Formulation and the antioxidant activity of Cincau hijau leaves (Cyclea barbata L. Miers) ethanolic extract

  

Formulation and the antioxidant activity of Cincau hijau leaves (Cyclea

barbata L. Miers) ethanolic extract

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  1 Yunahara Farida , Erlindha Gangga , Kartiningsih , Arsila 1)

  Faculty of Pharmacy, Pancasila University Jakarta Email: yunahara_farida@yahoo.com

  

Abstract

Cincau hijau (Cyclea barbata L.Miers) is a plant that has antioxidant activity. One of its secondary

metabolites is flavonoid. The research studied the extract of Cycleabarbata leaves in 4 different gel

formulas and investigated the accelerated stability for 3 months at room temperature and at 40 C 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 40

  C. Formula 2 has stronger antioxidant activity than formula 4, formula 1 and formula 3 with IC ^{50 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

  The dried powdered leaves of C. barbata was macerated with ethanol 70% at room temperature for 24 hours (three times), and the crude ethanolic solution was subsequently concentrated using rotary vacuum evaporator to obtain the respective extracts and stored in a freeze condition until used for further analysis (Depkes RI, 2000).

  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 0.0522 0,0522 0,0522 0,0522 barbata L.Miers)

  2 Carbomer 940 0,5 - - -

  3 Sepigel 305 - - 3 -

  4

• Triethanolamine (TEA) 0,5

  5 HPC-m - - -

  5

  6 HPMC - - -

  4

  7 Propylene glycol

  15

  

15

  15

  15

  8 Methylparaben 0,03 0,03 - -

  9 Propylparaben 0,01 0,01 - -

  10 Sodium benzoate 0,1 0,1 - -

  11 Edetate disodium 0,05 0,05 0,05 0,05

  12 Sodium metabisulfite 0,1 0,1 0,1 0,1

  13 Ethanol 40 - - -

  14 Distilled water qs 100 100 100 100 Formula 1 and 2

  : Dispersed carbomer 940 (gelling agent formula 1) and sepigel 305 (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.

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  Accelarated stability test for 3 months was done at room temperature and at 40 C ± 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:

  Absorbance of Control - Absorbance of Sample % DPPH radical scavenging = x 100 Absorbance of Control Then % inhibition was plotted against the respective concentrations to calculate IC 50.

RESULT AND DISCUSION

  The antioxidant activity test using DPPH free radical scavenging was done in triplicate. Table 1 showed IC value of ascorbic acid (standard) of 4.08 µg/mL. The extracts showed IC

  50

  50

  value of 47.94 µg/mL (Table 2.). The IC

  50 value was then considered in the calculation of the extract dosage in the gel formulation. o

  The results showed that the IC changed after 3 months of storage at 40

  C. This could be

  50

  due to the effect of temperature and time. However, the extract still have a high antioxidant activity with IC value range is between 50-100 µg/mL. Formula 2 is the strongest because

  50 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. IC

  50 value of vitamin C C A Absorbance of vit.C % Inhibition _control

  IC ₅₀ (ppm) (µg/mL)

  I II

  III

  I II

  III 0,5615 0,6004 0,5671 26,2640 21,1556 25,5286

  1 2 0,5353 0,5563 0,5449 29,7045 26,9468 28,4439

  0,7615 4,08

  0,4236 0,4240 0,4209 44,3729 44,3204 44,7275

  3 4 0,4008 0,4182 0,4164 47,3670 45,0821 45,3185

  0,3180 0,2219 0,3212 58,2403 70,8601 57,8201

  5 Table 2. IC value of cincau hijau (Cyclea barbata L Miers.) extract

50 C A blank Absorbance of extract % Inhibition

  IC ⁵⁰ (ppm) (µg/mL)

  I II

  III

  I II

  III 5 0,7363 0,7484 0,7316 10,4585 8,9870 11,0300 10 0,6504 0,6407 0,6383 20,9048 22,0844 22,3763

  0,8223 47,94 25 0,6060 0,5673 0,5619 26,3043 31,0106 31,6673

  50 0,4716 0,4807 0,4571 42,6487 41,5420 44,4120 100 0,3671 0,3954 0,3935 55,3569 51,9154 52,1464

  Table 3. IC ^{50 value of Cincau hijau (Cyclea barbata L. Miers) formulas after 3 months}

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at room temperature and at 40 C ± 2°Cand 75% ± 5% RH

  

IC value (µg/mL)

₅₀ Formula 1 Formula 2 Formula 3 Formula 4 Temp. Month

  Batch I Batch II Batch I Batch II Batch I Batch II Batch I Batch II 61,7630 60,9744 51,7821 59,4050 70,6755 68,0105 58,3833 62,8872 Room temp. _o

  (15-30

  C) _o 3 65,5141 61,2792 52,9465 61,7707 71,9485 74,5973 62,9523 67,2856

40 C ± 2°C

  3 67,6821 67,7251 57,6037 63,2377 72,1391 92,2392 59,2221 67,2856 Blank/control

  228,2685

  72,14

  80

67,73

  70 59,22 57,6

  60 47,94

  50 IC ₅₀

  40

  30

  20 4,08

  10 Vit.C Extract Formula 1 Formula 2 Formula 3 Formula 4

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

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  scavenging assay.Our study showed that 3 months storage at 40 C changing the IC of

  50

  extract formulas. Formula 2 has the strongest antioxidant activity with IC

  50 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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