTS, DPPH, and ^●NO-scavenging antioxidant activities (Table 3). This is suggestive of the effects of polyphenol and flavonoid constituents to the observed antioxidant activities of the PM leaf extracts. The polyphenol content showed a strong positive correlation with O₂^•‒ radical-scavenging activity, but negative correlation (r = −0.2 to −0.39) with FIC activity. Flavonoids demonstrated moderately positive correlation (r = 0.4–0.59) with O₂^•‒ radical-scavenging activity, as well as negative correlation with FIC activity.

Table 3. Pearson correlation analyses of antioxidant components and antioxidant activities.

	FRAP	ABTS	DPPH	O2^•‒	^●NO	FIC
TPC	0.993**	0.876	0.988*	0.783	0.886	−0.296
TFC	0.745	0.926	0.751	0.572	0.921	−0.202

Significant correlation at p < 0.05* and at p > 0.01**

Cellular Antioxidant Activity

The present study is the first report that measures the CAA activity of P. minus. CAA assay provides a complementary approach in assessing the antioxidant activity of compounds, since it takes into account biological factors in living systems such as membrane permeability, cellular uptake, metabolism, and antioxidant distribution.^[44] In the present assay, the ability of the PM-MeOH extract (and quercetin, as a positive control) to inhibit the oxidation of non-fluorescent 2ʹ,7ʹ-dichlorodihydrofluorescein (DCFH₂) to highly fluorescent 2ʹ,7ʹ-dichlorofluorescein (DCF) inside HCT116 cells was measured, while ABAP acted as a peroxyl radical generator.

The results indicated that PM-MeOH extract and quercetin were able to inhibit the oxidation of DCFH₂ in a dose-dependent manner (Figs. 2A and 2B). This suggests that the bioactive antioxidant(s) in the PM-MeOH extract was well absorbed into the HCT116 cells and exhibited antioxidant activity. The dose–response curves are shown in Figs. 2C and 2D, while the median effect plots are shown in Figs. 2E and 2F. The values of EC₅₀, which is the concentration at which ƒ_a/ƒ_u = 1 as calculated from the linear regression of the median effect plot,^[44] for PM-MeOH and quercetin were 263.92 ± 21.60 µg/mL and 93.76 ± 5.28 µg/mL, respectively. Although the EC₅₀ value of PM-MeOH extract was higher than that of quercetin, it was significantly lower than the values reported for extracts from common fruits (EC₅₀ > 2 mg/mL) and vegetables (EC₅₀ > 19 mg/mL).^[44,54] As for quercetin, its EC₅₀ value (310 ± 17.32 µM) in HCT116 cells was higher than HepG2 cells (EC₅₀, 5.09 ± 0.19 µM),^[44] suggesting that the antioxidant activity of quercetin inside HCT116 cells was less prominent.

Figure 2. Cellular antioxidant activity of PM-MeOH extract. Inhibition of peroxyl radical-induced oxidation of DCFH₂ to DCF in HCT116 cells by (A) PM-MeOH and (B) Quercetin. Dose–response curve for the inhibition of peroxyl radical-induced DCFH₂ oxidation by (C) PM-MeOH and (D) Quercetin. Median effect plots for the inhibition of peroxyl radical-induced DCFH₂ oxidation by (E) PM-MeOH and (F) Quercetin. The values shown are from a single experiment (mean ± SEM, n = 3).

Liquid chromatography mass spectrometry analysis

When PM-MeOH extract was subjected to LCMS-IT-TOF analysis, distinctive m/z values were detected (Figs. 3A and 3B), with eight of the components deduced (Table 4) based on published mass spectrometry data for five Polygonum species, including Persicaria hydropiper.^[30,49] The compounds identified were tannins and the flavonoids, including apigetrin, hyperoside, isoquercetin, astragalin, miquelianin, quercetin, and quercitrin. These compounds are known to exhibit antioxidant and free radical-scavenging activities^[49,52,53] and thus may, in part, explain the prominent antioxidant activity of PM-MeOH extract. Flavonoids have been reported to be important bioactive constituents of Polygonum species, which includes quercetin-3-O-rhamnoside (quercitrin).^[49] Quercetin and quercetin-3-O-rhamnoside in the EA extract of Persicaria hydropiper have been previously reported to show significant antioxidant activity, with IC₅₀ values of less than 20 µg/mL in the DPPH-scavenging assay.^[30]

Table 4. Liquid chromatography and mass spectrometry analyses (LCMS-IT-TOF) of PM-MeOH extract.

Peak (s)	Retention time (min)	m/z [M-H]^−	m/z [M-H]^+	Proposed compound
1	4.8	289	291	Tannins
1,5	4.8, 6.2	365	367	Unknown
1	4.8	431	433	apigenin-7-O-glucoside (apigetrin)
1,3,4	4.8, 5.5, 5.8	447	449	Unknown, kaempferol-3-O-glucoside (astragalin), quercetin-3-O-rhamnoside (quercitrin)
1,2,3	4.8, 5.2, 5.5	463	465	quercetin-3-O-galactoside (hyperoside), quercetin-3-O-glucoside (isoquercetin), Unknown
1	4.8	577	579	Unknown
2,3	5.2, 5.5	477	479	Unknown, Quercetin-3-O-β-D-glucuronide (miquelianin)
3	5.5	285	287	Unknown
4	5.8	301	303	Quercetin
4	5.8	381	-	Unknown
4	5.8	461	463	Unknown
5	6.2	285	287	Unknown
6	22.8	885	887	Unknown
7	23.6	870	872	Unknown

The table shows the m/z values of the proposed and unknown compounds detected by the analysis.

Figure 3. Ultrahigh-performance liquid chromatography (UHPLC) and mass spectrometry analyses of PM-MeOH extract. (A) UHPLC chromatogram for PM-MeOH extract. (B) LCMS-IT-TOF analyses. Representative PM-MeOH extract spectrum at a retention time of 4.8 min.

Anti-proliferative activity of P. minus leaf extracts

In the present study, the effect of P. minus leaf extracts on the viability of normal colon (CCD841) and colon cancer (HCT116) cell lines was evaluated through MTT assay. Two of the extracts (i.e. PM-MeOH and PM-W) appeared to show a dose-dependent stimulation of CCD841 cells proliferation (Fig. 4). At 333 µg/mL, PM-MeOH and PM-W stimulated the proliferation of CCD841 cells, increasing the cell viability percentage to 139% and 160%, respectively. When the experiment was repeated using HCT116 cells, the PM-W extract was again observed to stimulate cell proliferation, increasing the percentage of HCT116 cell viability to 134% and 157%, at 167 µg/mL and 333 µg/mL, respectively. These observations suggest that PM-MeOH contains compound(s) that could preferentially encourage the healing of normal colon cells, without stimulating the proliferation of HCT116 colon cancer cells. In contrast, PM-W extract contains compound(s) that could promote the progression of colon cancer.

Figure 4. MTT anti-proliferative assay of P. minus extracts on HCT116 and CCD841 cell lines. The numbers beside the legends indicate concentration of P. minus extracts in µg/mL. The percentage of cell viability is the ratio of cell viability for the treated over untreated cells x 100%. Values are mean ± SD (n = 6) . The PM-EA extract had lower solubility in DMSO, thus lower range of concentrations (26.86, 53.72 and 107.44 µg/mL) were used. The values with different letters (a-c) are significantly different, p < 0.05 (Tukey’s test).

In the same experiment, PM-HX and PM-EA extracts also appeared to show prominent anti-proliferative activity towards HCT116 cells, causing more than 50% reduction in the cell viability percentage (Fig. 5). The anti-proliferative activity of these extracts on HCT116 was investigated further by determining their IC₅₀ values, since these extracts were more cytotoxic towards HCT116 compared with CCD841. PM-HX and PM-EA were able to inhibit HCT116 cells proliferation in a dose-dependent manner (Fig. 5). The IC₅₀ values for these two extracts on CCD841 were markedly higher than HCT116, indicating their selective cytotoxicity towards colon cancer cells (Table 5). A “Selectivity Index” (SX) was then calculated based on the following formula: SX = IC₅₀ CCD841/IC₅₀ HCT116 x 100.^[55] An SX value of >100 implies that the cytotoxic effect is more selective on the cancer cells. Both PM-HX and PM-EA extracts showed SX value >100, implying selective toxicity towards colon cancer cells.

Table 5. Anti-proliferative activity of PM-HX and PM-EA extracts on HCT116 and CCD841 cell lines.

Extracts	HCT116 cell line, IC50 (µg/mL)	CCD841 cell line, IC50 (µg/mL)	Selectivity Index (SX)
PM-HX	40.00 ± 0.83^a	190.62 ± 6.41^a	478
PM-EA	43.18 ± 0.67^b	89.20 ± 2.09^b	207

The table lists IC₅₀ values of PM-HX and PM-EA extracts, with the corresponding selectivity index (SX). Values are mean ± SEM (n = 6). The mean values in each column with different letters (a and b) are significantly different (p < 0.05).

Figure 5. Cell viability assay. Dose–response curves for PM-HX and PM-EA extracts on HCT116 cells (48 h treatment).

A previous study has reported that the water and ethanol extracts of P. minus did not show any cytotoxicity towards normal lung cell line (Hs888Lu) at 500 µg/mL.^[25] Ghazali et al.^[24] reported that P. minus exhibited anti-proliferative activity on the following cell lines, with the corresponding IC₅₀ values: HepG2 (EA extract, 32.25 ± 3.72 µg/mL); HeLa (EA extract, 63.09 ± 6.70 µg/mL); WRL-68 (petroleum ether extract, 56.23 ± 3.2 µg/mL); and HCT116 (MeOH extract, 86.3 ± 3.5 µg/mL; EA extract, 199.52 ± 5.2 µg/mL). In the present study, the IC₅₀ of PM-EA extract on HCT116 cells was significantly lower compared to that reported by Ghazali et al.,^[24] suggesting that the sequential extraction employed in this study was able to produce PM-EA extract with a higher anti-proliferative activity.

In an attempt to elucidate the cytotoxicity mechanism induced by PM-HX and PM-EA extracts, further analysis was performed using ApoTox-Glo™ Triplex Assay, Muse® MultiCaspase, Muse® Annexin V, and Dead Cell Assay Kits. However, the results suggested that the cytotoxic effect of the extracts on HCT116 cells was not caspase-dependent (data not shown). Hence, the extracts’ anti-proliferative activity might have been achieved through other programmed cell death mechanisms such as necroptosis or paraptosis.^[56]

Conclusion

In the present study, PM-MeOH extract was generally observed to have the highest antioxidant potential, which is most likely attributed to its relatively higher phenolic and flavonoid contents. LCMS-IT-TOF analyses of the PM-MeOH extract suggested the presence of tannins, apigetrin, hyperoside, isoquercetin, astragalin, miquelianin, quercetin, and quercitrin. Results from the CAA assay indicated that the PM-MeOH extract was well absorbed into HCT116 cells and it was able to provide protection against oxidative damage at the cellular level, with an EC₅₀ value of 263.92 ± 21.60 µg/mL. PM-HX and PM-EA extracts showed selective cytotoxicity towards HCT116 with IC₅₀ values of 40.00 ± 0.83 µg/mL and 43.18 ± 0.67 µg/mL, respectively. The present study suggests that P. minus is a promising source of bioactive phytochemicals that may be useful in cancer therapeutics and nutraceutical industry. Further research is warranted to isolate the anticancer and antioxidant components, to understand their health-promoting mechanisms of action.

Funding

This study was supported by the University of Malaya research grants RG469-12HTM, PG086-2012B, and FRGS grant FP016/2013B.

Supplemental data

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This study was supported by the University of Malaya research grants RG469-12HTM, PG086-2012B, and FRGS grant FP016/2013B.