Indo. J. Phar. Scie. Tech. Vol. 5, No. 2, 43-48 (2018). https://doi.org/10.24198/ijpst.v5i3
Triyono Triyono, Anis Yohana Chaerunisaa, Anas Subarnas
Department of Pharmaceutical Technology, Faculty of Pharmacy, Universitas Padjadjaran, Jawa Barat – Indonesia
Korespondensi : firstname.lastname@example.org (Triyono Triyono)
Keywords : Antioxidant, Curcuma domestica, Cassia fistula, DPPH Method
There has been the attention toward the field of free radical chemistry in recent year. Free radicals such as reactive oxygen species and reactive nitrogen species are generated by our body by various endogenous systems, exposure to different pathological states or physiochemical conditions1. Free radicals generally form naturally as part of our body’s metabolic processes. However, free radicals can also be affected by environmental factors pesticide use in food including, smoking habits, radiation, and pollution2. Free radicals are molecules that have one or more unstable electron3. Research on antioxidant activity is conducted for the purpose of improvement of food quality and treatment.
Antioxidants have the ability to neutralize free radicals without becoming free radicals themselves4. When antioxidants neutralize free radicals by receiving or donating electrons, they will not turn into free radicals and remain stable. Antioxidants are widely found in vegetables, fruits, and medicinal plants5.
The use of natural product as traditional medicine is not enough only based on experience passed down from generation to generation, but also need to be proven scientifically. As we known medicinal plants contain active substances that can be efficacious for the cure of disease. Various studies have been conducted to prove the pharmacological activity and determine the chemical content of the natural materials. One of these natural materials has been reported some pharmacological activity is Cassia fistula L.6 and Curcuma domestica Val.7.
Trengguli plant is proven to have some pharmacological activity. Trengguli bark ethanol extract has antioxidant activity by inhibiting DPPH8,9. Trengguli plant studies show strong relation between hepatoprotective activity and antioxidant activity that can be seen from liver biochemical and oxidative stress parameters10. Meanwhile, the study of turmeric rhizome shows that the ethanol extract of turmeric rhizome has hepatoprotective activity in paracetamol-induced rats11. Curcumin as one of the active compound in turmeric rhizome has activity as antioxidant, anti-inflammatory, antidiabetic, anticarcinogenic, anticoagulant, antihypertensive, and antidislipidemia12. Research on the effectiveness of C. domestica Val. as hepatoprotectors and immunomodulators show that turmeric can decrease Serum Glutamic Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT) levels and may increase the activity of nonspecific immune systems in mice induced by CCl48. Other studies have shown that turmeric rhizome has hepatoprotective activity and prevent the increase of SGOT or SGPT due to toxic dosage of acetaminophen13.
Various studies above have demonstrated various pharmacological activities from turmeric rhizome and different parts of the trengguli plant. The aim of this research is to know the potency of in vitro antioxidant activity on combination of ethanol extract from turmeric rhizome and trengguli bark compare to each extract and ascorbic acid as standard with DPPH method because of its accuracy and ease.
Materials and Methods
2.1. Materials and Apparatus
Turmeric rhizome from species C. domestica Val. and trengguli bark from species C. fistula L. obtained from Manoko plantation, Lembang, Bandung. Determination of plants is evaluated at the Laboratory of Taxonomy, Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University. The chemicals used in this study include ethanol 70% (Merck), ethanol 96% p.a (Merck), DPPH (2,2-diphenyl-1-pycryl-hydrazil) (Sigma), ascorbic acid (Merck). Absorbance of sample is analyzed using UV-Vis spectrophotometer (TECAN M200Pro).
The extraction simplicia of turmeric rhizomes and simplicia of trengguli bark plant were done on different maserator by maceration with 70% ethanol solvent for 3 times 24 hours. Liquid extract concentrated with a rotary evaporator at a temperature of 60 ºC then steamed above a water bath with a temperature of 40 ºC until constant weight of the extract. The yield of the extract can be calculated by the formula:
Furthermore, the examination of organoleptic extract consisting of form, color, odor, and taste extract.
2.2.2. Thin Layer Chromatography Patterns
Thin layer chromatography was performed to determine the chemical content in the extract. In the thin layer chromatography analysis, silica gel phase GF254 was used in this experiment with mobile phase toluene : ethyl acetate : formic acid (5: 4: 1) for trengguli bark extract8 and chloroform : methanol (95 : 5) for turmeric rhizome extract. The extract was first dissolved in ethanol and then applied to a silica gel plate of size 10 cm x 2.0 cm and inserted into a chromatographic vessel previously saturated mobile phase. The chromatographic process was stopped when the mobile phase reached the finish line. The chromatogram pattern observed with visible and UV light (λ254 and λ366 nm), then calculated the value of Rf.
2.2.2. Determination of IC50 with DPPH Method
The ethanol extract of turmeric rhizome, trengguli bark and a combination of ethanol extract of turmeric rhizome – trengguli bark (1:1.5) were added 1 mL DPPH for each concentration, then vortex and incubated for 30 minutes at room temperature. The absorbance was measured at 516 nm. The inhibition percentage was calculated using the equation :
Ab = DPPH absorbance
As = Sample Absorbance
The % inhibition obtained then used for determined IC50 that showed the concentration of sample to inhibit 50% free radical
2.2.2. In vitro Antioxidant Activity Test of Extract
a. Sample Preparation
0.01% w/v ethanol extracts were prepared with a stock solution of 100 ppm. 100ppm stock solution is diluted to concentration 4, 6, 12, 16, 20 ppm.
b. Preparation of Comparative Solutions
0.01 % w/v Ascorbic acid was prepared with a solution of 100 ppm. Diluted a stock solution untill concentration 10 ppm. 10 ppm stock solution made standard solution 1, 2, 3,
4, 5 ppm.
c. Preparation of DPPH solution (2,2-diphenyl-1-pycryl-hydrazyl)
DPPH was weighed and dissolved in ethanol p.a at a concentration of 0.01% w/v (100 ppm) for immediate use and maintained in low temperatures and protected from light exposure.
d. Maximum Wavelength Determination
DPPH solution of 1 mL was dissolved with ethanol to 5 mL in 5-ml flask, measured at 500-530 nm wavelength to obtain an absorbance of ± 0.2-0.8.
The results of plant determination show turmeric rhizome belongs to the Family: Zingiberaceae, Genus: Curcuma, Species: Curcuma domestica Valeton. Trengguli bark
included in the Family: Fabaceae, Genus: Cassia, Species: Cassia fistula L.
Turmeric rhizome extraction (1100 g) was macerated with 70% ethanol solvent resulting rhizome extract of 250.25 g (rendement = 22.75%). While the extraction of trengguli bark (850 g) in maceration with 70% ethanol solvent resulting trengguli bark extract of 276.25 g (rendement = 32.50 %).
3.3. Phytochemical Extract Screening and Organoleptic Test Results
The results of phytochemical screening of extract can be seen in Table 1 and organoleptic test can be seen in Table 2.
3.4. TLC results
TLC results seen in visible light, UV λ254 nm and UV λ366 nm TLC results can be seen in Table 3.
3.5. Antioxidant Activity Test Results
The results of in vitro antioxidant activity test of ethanol extract of turmeric rhizome, ethanol extract of trengguli bark, combination both of them and ascorbic acid can be seen in Table 4 – 7. For comparasion, ascorbic acid used because known as a strong antioxidant.
The combination of both extract (1 : 1.5), chosen to illustrate the combination of the minimum dose of the two extracts, shows IC50 values is 13.70 μg/mL (Table 6). The minimum dose that still has antioxidant activity for turmeric rhizome extract is 100 mg/kg bw and trengguli bark extract is 150 mg/kg bw9. From the results of research that IC50 values of ethanol extract of trengguli bark is better than ethanol extract of turmeric rhizome which causes antioxidant activity becomes stronger than single extract of turmeric rhizome, but the combination ethanol extract of turmeric rhizome – trengguli bark (1: 1.5) has not been able to produce IC50 value smaller than ethanol extract of trengguli bark. This is probably due to the comparison between the two extracts based on the minimum dose can not reach the IC50 value under a single extract of the trengguli, but the combination can still prove that with a combination ethanol extract of turmeric rhizome – trengguli bark (1: 1.5) including a very strong antioxidant category.
The results showed that ascorbic acid, ethanol extract of turmeric rhizome, ethanol extract of trengguli bark and combination of ethanol extract of turmeric rhizome – trengguli bark (1:1.5) gave antioxidant activity by DPPH method. Ascorbic acid, as a comparasion, shows IC50 value 3.14 μg/ mL. While ethanol extract of trengguli bark has the best antioxidant activity with IC50 value 10.98 μg/mL compare to combination of ethanol extract of turmeric rhizome – trengguli bark (1:1.5) and ethanol extract of turmeric rhizome with IC50 value is 13.70 μg/ mL and 41.95 μg/mL, respectively.
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