Formulation and the antioxidant activity of Cincau hijau leaves (
Cyclea
barbata
L. Miers) ethanolic extract
Yunahara Farida1, Erlindha Gangga1, Kartiningsih1, Arsila1 1) formulas and investigated the accelerated stability for 3 months at room temperature and at 400C with a humidity of 75% and as well as evaluated the antioxidant activity using DPPH free radical scavenging assay. The results showed that all formulas showed different activity after 3 months storage at 400C. Formula 2 has stronger antioxidant activity than formula 4, formula 1 and formula 3 with IC50 value of 57.60; 59.22; 67.73 and 72.14 µg/mL respectively. All formulas demonstrated antioxidant activity and can be used to inhibit the premature aging of the skin.
Keyword : Cincau hijau (Cyclea barbata L. Miers), antioxidant activity, DPPH, gel formula.
INTRODUCTION
Indonesia is famous as an agricultural country with a variety of plants. In recent years, many researchs exploiting plants to obtain secondary metabolites to treat various diseases such as diabetes, high blood pressure, respiratory tract infections and other disease that related to the damage caused by free radicals. Free radicals contain one or more unpaired electrons and can generate reactive molecules. These molecules act as electron acceptor and steal electron from other molecules. In an attempt to prevent free radical damage, it would require the presence of antioxidants (Winarsi, 2007).
Antioxidant is a compound that slow or prevent damage caused by free radicals. Antioxidant is a powerful electron donor that react with free radicals that damage biomolecules.
Cincau hijau (Cyclea barbata L. Miers) leaves used for health, besides containing flavonoids, its also contains alkaloids, saponins, tannins and steroid/triterpenoids. These compounds work in synergy so as to increase its antioxidant activity (Chalid, 2003; Katrin et al., 2012). C. barbata plants are vines that grow in the area of West Java. The plants usually made drinks in gel form and some people used as medicine for fever or hearthburn empirically. This study was done to create formulas in gel dosage form of C. barbata leaves extract (Allen and Loyd, 2002), then the formulas tested its activity as an antioxidant after three month
MATERIALS AND METHODS
Materials. Cincau hijau leaves (C. barbata) were obtained from the Medicinal Plant Garden, Research Institute for Spices and Medicinal Plants (Balittro), Bogor. DPPH, methanol, carbomer 940, Sepigel 305, TEA, HPC-m, HPMC, Propylene glycol, methyl paraben, propyl paraben, sodium benzoate, edetate disodium, sodium metabisulfite, ethanol.
Methods.
Preparation of plant extracts
Preparation of gel formula containing extract
Adapted from Allen and Loyd, 2002; Barel et al., 2009) The ingredient on formula 1 – 4
No. Ingredient Formula % (w/w)
F1 F2 F3 F4
1 Cincau hijau extract (Cyclea barbata L.Miers)
(gelling agent formula 2) in distilled water at room temperature for 24 hours with continuous stirring at 2000 rpm in speed. Methyl paraben and propyl paraben were dissolved in propylene glycol. Sodium metabisulfite and edetate disodium were dissolved indistilled water and mixed it with Cyclea barbataextract. Finally full mixed ingredients were mixed properly to the carbomer 940 gel with continuous stirring 10-15 min, (2000 rpm) until homogen and triethanolamine was added wisely to the formulation to obtain the gel at required consistency.The same method was applied for preparation with basis gel formula in Table 1. Accelarated stability test for 3 months was done at room temperature and at 40oC ± 2°C and 75% ± 5% RH in climatic chamber.
Formula 3 and 4: Dispersed carbomer 940 with aquadest at room temperature for 24 hours
and then homogenized with 2000 rpm speed (as gelling agent). Triethanolamine (TEA) was dissolved in distilled water, then mix with carbomer 940. Sodium benzoate, sodium metabisulfite and edetate disodium were dissolved in distilled water and the mixed with extract of Cyclea barbata. Formula 3 used HPC-m whereas formula 4 used HPMC as basis gel. HPC-m and HPMC were dispersed with ethanol (as gelling agent).
Antioxidant activity Test
The antioxidant activity of C. barbata extract and formulas were tested using DPPH scavenging. Briefly, samples (extract dan four formulas) of various concentrations were prepared 5, 10, 25, 50, and 100 µg/mL. Each sample concentration was mixed with 1.0 mL of 1mM methanolic DPPH solution. All the solution were prepared with methanol to 5.0 mL. Experiment was done in triplicate. The test sample were incubated for 30 min. at room temperature and the absorbance measured at 517 nm (Blois, 1958; Gow-chin and Chen, 1995; Yunahara et al., 2009). Ascorbic acid was used as a standard and DPPH in methanol was used as a control. The different in absorbance between the test and the control was calculated and expressed as % radical scavenging activity. Percent radical scavenging activity was measured using following equation:
Then % inhibition was plotted against the respective concentrations to calculate IC50.
RESULT AND DISCUSION
The antioxidant activity test using DPPH free radical scavenging was done in triplicate. Table 1 showed IC50 value of ascorbic acid (standard) of 4.08 µg/mL. The extracts showed IC50 value of 47.94 µg/mL (Table 2.). The IC50 value was then considered in the calculation of the extract dosage in the gel formulation.
The results showed that the IC50 changed after 3 months of storage at 40oC. This could be due to the effect of temperature and time. However, the extract still have a high antioxidant activity with IC50 value range is between 50-100 µg/mL. Formula 2 is the strongest because it was using base gel modification (sepigel 305), that makes it more stable than the others. Figure 1 shows the antioxidant activity of cincau hijau, the Formulas and Vit.C as a control.
The gel formulation can be used as an antioxidant because it can inhibit the activity of free radicals (Zao et al., 2004), that can inhibit premature aging of skin.
Table 1. IC50 value of vitamin C
C (ppm)
Acontrol Absorbance of vit.C % Inhibition IC50
(µg/mL)
Table 2. IC50 value of cincau hijau (Cyclea barbata L Miers.) extract
Table 3. IC50 value of Cincau hijau (Cyclea barbata L. Miers) formulas after 3 months at room temperature and at 40oC ± 2°Cand 75% ± 5% RH
Ablank Absorbance of extract % Inhibition IC50
Figure 1. Graph of the antioxidant activity (µg/mL) of cincau hijau extract and as mean of batch I and II, the formulas, and vit C. as control
CONCLUSION
Cincau hijau (Cyclea barbata L. Miers) showed antioxidant activity in DPPH radical scavenging assay.Our study showed that 3 months storage at 40oC changing the IC50 of extract formulas. Formula 2 has the strongest antioxidant activity with IC50 value of 57.60 µg/mL. All the formulas demonstrated activity because it can scavenge DPPH free radicals and could be developed to prevent premature aging of the skin
Acknowledgments
We are grateful to DITLITABMAS DIT.JEN DIKTI for Research Grant of Hibah Bersaing (2013) and Faculty Pharmacy of Pancasila University for facilities of this research.
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