Abstract: The present study determined nutritional value in various parameters such as moisture content, carbohydrate, protein, lipid as standard methods and evaluated the antioxidative properties of the medicinal plant Gynura procumbens Extract by hydroxyl scavenging activity, ferrous ion-chelating activity and inhibition of lipid peroxidation. The highest extraction yield was obtained from extraction with distilled water (84.51%). While, Ethanolic Gynura extract (EGE) exhibited the highest in the antioxidative properties in every assay, EC50 = 1.63 in hydroxyl scavenging test, EC50 = 2.17 in chelating activity test and EC50 = 2.75 in inhibition of lipid peroxidation test. Although, the content still low (186.25±0.00018 to 300.18±0.0003 μg GAE g-1 of extract). According determination the nutritional values was found that Gynura procumbens Extract were containing the contents of moisture was 7.08%, carbohydrate was 0.0537 to 0.1968 μg glucose equivalent/100 g dry weight at EGE 0.1 μg mL-1, protein was 4.51 g/100 g dry weight and lipid was 0.023 g/100 g dry weight. These results suggest that Gynura Extract is a good protein source and that they may have positive effects on free radical scavenging and iron chelating which may use as preliminary information and develop further to be commercially useful in food industry or health products as medicinal food.
INTRODUCTION
Working on plants was a good start to a research career, since plants are of enormous importance in the free radical/antioxidant field. First, they supply us with the essential biradical, O2. Second, plants supply a range of antioxidants to humans. Third, diets rich in plants are associated with lower risk of developing many age-related diseases (e.g., some cancers, diabetes, atherosclerosis and dementia) (Halliwell, 2009). Because plant-based foods are complex mixtures of bioactive compounds, information on the potential health effects of individual polyphenols is linked to information on the health effects of foods that contain those polyphenols (Gharras, 2009). There is evidence that phenolic substances act as antioxidants by preventing the oxidation of LDL-lipoprotein, platelet aggregation and damage of red blood cells (Cheynier, 2005). Additionally, phenolics act as: (1) metal chelators, (2) antimutagens and anticarcinogens, (3) antimicrobial agents and (4) clarifying agents (Anli and Vural, 2009; Proestos et al., 2005). These compounds are a part of the everyday diet and also used as medicines or supplements. Research has shown that fruits and vegetables contain other antioxidant nutrients which significantly contribute to their total antioxidant capacity (Cao et al., 1998; Wang et al., 1996). Gynura procumbens (Compositae), a fast growing herbaceous plant is an annual evergreen shrub with a fleshy stem. Gynura procumbens is found in various parts of Asia and is widely used in Thailand and Southeast Asia as a traditional medicine. The plant has traditionally been used for treatment of eruptive fevers, rash, kidney disease, migraines, constipation, hypertension, diabetes mellitus and cancer (Perry, 1980). Recently, pharmacological studies have indicated that G. procumbens has antioxidant (Rosidah et al., 2008, 2009), anti-herpes simplex virus (Nawawi et al., 1999), anti-hyperglycemic (Li et al., 2009; Akowuah et al., 2002), anti-inflammatory (Iskander et al., 2002), anti-hyperlipidaemic (Zhang and Tan, 2000) and blood hypertension reduction capabilities(Hoe et al., 2006; Kim et al., 2006). The advantages of the traditional use of G. procumbens have also been supported by the isolation and identification of several possible active chemical constituents from this plant, including flavonoids, saponins, tannins, terpenoids and sterol glycosides (Akowuah et al., 2002). Even though the various uses over long time periods, little information is available regarding nutritional value and the antioxidative properties of G. procumbens from Thailand. Therefore, the aim of the present study was to provide scientific data on this point, focusing on the nutritional value in various parameters such as moisture content, carbohydrate, protein, lipid and evaluated the antioxidative properties of the medicinal plant Gynura procumbens extract.
MATERIALS AND METHODS
Plant material:
Gynura procumbens was collected from the local area of Mahasarakham Province, Mahasarakham, Thailand in February 2008. Fresh leaves (100-200 g) were air-dried and then 60°C oven. Each sample was extracted three times. The organic extract was then evaporated and stored at -20°C until ready to be tested.
Chemicals:
The 1,1,3,3-tetraethoxypropane (MDA) and 2-thiobarbituric acid (TBA) were purchased from Sigma Chemical Co., USA. Ferric chloride hexahydrate (FeCl3.6H2O) and ferrous sulfate heptahydrate (FeSO4.7H2O) were obtained from Fluka, Germany. Sodium azide (NaN3), absolute ethanol and trichloroacetic acid (TCA) were products of Merck, Germany. Hydrogen peroxide (H2O2) and chloroform was purchased from BDH, UK. All other chemicals used were in the purest form available commercially.
Moisture content:
Fresh G. procumbens leaves (100-200 g) were air-dried and then oven
drying at 60°C until a constant weight for determination of moisture content
(AOAC, 1990, method 985.14).
G. procumbens extraction:
After drying G. procumbens leaves, the solids were extracted with various
organic solvents (hexane, methanol, chloroform, acetonitrile, distilled water
and ethanol). The concentration and solvent with the highest efficiency of extraction
was used in subsequent experiments. The extraction protocol was done for 3 h
(3 times) as modified method (Owen et al., 2003).
Organic solvent was removed by rotary evaporation at 35°C in vacuo
(Buchi Rotavapor R-200, USA) until dry.
Determination of the yield of G. procumbens extracts:
The evaporative dried G. procumbens extracts were weighed for calculating
the yield by the following Eq. 1:
| (1) |
Equation 1 calculation of % yield.
where, W1 is the weight of extracts after evaporation and W2 is the solid weight of G. procumbens
Nutritional value determinations:
All Nutritional value determinations were performed at least in triplicate.
Values of different parameters were expressed as the Mean±Standard Deviation
(SD). Quantitative analysis of carbohydrate content was done as standard method
of Dubois et al. (1956). Total protein was determined
by the Kjeldahl method. Protein was calculated using the general factor (6.25)
(AOAC, 1990) and total lipid was measured by standard
protocol of AACC (1987) method.
Determination of the antioxidative properties
Hydroxyl radical-scavenging activity assay:
The effect of hydroxyl radical was assayed by using the 2-deoxyribose
oxidation method (Chung et al., 1997). 2-Deoxyribose
is oxidized by hydroxyl radical that is formed by the Fenton reaction and degraded
to malondialdehyde (MDA). The reaction mixture contained 0.45 mL of 0.2 M sodium
phosphate buffer (pH 7.4), 0.15 mL of 10 mM 2-deoxyribose, 0.15 mL of 10 mM
FeSO4-EDTA, 0.15 mL of 10 mM hydrogen peroxide, 0.525 mL of distilled
water and 0.075 mL of the Gynura extract solution in the tube. The reaction
was started by the addition of hydrogen peroxide. After incubation at 37°C
for 4 h, the reaction was stopped by adding 0.75 mL of 2.8% trichloroacetic
acid and 0.75 mL of 1.0% of thibarbituric acid. The mixture was boiled for 10
min, cooled in ice and then measured at 520 nm. The hydroxyl radical scavenging
ability was evaluated by the following Eq. 2:
| (2) |
Equqtion 2 calculation of % hydroxyl radical scavenging effect.
Then the hydroxyl radical scavenging capacity was expressed as Median Effective Concentration (EC50; mg mL-1) which represented the concentration of sample having 50% hydroxyl radical scavenging ability.
Metal iron-chelating assay:
The ferrous ion-chelating activity of the Gynura extract was measured
according to the modified method of Yen and Wu (1999).
The absorbance of the ferrous iron-ferrozine complex at 562 nm was measured
to determine the Fe2+-chelating ability of the extract. Briefly,
the reaction mixture, containing the extract (1-80 mg mL-1), FeCl2
(2 mM) and ferrozine (5 mM), was adjusted to 5 mL with distilled water. The
mixture was shaken well and incubated for 10 min at room temperature. The absorbance
of the mixture was measured at 562 nm against a blank. EDTA was used as positive
control. The ability of extracts to chelate ferrous ion was calculated using
the following Eq. 3:
| (3) |
Equation 3 calculation of % metal iron chelating activity.
The ferrous ion-chelating activity was expressed as Median Effective Concentration (EC50; mg mL-1) which represented the concentration of sample at which the chelating activity was 50%.
Lipid peroxidation assay:
The inhibition of Lipid peroxidation of the Gynura extract was assessed
by modified method (Lo and Cheung, 2005). Briefly, 0.1
mL of linoleic acid in 0.2 M Na-phosphate buffer, pH 7.0 was mixed with 0.2
mL of the Gynura extract (1-80 mg mL-1), followed by addition
of 0.1 mL of 10 μM FeSO4 and 0.1 mM L-ascorbic acid; the mixture
was incubated at 37°C for 1 h. The reaction was terminated by adding .5
mL trichloroacetic acid (TCA, 28%, w/v), followed by 0.38 mL thiobarbituric
acid (TBA, 2%, w/v), with heating at 100°C for 20 min. After centrifugation
at 3000 g for 10 min to remove the precipitated protein, the absorbance of the
supernatant containing the thiobarbituric acid-reactive substance (TBARS) was
measured at 532 nm by a spectrophotometer. Malondialdehyde (MDA) was used as
a standard and phosphate buffer was used instead of sample as the negative control.
The inhibition percentage of lipid peroxidation was calculated by the following
Eq. 4:
| (4) |
Equation 4 calculation of % inhibition activity of lipid peroxidation.
The inhibition of lipid peroxidation was expressed as median effective Concentration (EC50; mg mL-1) which represented the concentration of sample having 50% inhibition effect on lipid peroxidation.
Statistical analysis:
Data were expressed as Mean±SD Paired samples test and ANOVA were used
to perform statistical analysis for the comparison. Post hoc testing was carried
out using the Bonferroni test. The criterion for statistical significance was
p<0.05.
RESULTS
Extraction yield of the G. procumbens leaves:
Gynura procumbens leaves were extracted using distilled water, hexane,
methanol, chloroform, acetonitrile and ethanol. Distilled water produced the
highest yield of G. procumbens leaves (84.51%) which was statistically
different (p<0.01) from all of the Gynura extracts, following with
ethanol and methanol, respectively (14.73 and 7.02%, respectively, Fig.
1).
Nutritional value of G. procumbens leaves:
The moisture content of fresh G. procumbens leaves before drying was
approximately 7.08 g/100 g dry weight. The chemical compositions of the Gynura
extract possessed carbohydrate approximately 0.0537 to 0.1968 μg glucose
equivalent/100 g dry weight (at the concentration of the ethanolic Gynura
extract 0.1 μg mL-1), protein approximately 4.51 g/100 g dry
weight and lipid approximately 0.023 g/100 g dry weight (Fig.
2).
The antioxidative properties of the gynura extract:
We evaluated the antioxidative properties of the Gynura Extract
by using distilled water, methanol and ethanol, which composing highest yield
(Fig. 2), polyphenol and flavonoid (data not shown). The results
indicated that Ethanolic Gynura Extract (EGE) exhibited the highest in
the antioxidative properties in every assay, EC50 = 1.63 in hydroxyl
scavenging activity, EC50 = 2.17 in iron chelating activity and EC50
= 2.75 in inhibition of lipid peroxidation (Fig. 3).
| Fig. 1: | Percent yield (dry basis, db) of the Gynura extracts. Data are representative of three repeated extractions |
| Fig. 2: | Nutritional value of G. procumbens leaves |
| Fig. 3: | The antioxidative properties in three assays of the Gynura extracts |
In this study reveals that the Gynura extract has a strong antioxidant properties by radical-scavenging action, iron metal chelating and lipid peroxidation inhibition. Anyway, the Gynura extracts showed the highest activity in hydroxyl radical-scavenging than the other activities (Fig. 3). These results suggested that consumption the Gynura extract may have health benefits.
DISCUSSION
Nowadays, herbaceous plant is gaining popularity in developing countries as medicinal food. Although, medicinal plants may cause several biological activities in humans, very little is known, such as Gynura procumbens. Although, previous studies have indicated that the important bioactive ingredients are present in methanol extract (Akowuah et al., 2002) and the recently study which revealed that administration of the methanol extract from leaves at 1000-5000 mg kg-1 did not produce mortality or significant changes in the general behavior, body weight, or organ gross appearance of rats (Rosidah et al., 2009). Contrary with our present extraction method which found that ethanolic Gynura extract (EGE) exhibited the highest in the antioxidative properties in every assay; this activity might be mediated directly by the phenolic compounds in the extracts. The phenolic mixtures in EGE extract might contain several different antioxidant activities or act in complex multiphase systems. Anyway, it agree well with the study of Iskander et al. (2002) which claimed that the crude ethanolic extract of G. procumbens has anti-inflammatory action, similar to Hoe et al. (2006) which investigated the hypotensive and angiotensin-converting enzyme (AGE) inhibitory activities of a partially purified fraction of the leaves of G. procumbens by the crude ethanolic extract. The findings also suggest that the putative hypotensive agents seem to be glycoconjugated or peptidal substances in nature. This evidence may support by our study which found that Gynura extract is a good protein source; however it showed a bit carbohydrate content. We believe the Gynura extract shows potential as a functional food or value-added ingredient. However, there are no studies of reactive oxygen species or anti-oxidation in relation to these Gynura extracts. Several investigations showed differences between the test systems for the determination of antioxidant assay (Gahler et al., 2003; Schlesier et al., 2002). It was recommended to use at least two methods. In this study, we use free radicals, such as hydroxyl radical in assayed the hydroxyl radical-scavenging activity. Phenolic compounds scavenge the H2O2 radicals that initiate lipid peroxidation (Cotelle et al., 1996; Laughton et al., 1991). Moreover, the ferrous ion-chelating activity and the inhibition of lipid peroxidation were investigated. A previous study reported that phenolic molecules may behave as metal chelators due to their catechol structure (Packer et al., 1999). It is not clear whether the Gynura extract can chelate metal ions and inhibits lipid peroxidation, therefore we conducted the experiment and found that the Gynura extract showed antioxidative properties in all of the tested methods. Our present study is the first reveal that the mechanisms of antioxidant activities in the Gynura extract is likely free radical scavenging and ion chelating, this activity might be mediated directly by the polyphenolic compounds in the extracts. The polyphenolic mixtures in the extract might contain several different antioxidant activities or act in complex multiphase systems. This investigation suggested that the Gynura extract is a good source of natural antioxidant similar in the recent study of Rosidah et al. (2008) which suggest that phenolics in these plants provide substantial antioxidant activity. Thus, consumption of foods containing various antioxidants has been recommended to prevent or slow the oxidative stress caused by free radicals (Shahidi and Wanasundara, 1992), especially, above preliminary information of the Gynura extract supported the benefits of the traditional use. The Gynura extract may further help strengthen the observations that this plant does indeed possess active principles and develop further to be commercially useful in food industry or health products as medicinal food. However, there is no study so far concerning the characterization of the active antioxidative components in Thai G. procumbens leaves. Since phenolic compounds might be a possible class of antioxidant in this the Gynura, future work on the isolation and structural characterization of the active compounds is needed.
ACKNOWLEDGMENTS
Financial support from the Center for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education is gratefully acknowledged. We are also grateful to thank the Faculty of Science, Mahasarakham University for Research Grant.