Antioxidant potential of garlic and turmeric mixture A Traditional Indonesian formulation

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1 Indian Journal of Traditional Knowledge Vol. 14(4), October 2015, pp Antioxidant potential of garlic and turmeric mixture A Traditional Indonesian formulation E Y Sukandar *, I K Adnyana & R S Nurfitria School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia elin@fa.itb.ac.id Received 16 April 2015, revised 7 September 2015 Garlic (Allium sativum L.) and Turmeric (Curcuma longa L.) are known as cooking spices and as traditional drugs that can control several diseases such as diabetes and hyperlipidemia. Many studies on garlic and turmeric have been done previously in the laboratory. Antioxidant deactivates free radicals which involve in many diseases such as atherosclerosis, aging, immune suppression, cancer and diabetes. Herbal medicine usually used in combination to enhance the activity. Therefore, this study was aimed to determine the antioxidant activity of combination of garlic and turmeric. In this study, antioxidant activity of garlic bulb water extract and turmeric ethanol extract had been examined in vitro using DPPH method and activity of the extract and its combination had been examined using lipid peroxidation method in vitro and in Swiss Webster female rats ex vivo. The EC 50 of in vitro DPPH scavenging of garlic and turmeric extracts were and µg/ml and the lipid peroxidation scavenging of garlic, turmeric, and garlic-turmeric (1:1) extracts were , , and µg/ml, respectively. Garlic and turmeric extract of 200 mg/kg body weight (bw) each and combination of both and mg/kg bw scavenged lipid peroxidation significantly compared to control by 35.40, 22.71, 19.75, and % (p<0.05). Combination of garlic and turmeric extract showed higher antioxidant activity in vitro compared to each extract, but in ex-vivo study showed similar effect. Keywords: Antioxidant, DPPH, Lipid peroxidation, Garlic, Turmeric IPC Int. Cl. 8 : A61K 36/00, C09K 15/00, C07D 311/72, C01, C07 A study in Japan found that 90% of public diseases were caused by free radical which caused oxidative damage to the cells correlated with cardiovascular diseases, cancer, aging or many other degenerative diseases. Free radical can be formed endogenously or exogenously and can be reduced with antioxidant activity 1. Antioxidant acts in stabilizing or deactivating free radicals, usually before attack target in biological cell 2. The role of free radical deactivation in disease pathology is known to be involved in many diseases, such as diabetes, atherosclerosis, aging, immune suppression and neurodegeneration 3. Since 1950s, studies regarding free radicals which followed by natural antioxidant still on going and developing. Ascorbic acid, α-tocoferol, betacaroten, fenolic and flavonoid, can act as antioxidant 4-5. Garlic (Allium sativum L.) bulb and turmeric (Curcuma longa L.) rhizome are well known as cooking ingredients and traditional drugs. Aggarwal 6 documented traditional uses of turmeric in India, i.e., *Corresponding author for treating abdominal pain, fever, arthritis, anorexia, wrinkled skin, diabetic wounds, antihelminthic, anemia, cough and many other diseases. These plants can easily be found and widely studied as alternative treatment. Both garlic and turmeric have proven to variety pharmacological effects. Garlic showed biological effects for diabetic complication, hypertension, dyslipidemia and obesity 7. In previous study, the effects of combined extract of garlic and turmeric showed higher effect compared to single extract in lowering blood cholesterol level, triglyceride and glucose level in rat 8. The efficacy and safety of garlic-turmeric combination as antidiabetic and antidyslipidemia agent has also been evaluated in type-2 diabetes mellitus patients with hyperlipidemia at the optimum therapeutic dose of 2.4 gm daily in adult of around 60 kg 9. The combination showed to lower blood glucose level, total cholesterol, triglyceride, and LDL level Active component of turmeric is curcuminoid and active component of garlic are aliin, allicin, s-methyl cysteine. Garlic and its component allicin can suppress bioactivation of

2 SUKANDAR et al.: ANTIOXIDANT POTENTIAL OF GARLIC AND TURMERIC MIXTURE 633 carcinogen, inhibit tumor growth and prevent metastases Curcumin showed also anticancer activity pre-clinicaly and clinically 6, inhibited cell proliferation 7, showed activities to cervical cells carcinoma, head and neck squamous cell carcinoma 14. Our previous animal study has found that combination of S -methyl cystein and curcuminoid has synergistic effect on regulating cholesterol homeostasis 15 and the combination could reduce triglyceride level in rats 16. S methyl cysteine and curcuminoid also were proved to improve pancreatic langerhans islets damage of rats 17. Methodology Materials Garlic (Allium sativum L.) bulb water extract from Finzelberg herbal drug Industries, turmeric rhizome ethanol extracts (Curcuma longa L.) from PT. Chemindo Reksa. Other material: Sodium carboxymethylcellulose, 1,1-diphenyl-2-picrylhidrazyl (DDPH), methanol, tris buffered, potassium chloride, hydrochloric acid 0.1 N, 2-tiobarbiturate acid, sodium lauryl sulphate, ascorbic acid (vit C), vit E. Devices Sonde, surgical devices, water bath, reaction tube, cold temperature centrifuge (GS-15R centrifuge Beckman), micropipette, incubator, centrifuge tube, regular centrifuge, vortex, UV-Vis spectrophotometer (Hawlett-Packard AP 8452), cuvet. Animals Female Swiss Webster mice aged 6-8 weeks with gm of body weight from Animal laboratory of School of Pharmacy, Bandung Institute of Technology. Experimental procedure was conducted according to Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research NRC, 1996 Washington, DC: National Academy Press 18 and accepted by animal ethics committee in School of Pharmacy, Bandung Institute of Technology. Methods Preparation of extract Garlic (Allium sativum L.) bulb water extract was purchased from Finzelberg herbal drug Industry, this extract was prepared with maceration method using water and contained of 3.25% alliin. Turmeric rhizome ethanol extracts (Curcuma longa L.) was purchased from PT. Chemindo Reksa, this extract was prepared with reflux method using 95 % ethanol solution and contained of % curcuminoid. Free radical DPPH scavenging activity study Each sample was diluted in methanol to achieve the concentration of 2, 25, 50, 100, 200, and 500 µg/ml, DPPH was diluted in methanol to make a concentration of 40 mcg/ml, kept in low temperature and protected from direct light. A 1.5 ml sample mixed with 3.0 ml DPPH solution and incubated in room temperature for 30 min then measures at 516nm. As control, 1.5 ml methanol mixed with 3.0 ml DPPH solution. Free radical scavenging activity percentage was calculated by the following equation: scavenging activity (%) = (A control A study ) A control X 100 % Lipid peroxidation scavenging activity in vitro study Samples were diluted in 0.5% carboxymethylcellulose sodium to achieve the concentration of 50, 100, 250, 500, 750, and 1000 µg/ml. A 1 gm female rat liver homogenized in 10ml 150mM: 50mM tris-potassium chloride ph 7.2, kept in 20 0 C then centrifuged with 300 rpm at 4 o C for 10 min. A 0.2ml homogenized supernatant incubated in 37 o C for 1 hr, then added 2 ml 0.1N chloric acid, 0.1 ml 10% b/v sodium lauryl sulphate then mixed by Vortex mixer for 2 min. To this mixture added 2mL 0.5% b/v 2-thiobarbituric acid and mixed for 30 seconds, heated for 1 hr in water bath of 95 o C. Sample then centrifuged at 3000rpm for 10 minutes, then, measured at 532nm. The more TBARS (Thiobarbituric Acid Reactive Substance), i.e., the reaction of malondialdehyde with 2-thiobarbituric acid formed then the higher the absorbance, and the extract with highest antioxidant effect was shown with the lowest absorbance. Lipid peroxidation scavenging activity percentage was calculated with the equation similar to DPPH scavenging activity study. The concentration that can reduce 50% of free radicals lipid peroxidation is concluded as effective concentration 50 (EC50). Lipid peroxidation scavenging activity ex vivo study Mice were divided into ten groups, i.e., garlic extract of 100 mg/kg and 200 mg/kg bw, turmeric extract of 100 and 200 mg/kg bw, combination of garlic-turmeric of mg/kg bw

3 634 INDIAN J TRADIT KNOWLE, VOL. 14, NO 4, OCTOBER 2015 and mg/kg bw, ascorbic acid of 6.5 and 130 mg/kg bw, vit E of 52 IU/kg bw and control group that received only vechicle (0.5 % CMC Na solution).. Samples were given orally once a day for 7 days. Liver tissue homogenate was prepared on day 8. The next procedure is similar with lipid peroxidation in vitro study. The data from this study were analyzed with ANOVA. Results and discussion In this study, the DPPH (1,1-diphenyl-2- picrylhydrazyl) scavenging activity in vitro showed by garlic and turmeric extract. DPPH is a synthetic free radicals that easily accept an electron or hydrogen to form a stabile diamagnetic molecule. Associated with it, we can see that the ability of the substance in the extract to reduce DPPH by donating hydrogen. The activity was verified by the formation of yellow coloured DPP-hydrazine. The scavenging activity of DPPH is represented as extract concentration that can scavenge 50% DPPH or known as 50% effective concentration (EC 50 ). Garlic and turmeric extract have the free radical scavenging activity. Turmeric has a higher free radical scavenging activity (EC mcg/ml), on the other hand garlic has a low scavenging activity (EC mcg/ml), whereas ascorbic acid as standard showed strong antioxidant activity (EC mcg/ml). Turmeric extract is also included in a strong antioxidant activity with EC 50 value less than 200µg/mL, as shown in Fig. 1. In vitro study in lipid peroxidation scavenging activity is done according to the Xiu-Wei Yang s method, which was modified to figure out the strength of the extracts, the combination, and standard to scavenge the lipid peroxidation in the liver. Lipid peroxidation products such as malondialdehyde always produced when lipid hydroperoxide was metabolized in body s biology systems and higher concentration of malondialdehyde indicates higher oxidative damage. The malondialdehyde is the product of LDL oxidation, that has an important role in the atherosclerotic process. Malondialdehyde can react with 2-thiobarbiturate to form the pink color and can be measured by spectrophotometry. Turmeric extract has a higher lipid peroxidation scavenging activity (EC mcg/ml) compare to garlic extract (EC mcg/ml). Combination of garlic and turmeric extract has a higher scavenging activity (EC mcg/ml) compared to each single extract. As standard substance, lipid soluble antioxidant was chosen, i.e., α-tocopherol which showed EC50 of mcg/ml. Tocopherol is also deposited in liver organ and has a stronger scavenging activity compared to ascorbic acid (EC mcg/ml). The comparison of lipid peroxidation scavenging activity of the extract and standard can be seen in Fig. 2. Ex vivo study of lipid peroxidation scavenging activity was done in mice that have treated with the extract. The objective of this study is to compare the ability of combination of garlic and turmeric extract to each extract alone. As standard, 2 dosages of ascorbic acid were used, i.e., 6.5 and 130 mg/kg bw. This dosages were chosen based on recommended daily allowance in human, i.e., 50 mg/day (conversion dose in mice is 6.5 mg/kg bw) and high dosage of ascorbic acid, i.e., 1000 mg (conversion dose in mice Fig 1 Scavenging activity of DPPH of garlic, turmeric and ascorbic acid. Fig 2 Lipid peroxidation scavenging activity in vitro, EC 50 diagram

4 SUKANDAR et al.: ANTIOXIDANT POTENTIAL OF GARLIC AND TURMERIC MIXTURE 635 is 130 mg/kg bw). High dose of ascorbic acid was believed to have an antioxidant activity and were used to prevent cancer, which caused by cell oxidative damage that lead to genetic mutation. Ascorbic acid is the primary antioxidant in plasma and suggested to have a better activity in high dose. The dosage of vit E was calculated by converting human dose (400 IU/day) to mice dose (52 IU/kg bw). The results can be seen in Table 1 and Fig. 3. The result showed that 100mg/kg bw garlic extract or turmeric for 7 days treatment showed no antioxidant effect, because there was no positive scavenging activity to lipid peroxidation and showed no significant difference compared to control. Dosage of single extract of garlic and turmeric of 200 mg/kg body weight showed a positive scavenging activity and had a significant difference compared to control (p<0.05). Garlic had a higher scavenging activity than that of turmeric (35.40 vs %). Dosage of 200 mg/kg bw of garlic extract had a higher antioxidant effect compared to 130 mg/kg bw ascorbic acid, but not significantly different statistically. Generally, the combination of garlic-turmeric extract had an antioxidant effect both in lower dose (50-50 mg/kg bw) and higher dose ( mg/kg bw) and also showed a positive scavenging activity that significantly different to the control (p<0.05). Although the combination mg/ kg bw did not show a higher scavenging activity compared to the single extract. Ascorbic acid at a dose of 6.5 mg/kg bw showed no lipid peroxidation scavenging activity after 7 days of treatment, while 130 mg/kg bw dosage showed scavenging activity of 28.44%. The highest antioxidant activity was shown by α-tocopherol. The differences of lipid peroxidation scavenging activity can be seen in Fig. 3. In vitro study showed that garlic had low antioxidant effect, but in ex vivo study garlic can reduce the lipid peroxidation significantly different compared to control depends on the dosage (200 mg/kg bw). This showed that garlic antioxidant effect related to the initiation mechanism, i.e., preventing free radicals formation. Garlic extract had the ability to reduce the formation of hydrogen peroxide that could cause hydroxyl radicals formation, increasing superperoxide disynthase, glutation peroxidase, and catalase enzymes activity in vascular endothelial cells. Cardiovascular disease and cancer can be prevented with garlic extract due to this mechanism. Fig 3 Lipid peroxidation scavenging activity ex vivo study diagram G= garlic, T=turmeric Group Table 1 Lipid Peroxidation Ex vivo study results Doses (mg/kg body weight) Scavenging activity (%) n=5 Garlic bulb water ± 3.00 extract ± 9.87* Turmeric turmeric ± extract ± 19.09* Combination garlicturmeric ± 5.16* extract ± 18.83* Ascorbic acid ± (vit C) ± 10.82* α-tocopherol 52 (IU/kg bw) ± 6.24* Control 0 Note: * significantly different compared to control (p<0.05) Turmeric extract can capture free radicals in vitro and ex vivo depends on the dosage used. Combined extract has the ability to capture free radicals better than each single extract, based on the lipid peroxidation in vitro study. This combination also reduced lipid radical peroxidation in ex vivo experiment at doses mg/kg bw and mg/kg bw. The combined extract showed higher free radicals scavenging activity compared to each single extract either at in vitro study or at in vivo study. Conclusion Combination of garlic and turmeric had higher free radicals scavenging activity compared to each single extract. Ex vivo study in mice showed that single extract of garlic and turmeric at doses of 200 mg/kg body weight, combined extract of garlic and turmeric at doses of and gm/kg body weight

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