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The Natural Products Journal, 2012, 2, 247-251 247

Effects of Gynura procumbens Leaf Extracts on Plasma Lipid Peroxidation and Total Antioxidant Status in CCl4-Treated Rats

Gabriel A. Akowuah1,*, Mariam Ahmad

2 and Yam M. Fei

2

1Faculty of Pharmaceutical Sciences, UCSI University, 56000, Kuala Lumpur, Malaysia;

2Faculty of Pharmaceutical

Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

Abstract: The effect of extraction solvents (methanol, 95%ethanol, and water) on total phenolic contents and free radical

scavenging activity (FRSA) of Gynura procumbens leaf was investigated. The contents of total phenolic and in vitro

FRSA were significantly higher in the methanol extract compared to the 95% ethanol and water extracts. Consequently, the

effect of the methanol extract on plasma lipid peroxidation and plasma total antioxidant status in carbon tetrachloride

(CCl4) -induced oxidative stress in rats was determined. The administration of CCl4 to rats showed significant increase in

plasma thiobarbituric acid reactive substances (TBARS) levels. The plasma lipid peroxidation elevation produced by CCl4

was reversed in rats fed with the methanol extract of G. procumbens leaf for 14 days before CCl4 challenge.

Administration of CCl4 to rats did not showed significant alteration in plasma total antioxidant status (TAS).

Keywords: Antioxidant, extraction solvents, Gynura procumbens, lipid peroxidation, plasma.

INTRODUCTION

Gynura procumbens (Merr., Compositae) is a medicinal plant native to Southeast Asia, where it has been used to treat urinary lithiasis, edema, eruptive fever, influenza, rheumatism, colon cancer, haemorroids and diabetes [1]. The leaf extract of G. procumbens was reported to show in vitro antioxidant activity [2], anti-hyperlipidaemic [3], and anti-hypertension properties [4, 5]. The active chemical constituents of G. procumbens leaves include, flavonoids, sterols and their glycosides [6, 7].

Polyphenols are bioactive constituents present in food plants which are very important in the control and prevention of tissue damage by activated oxygen species due to their antioxidant effects [8]. The stability of polyphenols in plant extracts depends on many factors including the drying, extraction method, solvent, temperature, and pH. These factors can markedly influence the levels and efficacy of bioactive compounds of dietary supplements such as polyphenols. Therefore, objectives of this study were to evaluate the effectiveness of different extraction solvents on total phenolic contents and free radical scavenging activity (FRSA) of the leaf extracts of G. procumbens, and the effect of the extract with the highest FRSA activity on oxidative stress biomarkers in carbon tetrachloride treated (CCl4)-treated rats.

MATERIAL� AND METHODS

Chemical and Reagents

Gallic acid, butylated hydroxytoluene (BHT), quercetin, 2,2

’-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS),

*Address correspondence to this author at the Faculty of Pharmaceutical

Sciences, UCSI University, 56000, Kuala Lumpur, Malaysia; Tel: 603-9101

8880 3072; Fax: 603 9102 3606; E-mail: wuahmy@yahoo.com,

agabriel@ucsi.edu.my

thiobarbituric acid, trichlroacetic acid, 1,1,3,3-tetraethoxypropane, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Company (St. Louis MO, USA). Methanol was obtained from Merck. All other solvents were analytical grade or HPLC grade.

Plant Materials

The Gynura procumbens was obtained from Penang Island, Malaysia. A voucher specimen (10117) was deposited at the herbarium of the School of Biological Sciences, Universiti Sains Malaysia. The leaves of G. procumbens were dried at 35

oC in an air oven and finely

powdered.

Extraction

Powdered leaf sample (100.0 g) was weighed and transferred into conical flasks. The leaf sample was extracted with de-ionized water (1.0 L), methanol, and 95% ethanol respectively, at 40

oC for 6 h with intermediate shaking. The

extracts were vacuum filtered (Whatman No. 1) and the solvent evaporated using rotary evaporator.

Contents of Total Phenolics and Soluble Solids

The total phenolic content in extracts was determined by using Folin-Ciocalteu reagent and external calibration with gallic acid. Briefly, 0.2 mL of extract solution in a test tube and 0.2 mL of Folin-Ciocalteu reagent were added and the contents mixed thoroughly. After 4 min, 1 mL of 15% Na2CO3 was added, and then the mixture was allowed to stand for 2 h at room temperature. The absorbance was measured at 760 nm by using Lambda 45 UV-Vis spectrphotometer (Perkin-Elmer, USA). The content of total phenolics was calculated by using gallic acid calibration curve. The results were expressed as mg GA / g dry material.

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248 The Natural Products Journal, 2012, Vol. 2, No. 4 Akowuah et al.

The content of total soluble solids of an aliquot (20 ml) of extract was determined in triplicate [9].

In Vitro Free Radical Scavenging Activity

The free radical scavenging activity of the extracts was measured by 1,1,-diphenyl-2-picryl-hydrazil (DPPH) method

[10]. Briefly, DPPH solution (0.1 mM) was prepared in methanol and to 2 mL of this solution was mixed with 0.2 mL of samples of extracts. The volume of the solution was adjusted with methanol to a final volume of 3 mL. After

incubation at room temperature for 30 min, the absorbance of the mixture was measured at 517 nm against methanol as blank using UV-Vis spectrphotometer. Quercetin (0.1 mg/mL) was used as positive reference compound. The

activities of the samples were evaluated by comparison with a control (containing 2 mL of DPPH solution and 1 mL of methanol). Each sample was measured in triplicate and averaged. The activity was calculated according to the

formula:

% Inhibition = [(AC - AS) / AC ] x 100

where AC is the absorbance value of the control and AS is the absorbance value of the test solution.

IN VIVO ANTIOXIDANT ACTIVITY

The methanol extract demonstrated the highest free

radical-scavenging activity for this reason it was selected for the in vivo antioxidant study.

Animals

Experimental animals used were Sprague-Dawley rats, weighing 200-220g. The rats were divided into 3 groups

with each group containing six animals (n=6). The animals of the first and second groups were administered distilled water for 14 days. The animals of the third group were administered a daily single dose of freeze-dried methanol

extract suspended in distilled water orally at 1.0g / kg body weight for 14 days. Food and drinking water were provided ad libitum. The animals in the second and third groups were given a single dose of CCl4 (1:1 in olive oil) at 2.0

kg of body weight 2 h after the last dose of administration of distilled water and extract, respectively. On the 15

th

day blood was collected from all the groups by cardiac puncture and immediately transferred to heparinized tubes

for the separation of plasma. Plasma was separated by centrifugation at 3000 x g for 15 min at 4

oC and stored at -

80oC analyzed subsequently for thiobarbituric acid reactive

substances (TBARS) content and total antioxidant status

(TAS).

Measurement of Plasma Lipid Peroxidation Levels

Plasma samples were assayed for thiobarbituric acid reactive substances (TBARS) content by the method of Draper et al. [11] with slight modifications. Plasma (1 mL)

was mixed with 30% trichloroacetic acid (1 mL) and 0.67% thiobarbituric acid (1 mL) in the presence of 200 mM butylated hydroxytoluene (0.1 mL). The mixture was heated at 90

oC for 45 min in a tightly closed tube in boiling water.

After cooling, the tubes were centrifuged at 3000 rpm for 10

min at 5oC and the absorbance of the supernatant was

measured at 532 nm at room temperature using UV-Vis spectrophotometer. The amount of TBARS produced was calculated using 1,1,3,3-tetraethoxypropane as a standard. Lipid peroxidation was expressed in nmoles of TBARS

formed per mL of plasma sample.

Estimation of Plasma Total Antioxidant Status

Plasma total antioxidant status of plasma was determined

by the method described by Miller et al. [12] with slight modifications. Briefly, 10 mM phosphate buffer, pH 7.4 (650 μL), 250 μM ABTS (100 μL) and 5 mM metmyoglobin (100 μL) diluted in phosphate buffer (10 mM, pH 7.4) were added

to a quartz cuvette containing plasma (50 μL). ABTS radical cation (ABTS

o+) was generated by adding 250 μM hydrogen

peroxide (100 μL) to the cuvette and absorbance at 734 nm was measured immediately (A0) and 5 min (At) after addition

of hydrogen peroxide with UV-Vis spectrophotometer. Absorbance of a blank containing phosphate buffer instead of plasma was also measured to calculate the percentage inhibition. The inhibition activity was calculated by using the

formula:

Inhibition activity = [(A0 – At )blank - (A0 – At )plasma / (A0 – At )blank ] x 100

The inhibition activity was converted to trolox equivalent antioxidant capacity (TEAC) by trolox standard curve. The

result was expressed as mmoles of TEAC per litre of sample.

RESULTS

Effect of Extraction Solvent on Total Soluble Solids and

Total Phenolic Contents

Table 1 shows the effect of extraction solvents on total soluble solids and total phenolic contents of G. procumbens leaf. The de-ionised water extract showed the highest total soluble solids followed by the methanol extract. The 95%

ethanol extract showed the lowest total soluble solids. There was a significant difference (P < 0.5) between the total soluble solids obtained by each of the extraction solvent.

The methanol extract demonstrated the highest total

phenolic levels. There was significant difference (P < 0.5) between the total phenolic content of the methanol extract compared to the values of the ethanol and water extracts. The content of total phenolic of the extracts decreased in the

order methanol > 95% ethanol > water.

The Influence of Extraction Solvents on Free Radical

Scavenging Activity

Table 1 shows the results of FRSA of the extracts. All the

extracts demonstrated inhibitory activity against the DPPH radical. For the extraction solvents studied, methanol extract showed the highest free FRSA which was comparable to that of quercetin. The water and ethanol extracts showed

significantly ( p < 0.05) lower FRSA compared to the value of the methanol extract. The order of FRSA of extracts and reference standard was: Quercetin > methanol > 95% ethanol > water.

Effects Gynura procumbens Leaf Extracts on Plasma Lipid Peroxidation The Natural Products Journal, 2012, Vol. 2, No. 4 249

Plasma Lipid Peroxidation Levels and Total Antioxidant

Status

The effect of treatment of rats with CCl4 and treatment of rats with G. procumbens methanol extract followed by CCl4 challenge on plasma TBARS levels is shown in Fig. (1A). Treatment of rats with CCl4 significantly increased (p < 0.05) plasma TBARS levels by 97.19% as compared to values of the control group. Treatment of rats with G. procumbens methanol extract (1g/kg p.o.) for 14 days followed by CCl4 challenge preserved the plasma TBARS levels, which were comparable to the control values.

The effect of treatment of rats with CCl4 and treatment of rats with G. procumbens methanol extract followed by CCl4 challenge on plasma TAS levels is shown in Fig. (1B). Treatment of rats with CCl4 did not showed significant alteration (p > 0.05) in plasma TAS as compare to the values of control group. Reduction in plasma TAS of 9.52% relative to control group was produced by CCl4 treatment. Plasma TAS values of rats fed with the extracts (1g/kg) for 14 days

followed by CCl4 administration were comparable to the values of the control group.

DISCUSSIONS

Polyphenols are unstable compounds and their degradative reactions occurred throughout the stages of formulation process of a dietary supplement. The content of polyphenols in different extracts from the same plant sample depends on the extraction solvent used for the removal of the polyphenolic compounds, and it is apparent that extracts from the same plant material may vary widely with respect to their antioxidant concentrations and activities. Water, organic liquids and mixtures of organic liquids are used as extraction solvents for polyphenolic compounds from natural source. The selection of effective solvent depends on the number of hydroxyl moiety of the phenolic compounds in the sample. However, other factors including economy and safety are also considered in the choice of suitable solvents for extraction of polyphenols.

Table 1. Total Soluble Solids, Total Phenolic Contents and Free-radical Scavenging Activities of Gynura procumbens Leaf

Extracted with Various Solvents

Extraction

Solvent

Total Soluble Solids

(%,w/w)

Total Phenolics

(mg GA/ g Dry Extract)

DPPH· Activity

(%)

Water 10.28± 0.69a 16.74 ± 0.25a 56.84 ± 1.37a

Methanol 8.63 ± 0.40b 24.46 ± 0.28b 93.50 ± 1.22b

95% ethanol 7.45± 0.32c 20.15 ± 0.19c 57.21 ± 1.15a

Quercetin NA NA 94.86 ± 1.48b

Data are presented as means ± standard deviation (n = 3). NA, not applicable. The results were evaluated according to one-way analysis of variance (ANOVA). The significance of difference was evaluated by using Dunnett’s post hoc test. Means with different letters in columns indicate significantly different at p<0.05.

Fig. (1). The effect of treatment of rats with methanolic leaf extract of Gynura procumbens (1.0 g/kg p.o.) followed by carbon tetrachloride

(CCl4) administration on (A) plasma lipid peroxidation and (B) plasma total antioxidant status. Data are means ± S.D. (n = 6). The results

were evaluated according to one-way analysis of variance (ANOVA). Student's t-test was used for statistical significance between groups.

* Statistical significant (p < 0.05) as compared to control group. # Statistical significant (p < 0.05) as compared to CCl4 treated group.

0

1

2

3

4

Control CCl4 Extract

TB

AR

S (n

mol

/ml)

0

0.5

1

1.5

2

2.5

Control CCl4 Extract

TAS

( (m

mol

TEA

C/l)

A B

*

#

250 The Natural Products Journal, 2012, Vol. 2, No. 4 Akowuah et al.

In our previous studies, the effect of extraction temperature on total phenolic contents and free radical scavenging activity of Gynura procumbens leaf extract was investigated [13]. Extraction of G. procumbens leaf with 80% methanol at temperature below 60°C, gave a greater retention of polyphenolic compounds and greater expression of free-radical scavenging activity [13]. In the present study, methanol is shown to be the most effective solvent for extraction of phenolics from G. procumbens leaf powder compared to water and 95% ethanol. Free radical-scavenging activities of water, methanol, and 95% ethanol extracts were evaluated using in vitro method. Significant reduction in FRSA of the samples extracted with water and 95% ethanol compared with the values of the methanol extract was observed. This was expected due to reduction in total phenolic contents of the 95% ethanol and water extracts.

The methanol extract demonstrated the highest FRSA hence it was selected for the in vivo antioxidative studies. The in vivo study was focused on the effect of G. procumbens leaf on plasma lipid peroxidation and plasma TAS in CCl4-induced oxidative stress in rats. It is well known that CCl4 is bioacitivated by cytochrome P450 to trichloromethyl free radical (

.CCl3) and chloride free radical

(.Cl) which can initiate lipid peroxidation process [14].

Formation of TBARS is commonly used as index of lipid peroxidation. Administration of CCl4 to rats did not showed significant alteration in plasma TAS. On the other hand, CCl4 administration to rats showed significant increase in plasma TBARS levels. The plasma lipid peroxidation elevation produced by CCl4 was reversed in rats fed with the methanol extract of G. procumbens leaf for 14 days before CCl4 challenge. This suggests that, the extract has the capability to stimulate natural antioxidant defence systems to increase removal of oxidatively generated harmful substance under oxidative stress conditions.

Free radical scavenging is established as the means by which antioxidants inhibit lipid peroxidation. In the in vitro section of this study, the methanolic extract exhibited FRSA comparable to quercetin which was used as positive standard. Our previous investigations have shown that polyphenols and their glycosides (rutin and kaempferol-3-O-rutinoside) are the major bioactive components in G. procumbens leaves [6]. These phytochemicals have been reported to possess high antioxidant activities [15]. The antioxidant properties of polyphenols are generally accepted as the basis of their therapeutic effect. Polyphenols reduce oxidative stress; possibly by inhibiting the formation of lipid peroxidation products in biological systems [16, 17].

CONCLUSIONS

In conclusion, extraction of G. procumbens leaf with methanol gave a greater retention of polyphenolic compounds and greater expression of free-radical scavenging activity in vitro which was comparable to that of quercetin. On the other hand, lesser expression of antioxidant activity occurred with water and ethanol extracts. This shows that selective extraction of polyphenolic compounds from natural sources by suitable solvent can provide extracts

with high antioxidant activity. Antioxidative effects of the methanol extract against CCl4-induced oxidative stress in rats indicates that the extract is capable of enhancing and maintaining activity of antioxidant enzymes involved in combating free radicals. The development of simple and rapid extraction methods for analysis of polyphenols in plant extracts is of great significance in the quality control of herbal medicine and botanical supplements. The effect of extraction solvent on total phenolic and antioxidant activity of plants is important to ensure efficacy of dietary supplements which is the most common form of plant phenolics. The in vivo antioxidative data are useful indication of the ability of the extract to regulate damage caused by free radicals.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

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Received: April 02, 2012 Revised: June 18, 2012 Accepted: July 04, 2012