Phenolic Composition and Radical Scavenging Capacity of Vaccinium Bracteatum Thunb. Leaves

Abstract

The water, ethanol, and methanol extracts of Vaccinium Bracteatum Thunb. leaves (VBTL) were investigated for their potential antioxidant activity. The total phenolic content was determined, and the antioxidant activity of the extracts was assessed by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging analysis. Five phenolic compounds including quercetin, kaempferol, luteolin, apigenin, and chrysin, were identified in the ethanol extract by high pressure liquid chromatography (HPLC).

Keywords:

• Vaccinium Bracteatum Thunb.
• Antioxidant
• Phenolic compounds
• Radical scavenging

INTRODUCTION

Vaccinium Bracteatum Thunb., a resource of Chinese traditional medicine, belongs to the genus Rhododendron. It is grown massively in China but can be found worldwide. Vaccinium Bracteatum Thunb. leaves (VBTL) are rich in phenolic compounds. Some properties and functions of the extracts of VBTL including UV absorption, stability, and dyeing ability were determined.[1–3] The pigments of extract were stable even at 100°C for 2 h and could protect the retina from light damaging.[3] The fatty acids were also extracted from VBTL by supercritical CO₂ and six components were identified.[4, 5] The fatty acids had significant action on adjusting blood-lipid without toxicity. Recently, the chemical constituents from the leaves of Vaccinium Bracteatum were investigated.[6] Twelve compounds were isolated and identified as chrysoeriol, scopoletin, trans-p-hydroxycinnamic acid, trans-p-hydroxycinnamic acid ethyl ester, cafeic acid ethyl ester, β-sitosterol, luteolin, quercetin, esculetin, cafeic acid, isolariciresinol-9-O-β-D-xyloside, and 10-O-trans-p-coumaroylsandoside. Although the chemical constituents and several biological activities of this plant have been reported, negligible study examined its antioxidant activity. The aim of this study is to investigate the phenolic composition and antioxidant activity of various extract from VBTL using water, ethanol and methanol. The radical scavenging capacity was measured and compared to the synthetic antioxidants of butylated hydroxytoluene (BHT) and ascorbic acid (Vit. C).

MATERIALS AND METHODS

Plants, Chemicals, and Reagents

The Vaccinium Bracteatum Thunb. leaves were collected in October from Liyang, Jiangsu Province, China. The leaves were separated from the other parts and dried at room temperature. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and Folin-Ciocalteau reagent were purchased from Sigma Co. (St. Louis, MO, USA). Butylated hydroxyluene (BHT) and ascorbic acid (Vit. C), food grade antioxidants, were purchased from the Guangzhou Chemical Company, P. R. China.

Preparation of extracts

The leaves were ground to pass through a 1.0-mm sieve, and then were defatted by n-hexane at room temperature. The obtained powder of 10 g was extracted for 1.5 h in 100 mL of distilled water, 60% ethanol (v/v), and 60% methanol (v/v), respectively. After extraction, the extracted slurry was filtered off to collect the filtrate alone. The filtrate was concentrated and dried. The dried extract was weighed and stored in darkness at 4°C.

Determination of the total phenolic content

The total phenolic contents (TPC) were determined by the Folin-Ciocalteu colorimetric method.[7–9] The ethanol solution of each extract (0.2 mL, 500 μg/mL) was taken in a test tube. To this solution, 0.5 mL distilled water and 0.5 mL Folin-Ciocalteu reagent were added and the tubes were shaken thoroughly. After 1 min, 0.8 mL of sodium carbonate solution (7.5%) was added and the mixture was allowed to stand for 30 min with intermittent shaking. Absorbance was measured at 760 nm using a Shimadzu UV-Vis spectrophotometer. The total phenolic content was expressed as Gallic acid equivalents (GAE) in mg per g extract.

DPPH Free-Radical scavenging assay

The free radical scavenging activities of the extracts were evaluated according to the reported method,[10, 11] i.e., loss of absorbance at 517 nm of an ethanol solution of DPPH in the presence and absence of increasing concentrations of the extracts, BHT or Vit C (50–200 μg/mL dissolved in a 1:1 water/ethanol mixture). The DPPH scavenging ability was calculated as percentage of inhibition of DPPH absorbance, by the following equation

(1)

where A₀ is the absorbance of the control, and A_e is the absorbance of the sample.

HPLC Analysis of the ethanol extract

Phenolic compounds of the ethanol extract were measured at 280 nm by high pressure liquid chromatography (HPLC), using a Lichrospher C-18 analytical column (2.1 × 250 mm). The column was maintained at 30°C. A gradient elution was performed by varying the proportion of solvent A (water-acetic acid, 97:3 v/v) to solvent B (methanol) as follows: 0 min, 100:0; 5 min, 80:20; 10 min, 70:30; 15 min, 60:40; 20 min, 40:60; 25 min, 30:70; 30 min, 20:80. The flow rate was 0.3 mL/min and the injection volume was 10μL. The detector signal was electronically monitored with a WATERS 996 detector. Identification of the phenolic compounds was carried out by comparing their retention times to those of standards.

Statistical analysis

Experiments were repeated three times and the results were averaged. The variance analysis procedure of Statistical Analysis System (SAS Institute, Inc., version 8.0) was used to determine differences among the data. A significant level of p < 0.05 was chosen.

RESULTS AND DISCUSSION

The results of total phenolic contents and the yields of total phenolic compounds are reported in Table 1. It is evident that the recovery of phenolic compounds was dependent on the solvent used and its polarity. Significant difference in total phenolic content (p < 0.05) was detected among these extracts. The phenolic content varied from 10.86 to 25.87 mg GAE/g extract depending on the extraction solvent with the following order: ethanol > methanol > water. The extraction yield varied from 0.93 to 4.62 mg GAE/g VBTL depending on the extraction solvent with the following order: ethanol > methanol > water. However, no significant difference (p > 0.05) was found between the yield of ethanol extract and methanol extract. Compared to methanol, ethanol has acceptability for human consumption models.

Table 1 Total phenolic contents (TPC) and yields of VBTL extracts.*

Parameters	TPC (mg GAE/g extract)	Yield (mg GAE/g VBTL)
Water extract	10.86 ± 0.11^a	0.93 ± 0.05^a
Ethanol extract	25.87 ± 0.12^c	4.62 ± 0.03^b
Methanol extract	21.09 ± 0.23^b	4.50 ± 0.05^b
*Values were the means of three replicates ± standard deviation (SD). Values with different letters in the same column were significantly (p < 0.05) different.

The capacity for scavenging free radicals was evaluated for the water, ethanol, and methanol extracts and the reference antioxidants (BHT and Vit C). The results are shown in Table 2. The scavenging effect of different extracts on DPPH radical decreased in the following order: ethanol extract > methanol extract > water extract. The shown antioxidant activitives are in agreement with the total phenolic contents in the extracts under study. These results indicated that all the extracts had a noticeable effect on scavenging free radical. The ethanol extract had similar scavenging activity to BHT.

Table 2 DPPH free radical scavenging activities (I%) of VBTL extracts.*

Concentration (μg/mL)	50	100	150	200
Water extract	17.66 ± 0.80^a	30.49 ± 0.72^a	37.54 ± 0.81^a	49.77 ± 0.66^a
Ethanol extract	33.61 ± 0.57^c	47.29 ± 0.84^c	59.18 ± 0.67^c	73.34 ± 0.53^d
Methanol extract	26.11 ± 0.71^b	40.38 ± 0.64^b	48.68 ± 0.64^b	61.28 ± 0.68^b
BHT	34.01 ± 0.72^c	48.37 ± 0.69^c	60.85 ± 0.72^c	67.80 ± 0.84^c
Vc	40.03 ± 0.64^d	65.48 ± 0.64^d	77.19 ± 0.60^d	88.26 ± 0.51^e
*Values were the means of three replicates ± standard deviation (SD). Values with different letters in the same column were significantly (p < 0.05) different.

Since the ethanol extract contained the highest phenolic content and showed the strongest antioxidant activities, it was therefore analyzed by HPLC to determine its chemical composition that may contribute to this activity. The results are shown in Table 3. Five phenolic compounds including luteolin, apigenin, quercetin, chrysin, and kaempferol, were identified and their contents were quantified. The most abundant phenolic compound in the extract was quercetin, which has already been demonstrated to be a very strong natural antioxidant. This is one of the first study concerning total phenolic content, phenolic composition, and radical scavenging capacity of VBTL extract. Our work suggests that VBTL may be utilized as effective and safe antioxidant source.

Table 3 Phenolic compound contents (mg/g extract) of ethanol extract of VBTL by HPLC

Compound	Retention time (min)	Content mg/g extract
Quercetin	20.91	9.70 ± 0.12
Kaempferol	22.12	1.22 ± 0.07
Luteolin	23.89	3.92 ± 0.23
Apigenin	24.47	2.48 ± 0.18
Chrysin	27.61	1.36 ± 0.15
*Values were the means of three replicates ± standard deviation (SD).

CONCLUSIONS

The water, ethanol, and methanol extracts of VBTL showed antioxidant activity based on DPPH radical. The ethanol extract, which had the highest total phenolic content and the most active radical scavenging capacity in the extracts, can be used as an accessible source of natural antioxidants and as a possible supplement of synthetic antioxidants. Five phenolic compounds including quercetin, kaempferol, luteolin, apigenin, and chrysin, were identified in the ethanol extract. VBTL can be of use as an easily accessible source of natural antioxidants.

ACKNOWLEDGMENTS

This work was supported by Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT0627) and Natural Science Foundation of Jiangnan University (2008LYY021).