Mutiara Nugraheni, Badraningsih Lastariwati

Comparative Study Chemical Tube And Biologycal System Of Antioxidant Activity On Ethanolic Extract Of Coleus Tuberosus

Mutiara Nugraheni, Badraningsih Lastariwati

Department of cookery vocational of education, Yogyakarta State University, Yogyakarta, Indonesia

Email:

Received **** 2014

This work is licensed under the Creative Commons Attribution International License (CC BY).

Abstract

Coleus tuberosusis a minor tubers are included in family of Lamiaceae, sub-family of Ocimeae and Tribe of Nepetoide. This study Compared to the ability of the scavenging of free radicals with chemical tubes and in biological systems. The method used by the chemical of tube is 1.1-diphenyl-2-picrylhydrazyl (DPPH) and biological systems by the method of cellular antioxidant activity based on the oxidation of 2 ', 7'-dichlorofluorescein-diacetate (DCFH-DA) by reactive oxygen species (ROS). The results showed that the ethanol extract peel of Coleus tuberosus have higher antioxidant activity than ethanolic extract flesh of Coleus tuberosusthatevaluated by DPPH and cellular antioxidantmethod. This shows that the extract of Coleus tuberosus has potential as a source of natural antioxidants.

Keywords

Coleus tuberosus, celullar antioxidant

1. Introduction

Damage of proteins, fats and nucleic acids in the human body can be caused by free radicals. Such damage can result in degenerative diseases including cancer [1]. Efforts to protect the body against free radicals is by increasing plasma antioxidant capacity. This can be done by consuming vegetables and fruits that contain phytochemical compounds that have the ability as an antioxidant [2],[3].

Antioxidants in several studies to demonstrate its ability to minimize the occurrence of degenerative disease. Tests conducted so far is by in vitro chemical that is in a test tube chemical that has not been thought of uptake in the body. The analysis of antioxidants using the in vitro biological system can describe the complexity of biological systems and is an important tool to check the food, phytochemicals and dietary supplements potential for biological activity, because the model considers the cellular antioxidant activity of the compound by the cell retrieval, distribution and efficiency of protection against radicals free under physiological conditions [4]. This study aims to describe the comparative evaluation of antioxidants in-vitro chemical and biological in- vitro system.

2. Materials and Methods

Chemicals

Ethanol, 1.1-Diphenyl-2-picryl hydrazyl (DPPH), Ethanolic extraxt peel of Coleus tuberosus (EEPC) dan ethanolic extract flesh of Coleus tuberosus (EEFC), RPMI, 2,7-diacetate dichlorofluorescein (DCFH-DA), PMA from Sigma-Aldrich, Fetal Bovine Serum (FBS) from Gibco. HeLa were obtained from ATCC. All other reagents and solvents were of analytical reagent grade.

Free radical scavenging activity determination

Evaluation by DPPH refers [5]. 2 ml of DPPH (0.1 mM in methanol), 300μL extract of peel and flesh of Coleus tuberosus (100, 200 and 400 μg / ml) in methanol,after 30 minutes monitoring at λ 517nm. Ascorbic acid and BHT at concentration 10, 20 and 40 μg /ml used as a control standard. Experiment three replicates. IC50 values for determining the concentration required to scavenging of 50% DPPH free radicals.

Cell Culture

Human cervix HeLa cells was obtained from ATCC. Cells were cultured in the RPMI, supplemented with 10% heat-inactivated Fetal Bovine Serum and penicillin (100 units/ml-streptomycin (100µg/ml), using 75 cm2 flasks in a 37oC in humidified 5% CO2 incubator.

Cellular antioxidant activity in HeLa Cells.

In this study the effect of Ethanolic Extract flesh of Coleus tuberosus (EEFC), ethanolic extract peel of Coleus tuberosus (EEPC)on reduction of oxidative stress in HeLa cells was evaluated on the basis of the method reported by [6], 2-,7-Dichlorofluorescin diacetate (DCFH-DA), a peroxide-sensitive dye, was used for the evaluation of oxidative stress in cells based on oxidation of DCFH-DA by Reactive Oxygen Species. In this study, HeLa cells (cervix cancer cell line) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and streptomycin in an incubator at 37 °C, 5% CO2, 95% air, and humidity. The cell suspensions (200 µl at the concentration of 105 cells/well) were seeded in and incubated with EEFC and EEPC (100, 200, 400 and 800 µg/ml) for 20 min. Then cells were co-incubated with 25 íM DCFH-DA in the absence or presence of 100 ng PMA in darkness at 37 °C for 30 min. After incubation,cells were collected and washed once with ice-cold phosphatebuffered saline (PBS), resuspended in 200 ul of the same PBS, and placed on ice in darkness until flow cytometry was carried out. The amounts of intracellular hydrogen peroxide were detected by BD flow cytometer . At least 10000 cells were analyzed for each test, and the observed fluorescence reflects the intracellular hydrogen peroxide level.

In this test, oxidative stress is induced by addition of PMA in the extracellular medium of the HeLa. The antioxidant activity express on the reduction percentage of ROS generated in HeLa by exogenous PMA was calculated by the monitoring of the emitted fluorescence intensity (Fi).

The following relation was used (Fito− Fit1) × 100/(Fito− Fit2)

with Fito: control with oxidative stress; Fit1: treat cells; Fit2: control without oxidative stress [7].

3. Result and discussion

3.1. The antioxidant activity with DPPH method

The ability of antioxidants with DPPH method on EEPC and EEFC indicated by the IC50 (Table 1). Inhibitory concentration 50 (IC50) demonstrated ability to capture free radicals (DPPH) by 50%, the smaller the IC50 showed higher antioxidant activity. Based on the IC50ethanolic extract peel of Coleus tuberosus in this study is higher than in the flesh.

Table 1. IC50ethanolic extract peel and flesh of Coleus tuberosus with DPPH method

Compound / IC50 (µg/ml)
Ethanolic extract flesh of Coleus tuberosus (EEFC) / 1290.00±1.58a
Ethanolic extract peel of Coleus tuberosus (EEPC) / 310.97±0.32b

Note:different notation means significant different p < 0.05.

This study proved that the antioxidant activity (IC50) ethanolic extract peel is higher than ethanolic extract flesh of Coleus tuberosus. Differences in antioxidant activity peel and flesh caused by other bioactive compounds such as ursolic acid and oleanolic acid [8], maslinic acid and phytosterols (stigmasterol, beta-sitosterol, campesterol) and phenolic compounds that contain in ethanol extract peel and flesh of Coleus tuberosus[9]. Research [10-11] proved that maslinic acid and phytosterol contribute in capturing the radical DPPH. Phenol compounds have the ability to capture free radicals, it is evidenced by the strong correlation between the content of phenolic compounds and radical scavenging activity[12-14]. Differences in antioxidant activity in peel and flesh related to differences in the content of bioactive compounds [15-16]. The peel contains more bioactive compounds have a greater ability to transfer hydrogen atoms to free radicals (DPPH), so that the formation of diphenyl picrylhydrazylcompound higher than in the flesh. The greater the picrylhydrazyldiphenyl compound formed showed greater antioxidant ability, especially catching free radicals.

3.2. Cellular antioxidant activity

Cellular antioxidant activity in HeLa cells by treatment ethanolic extract peel and flesh of Coleus tuberosus in Figure 1.

A / B

Figure 1. Percentage reduction in reactive oxygen species (ROS) by treatment EEFC (A) and EEPC in HeLa cells induced by Phorbol Miristate Acetate

Note: different notations indicate significant difference (p <0.05).

Ethanolic extract peel and flesh of Coleus tuberosusable to reduce the formation of reactive oxygen species (ROS) in HeLa cells induced by PMA depends on the concentration (dose dependent manner). This study proves that the ethanolic extract peel and flesh of Coleus tuberosus able to reduce ROS on PMA-induced HeLa cells. The ability to reduce ROS presumably related to the content of bioactive compounds in Coleus tuberosus, such as ursolic acid and oleanolic acid, which has the activity of increasing the antioxidant defense system within the cell. The results are consistent with the results of research [17] which prove that the treatment Olive leaf extract containing triterpenic acid is oleanolic acid and maslinic acid can increase the antioxidant enzymes SOD and catalase compared with controls.

In addition to ursolic acid and oleanolic acid, ethanolic extract peel and flesh of Coleus tuberosus have some kind of bioactive compounds such maslinic acid, and phytosterols, such as stigmasterol, beta-sitosterol and campesterol [9] and phenolic compounds. Maslinic acid and phytosterols can increase cellular antioxidant activity both enzymatic and non-enzymatic [17-20]. Phenol compounds have the ability to increase the antioxidant defense system [21-22], [12]. so able to prevent 2,7-diacetate dichlorofluorescein (DCFH) oxidation and reduce the formation of fluorescent 2,7-diacetate dichlorofluoresceinDCF [23], [7].

ROS reduction mechanism in the ethanol extract peel and flesh of Coleus tuberosus expected as a mechanism of bioactive compounds contained therein (ursolic acid, oleanolic acid, maslinic acid and phytosterol) that captures the ROS attack the cell membrane. Increased ROS in the cells causing the cell membrane lipid undergoes oxidation so that the cell membrane permeability and fluidity changes [24]. Ursolic acid, oleanolic acid, maslinic acid, phytosterols have the ability to maintain the fluidity of cell membranes to capture ROS, so the cellular communication signal level can run well including berakaitan signal with activation of antioxidant enzymes (NRF-2-ARE).

Increased expression of NRF-2-ARE increasing role in the cell's antioxidant defense system (SOD, CAT, GPx, glutathione, vitamin C, vitamin E and carotene). An increase in the cell's antioxidant defense system (SOD, CAT, GPx) give effect to the increased ability to neutralize superoxide anion radicals (O2• -), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) induced by PMA. Increased glutathione, vitamin C, vitamin E and carotenoids in the cell can increase the capture of free radicals present in the cell, so as to decrease the number of free radicals in the HeLa cells.

This study proves that the ethanol extract peel of Coleus tuberosus able to reduce ROS is greater than the ethanol extract flesh of Coleus tuberosus. Differences in the ability to reduce ROS, one of them allegedly associated with differences in the content of ursolic acid, oleanolic acid, maslinic acid, phytosterols and phenolic compounds.The amount of ursolic acid and oleanolic acid in EEPC higher than EEFC. In EEPC the amount ursolic acid and oleanolic acid were calculated to be 13.78± 0.15, and, 19.75±0.30, respectively. EEFC had 3.41 ±0.04 µg/g sample, 3.71±0.06 µg/g sample, respectively [8].

3.3. Discussion

Coleus tuberosus of the family Lamiaceae, sub-family of Nepetoideae and tribe of Ocimeae. One characteristic is the presence of triterpenic acid compounds. The ethanolic extract of Coleus tuberosus contains triterpenic acid compounds such as ursolic acid, oleanolic acid [8]. Comparative evaluation of antioxidant with DPPH method and cellular antioxidant activity showed that the DPPH method (in vitro chemical) similar tendency, where the ability antioxidant activity of ethanolic extract peel of Coleus tuberosus greater than the ethanolic extract flesh of Coleus tuberosus.

Evaluation of antioxidant activity in vitro chemical (DPPH), the reaction tends to be on antioxidant compounds tested and free radicals are added. So the ability to captur of free radicals is highly dependent on the number of OH groups in its structure. While the evaluation of antioxidant activity using a biological system in this case is the HeLa cells, the antioxidant ability of a compound is not only a reaction between antioxidant compounds were tested by free radical compounds (PMA), but also involves other cellular mechanisms in the cell such as the cell membrane fluidity, antioxidant defense system. Thus, in vitro biological evaluation in more cells can describe the complexity of biological systems and is an important tool to check the food, phytochemicals and dietary supplements potential for biological activity, because the model considers the cellular antioxidant activity of the compound by the cell retrieval, distribution and efficiency protection against free radicals under physiological conditions [4]. This study proves that the bioactive compounds that ursolic acid, oleanolic acid, maslinic acid, flavonoids, phenolic compounds contribute to the antioxidant activity of ethanol extract of black meat and potato skins. A decrease in oxidative stress in cells may provide a positive effect on its potency in a biological system, especially the anti-proliferation of cancer cells [8],[25].

3.4. Conclusion

Evaluation of antioxidant activity for screening free radical scavenging ability of a compound can be done by combining the two methods, in vitro chemical (DPPH) and in vitro in biological systems (cell). This is done to provide a clear picture of the antioxidant potential of a compound. Variations in having antioxidant method in strengthening the information obtained. This research is expected to provide information, that Coleus tuberosus as a vegetable is not only a source of carbohydrates but also have potential as natural antioxidants.

Acknowledgements

Authors thank to the Directorate General of Higher Education Republic of Indonesia that has funded this research.

References

[1] Borek C. (2004). Dietary Antioxidant and human cancer. Integrative cancer therapies, 3 (4), 333-341.

[2] Garinstein S, Park YS, Heo BG, Namiesnik J, Leontowicz H, Leoontowicz M., Ham KS, Cho JY, Kang SG. A. (2009). Comparative Study of Phenolic Compound and Antioxidant and Antiproliferative Activities in Frequently Consumed Raw vegetables. Journal European Food Research and Technology 28: 903-911.

[3] Opata MM, Izevbigie EB. (2006). Aqueous Vernomia amigdalina extracts alter MCF-7 cell memrane permeability and efflux. International Journal of Environmental Research and Public Health. 3(2): 174-179.

[4] Liu RH, Finley J. Potential cell culture models for antioxidant research. (2005). Journal of Agricultural and Food Chemistry; 53: 4311-4314.

[5] Singh R, Singh B, Singh S, Kumar N, Kumar S, Arora S. (2009). Investigation of Ethyl Acetate Extract/fractions of Acacia nilotica Willd. Ex.Del. as potent antioxidant. Record Natural Products, 3:3: 131-138. ACG Publication

[6] Chang, S.T., Wu, J.H., Wang, S.Y., Kang, P.L., Yang, N.S., and Shyur, L.F., 2001. Antioxidant activity of extracts from Acacia confuse Bark and heartwood. Journal of Angricultural and Food Chemistry, 49, 3420-3424.

[7] Muanda FN, Bouayed J, Djilani A, Yao C, Soulimani R, Dicko A. Chemical composition and, cellular evaluation of the antioxidant activity of Desmodium adscendens leaves. Evidence-Based Complementary and Alternative medicine, 2011; Article ID 620862.

[8] Nugraheni M, Santoso U, Suparmo, Wuryastuti H (2011). Potential of Coleus tuberosus as an antioxidant and cáncer chemoprevention agent. International Food Research Journal, 18(4):1471-1480.

[9] Mooi, L.Y, Wahab, N.A, Lajis, N.H., and Ali, A.M. (2010). Chemopreventive properties of phytosterols and maslinic acid extracted from Coleus tuberosus in inhibiting the expression of EBV early-antigen in Raji cells. Journal Chemsitry and Biodiversity, 7(5), 1267-1275.

[10] Aladedunye, F.A., Okorie, D.A., and Ighodaro, O,M. (2008). Anti-inflammatory and antioxidant activities and constituents of Platostoma africanum P. Beauv.Natural Product Research, 22(12), 1067-1073.

[11] Gavani U., and Paarakh, P.M., (2008). Antioxidant activity of Hyptis suaveolens Poit. International Journal of Pharmacology, 4(3), 227-229.

[12] O’Sullivan, A.M., O’Callaghan, O’Grady, M.N., Quequineur, B., Hanniffy, D., Troy, D.J., Kerry, J.P., O’Brien, N.M., (2011). In vitro and cellular antioxidant activities of seaweed extracts prepared from five brown seaweeds harvested in spring from the west coast of Ireland. Food Chemistry, 126: 1064–1070

[13] Jung, J.K., Lee, S.U., Kozukue, N., Levin, C.E., Friedman, M., (2011). Distribution of phenolic compounds and antioxidative activities in parts of sweet potato (Ipomoea batata L.) plants and in home processed roots. Journal of Food Composition and Analysis, 24: 29–37.

[14] Ismail, H.I., Chan, K.W., Mariod, A.A., Ismail, M. (2010). Phenolic content and antioxidant activity of cantaloupe (cucumis melo) methanolic extracts. Food Chemistry, 119: 643–647.

[15] Nurliyana, R., Syed Zahir, I., Mustapha Suleiman, K., Aisyah, M.R. and Kamarul Rahim, K. (2010). Antioxidant study of pulps and peels of dragon fruits: a comparative study. International Food Research Journal, 17, 367-375.

[16] Wolfe, K., Wu, X., and Liu, R.H., (2003). Antioxidant activity of apples peels. Journal of Agricultural and Food Chemistry, 51(3), 609-614.

[17] Dekanski, D., Janicijevic-Hudomal, S., Ritic, S., Radonji, N.V., Petronijevic, N.D., Piperski, V., and Mitrovic, D,M. (2009).Attenuation of cold restraint stress-induced gastric lesions by an olive leaf extract. Gen. Physiology and Biophysics Journal Special Issue, 28, 135–142.

[18] Yoshida, Y., and Niki, E., (2003). Antioxidant effects of phytosterol and its components. Journal of Nutritional Science and Vitaminology, 49(4), 277-80.

[19] Wang, T., Hicks, K.B., and Moreau, R., (2002). Antioxidant activity of phytosterols, oryzanol, and other phytosterol conjugates. Journal of the American Oil Chemists Society, 79(12) Journal of the American Oil Chemists' Society,79(12), 1201-1206.

[20] Vivancos, M., and Moreno, J.J., (2008). Effect of resveratrol, tyrosol and b-sitosterol on oxidised low-density lipoprotein-stimulated oxidative stress, arachidonic acid release and prostaglandin E2 synthesis by RAW 264.7 macrophages. British Journal of Nutrition, 99, 1199–1207.

[21] Giovannini, C., Scazzocchio, B., Matarrese, P., Vari, R., D’Archivio, M., Benedetto, R.D., Casciani, S., Dessi, M.R., Straface, E., Malorni, W., Masella, R., (2008). Apoptosis induced by oxidized lipids is associated with up-regulation of p66Shc in intestinal Caco-2 cells: protective effects of phenolic compounds. Journal of Nutritional Biochemistry, 19: 118– 128.

[22] Verma, A.R., Vijayakumaar, M., Mathela, C.S., and Rao, C.V. (2009). In vitro and in vivo antioxidant properties of different fractions of Moringa oleifera leaves. Food and Chemical Toxicology, 47: 2196–2201.

[23] Salawu, S.O., Akindahunsi, A.A., Sanni, D.M., Decorti, G., Cvorovic, J., Tramer, F., Passamonti, S., and Mulinacci, N. (2011). Cellular antioxidant activities and cytotoxic properties of ethanolic extracts of four tropical green leafy vegetables. African Journal of Food Science, 5(4): 267 – 275

[24] Prades J, Vogler O, Aleman R, Gomez-Florit M, Funari SS, Ruiz-Guiterrez V, Barcelo F (2011). Plant triterpenic acid as modulators of lipid membran physical properties. Biochimica et Biophysica Acta , 1808: 752-760.

[25] Shyu MH, Kao TC, Y GC. (2010). Oleanolic acid and ursolic acid induce apoptosis in HuH7 human hepatocellular carcinoma cells through a mitochondrial-dependent pathway and downregulation of XIAP. Journal of Agricultural and Food Chemistry, 58 (10): 6110-6118.