Phytochemical Content and Antioxidant Activity in Wild Grape (Ampelocissus martini Planch.) Root Extracts

Introduction

Free radicals are occurred in life body whose using oxygen by metabolism, especially, a reactive oxygen species (ROS)^{1, 2}. They confer both advantages and disadvantages depending on their concentration in the living organism. At low or moderate concentrations, they are involved in the immune system, cellular signaling pathways and mitogenic response. However, at high concentration, free radicals lead to damage nucleic acids, lipids and proteins and are involved in many diseases including diabetes mellitus, various cancers and cardiovascular diseases².

An antioxidant is a molecule which can stabilize or deactivate free radicals before they destroy cells¹. The antioxidants can be produced by the body or obtained from the diet. Endogenous antioxidants (enzymatic and non-enzymatic) found in the body are important for maintenance of optimal cellular functions, but exogenous antioxidants from diets or dietary supplements may be needed to protect cells of the body from free radical attack under conditions which can cause oxidative stress³.

Plants are well known as a source of phytochemicals including vitamin C, b-carotene and polyphenols or phenolic compounds¹. Phenolic compounds are plant secondary metabolites, and universally occur in many plant parts including fruits, vegetables, nuts, seed, leaves, roots, and barks⁴. These compounds can be grouped into phenolic acids, flavonoids, proanthocyanidins, stilbenes, and lignans⁵.

One of popularly consumed fruits is grape. Different parts of the plant contain phenolic compounds which show antioxidant, anti-cancer, anti-inflammatory, antibacterial, and antihypertensive activities^5-11. Another plant namely “wild grape (Ampelocissus martini Planch.)” has similar characters including stem, leaf and fruit to table grape. This plant has been recognized as a Thai medicinal plant and applied in folk medicine. Many studies relating to antioxidants and phytochemicals in grape have been reported. Almost all of the researches are from extracts of seeds, leaves^12-13, juice^14-15, pomace¹⁰, skin and pulp^5,16, stem^8-9,17-18 and fruits¹⁹. However, there are only a few studies of grape root extract^20-21.

In this study, crude extract and fractions obtained from silica gel chromatography of the wild grape root were assessed for phytochemical content and antioxidant activity by spectrophotometric assay. The correlation coefficient (r) of phytochemical and antioxidant activity was also determined.

Materials and Methods

Material and Reagents

The wild grape roots were gathered in local forest in Roi-ET province, Thailand in June 2018. The analytical grade of all chemicals and reagents were chosen.

Preparation of Wild grape Root Powder

The wild grape roots were firstly washed with tap water to exclude some dirties, and then dried at room temperature. The dried roots were chopped into small pieces, ground to powder, and kept in sealed plastic bag until use.

 Extraction of Crude Extract

Crude extract was done following the previously published method⁹.

Fractionation of Compounds

Silica gel column chromatography was used to fractionate the crude extract was done following the previously published method ¹³. The solvent mixtures used in the elution step were ethyl acetate:methanol (100:0, 75:25), and methanol:water:acetic acid (87:10:3).

Determination of Total Phenolic Content

Total phenolic content (TPC) of the extracts were analyzed following previously reported method¹². A gallic acid was used as standard. The TPC was showed as mg GAE/g DW.

Determination of Total Flavonoid Content

The total flavonoid content (TFC) was measured following the previously published method²² except that quercetin was used as standard. The TFC was expressed as mg QE/g DW.

Determination of Total Proanthocyanidin Content

The total proanthocyanidin content (TPAC) was determined based on a previous report²³ except that ethanol was used as solvent for preparation of vanillin. A catechin was used as standard. The TPAC was reported as mg CE/ g DW.

DPPH Assay

The ability of extracts to scavenge DPPH radicals was evaluated following previous report¹² except that the absorbance measurement was done at 517 nm. The scavenging activity was expressed as IC₅₀ value indicating the concentration required to cause 50% inhibition.

ABTS Assay

The ability of extracts to scavenge ABTS was evaluated following previous report²⁴ except that sample was prepared in various concentrations using methanol instead of ethanol. The scavenging activity was reported as IC₅₀ value.

FRAP Assay

The ferric reducing antioxidant power of the extracts was determined following previous report²³. A standard curve was constructed from different concentrations of FeSO₄ solution and absorbance values at 593 nm. The FRAP value was reported as mmol Fe²⁺ /g DW.

CUPRAC Assay

The cupric reducing antioxidant capacity of the extracts was performed following previously published method²⁵. The Trolox was used as standard. The CUPRAC value was reported as mg TE/g DW.

Statistical Analysis

The mean + standard deviation (SD) and Duncan’s new multiple range test were used to evaluate the significant differences with P < 0.05. The Pearson’s correlation coefficients (r) was used to indicate data correlation.

Results and Discussion

Crude Extract and Fractionation by Silica Gel Column Chromatography

The crude extract of the wild grape root was passed through silica gel column chromatography to partially resolve components of the crude extract. Based on using different mobile phases in the elution steps and A₂₈₀ of each fraction, all fractions could be pooled into 3 major fractions designed as F1, F2 and F3 that were eluted with ethyl acetate, ethyl acetate: methanol (75:25) and methanol:water:acetic acid (87:10:3), respectively.

Phytochemical Contents (TPC, TFC, TPAC)

Table 1 shows the phytochemical contents. TPC was highest in the fraction eluted by solvent mixtures of ethyl acetate: methanol (75:25) (F2), while the fraction F3, eluted by methanol:water:acetic acid (87:10:3) had the lowest content. The crude extract and the fraction eluted by ethyl acetate (F1) had moderate content of the tested phytochemicals, and was not significantly different (P < 0.05) compared to crude extract. The variations trend of TFC was similar with TPC, which had the highest and lowest contents in F2 and F3, respectively. The TFC of F2 was almost 15-fold higher than that of F3. Crude extract had the highest TPAC while F3 showed the lowest content.

The results indicated that polarity of solvent influenced the phytochemical content of chromatographic fractions of wild grape root extract. The solvent mixture of ethyl acetate: methanol (75:25) was the best mobile phase to elute high TPC and TFC from silica gel chromatography. This result was in agreement of previous work about effect of solvent elution on fractions of wild grape leaves¹³.

Table 1: TPC (GAE/g DW), TFC (mg QE/g DW) and TPAC (mg CE/ g DW) of crude and different fractions from wild grape root (mean + SD, n = 3)

Samples	TPC	TFC	TPAC
crude	468.90 + 2.71 b	873.14 + 2.40 c	40.91 + 0.40 a
F1	471.23 + 1.56 b	974.15 + 8.33 b	15.68 + 0.04 c
F2	552.42 + 3.18 a	1235.95 + 4.13 a	24.84 + 0.15 b
F3	29.21 + 0.17 c	83.33 + 0.42 d	0.46 + 0.03 d

^a-d Superscripts in each column indicates significant difference (P < 0.05).

DPPH, ABTS, FRAP and CUPRAC assays

Plant phenolic compounds are important secondary metabolites. They have a variety of mechanisms for antioxidant activity²⁶. The scavenging activity of the extracts for DPPH and ABTS radicals and an ability to reduce Fe and Cu ions are shown in Table 2.

The reduction of DPPH^• resulting from the ability of the test samples to trap radicals caused the loss of absorbance at 517 nm^26-27. A low IC₅₀ value reflected high radical scavenging activity²⁷. The results showed that F2 has the highest scavenging activity followed by crude > F1 > F3, respectively. It was interesting to find that F2 has almost 21-fold more potency as a scavenger against DPPH^• than did F3. For the ABTS assay, all samples possessed different levels of radical-scavenging activity which were in the following ranked order: F1 > F2 > crude > F3.

The FRAP assay has also been widely used for reducing metal ion of the antioxidants²⁶ which reduced Fe³⁺ to Fe²⁺ to form the blue color²⁸. In this study, F2 showed the highest FRAP value while F3 showed the lowest FRAP value. Moreover, the CUPRAC assay was used for reducing power of Cu(II) to Cu(I) by antioxidants ²⁶. In the reaction mixture, the Cu(II)-neocuproin complex is reduced to the Cu(I)-neocuproin form which shows an absorption maximum at 450 nm²⁸. The variation in CUPRAC activity followed similar trends to that found in FRAP activity. The effect of solvents for extraction and elution of crude extract on antioxidant activity was also reported^13,29.

Table 2: Scavenging activity by DPPH and ABTS assays and reducing power by FRAP and CUPRAC assays of wild grape root crude and fraction extracts (mean + SD, n = 3).

Samples	DPPH assay	ABTS assay	FRAP assay	CUPRAC assay
	IC50 (mg polyphenols/L)	IC50 (mg polyphenols/L)	(mol Fe2+/ g DW)	(mg TE/g DW)
crude	21.61 + 0.00 c	13.35 + 0.02 b	413.02 + 8.04 c	706.23 + 1.87 c
F1	24.40 + 0.17 b	7.53 + 0.02 d	472.02 + 2.86 b	957.58 + 4.30 b
F2	14.30 + 0.07 d	8.94 + 0.03 c	548.05 + 4.91 a	1134.76 + 2.75 a
F3	303.21 + 0.49 a	287.30 + 0.25 a	31.76 + 1.47 d	N.D.

^a-d Superscripts in each column indicates significant difference (P < 0.05).

N.D. indicates not detected

Correlations Analysis

The correlation coefficient (r) of phytochemical types and antioxidant activity from tested methods were presented in Table 3.

TPC showed highly positive correlation coefficient (P < 0.01) with TFC and TPAC, and TFC showed moderate correlation coefficient with TPAC. High correlation coefficients between TPC and TFC have been reported by other researchers^13,29. Highly positive correlation coefficients were also found between DPPH and ABTS, and between FRAP and CUPRAC. Moreover, FRAP and CUPRAC showed high correlation coefficient to TPC and TFC, but moderate correlation coefficient to TPAC. The results suggested that high content of phenolics and flavonoids resulted in high metal reducing power. IC₅₀ values were inversed meaning of antioxidant activity²⁰; therefore, the obtained correlation coefficients (r) values where be negative. From the correlation coefficients values, the phenolics and flavonoids have antioxidant mechanism through the scavenging of free radical, but has lower capacity than FRAP assay. Therefore, the FRAP assay would be an appropriate technique for antioxidant activity determination in wild grape root. The result was in accord with the data of Feng and coworkers³⁰, who found that the FRAP technique could be a good technique to determine antioxidant activity in sugarcane extract since the technique had a higher correlation coefficient with phenolics and flavonoids, compared to the ABTS technique.

Table 3: Pearson’s correlation coefficient (r) of wild grape root extracts.

Trait	TPC	TFC	TPAC	DPPH	ABTS	FRAP	CUPRAC
TPC	1	0.987**	0.759**	-0.991**	-0.987**	0.993**	0.971**
TFC	–	1	0.671*	-0.959**	-0.954**	0.996**	0.992**
TPAC	–	–	1	-0.790**	-0.777**	0.680*	0.585*
DPPH	–	–	–	1	0.999**	-0.975**	-0.942**
ABTS	–	–	–	–	1	-0.973**	-0.941**
FRAP	–	–	–	–	–	1	0.992**
CUPRAC	–	–	–	–	–	–	1

*P < 0.05, ** P < 0.01 indicate significant differences values.

Conclusions

Silica gel chromatography could partially separate crude extract of wild grape root. The fraction eluted by ethyl acetate:methanol at 75:25 (v/v) (F2) showed highest values of TPC and TFC, while highest TPAC was found in the crude extract. Fraction F2 was also had the highest scavenging free radicals and reducing power antioxidant activity from all assays, except ABTS assay which showed the highest antioxidant activity in the ethyl acetate fraction (F1). High correlation coefficient between phytochemicals and antioxidant activity suggests that the substances in the wild grape root that play a role in free radical inhibition through radical scavenging activity, and metal reducing ability are phenolics and flavonoids. The results obtained from this work suggest that wild grape root is a good exogenous antioxidant because it contains high phytochemicals with high antioxidant activity. Further study might be quantification of phenolic components in each fraction and in vivo assay for biological activity to obtain more information of these phytochemicals.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The research was financially supported by Faculty of Science (Grant year 2019), Mahasarakham University, Thailand.

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