SCREENING ACTIVE FRACTION OF ETHANOLIC EXTRACT OF PASAK BUMI (Eurycoma longifolia Jack) ROOT AS ANTIOXIDANT Skrining Fraksi Aktif Ekstrak Etanol Akar Pasak Bumi (Eurycoma longifolia Jack) Sebagai Antioksidan

  

SCREENING ACTIVE FRACTION OF ETHANOLIC EXTRACT OF PASAK BUMI

(Eurycoma longifolia Jack) ROOT AS ANTIOXIDANT

Skrining Fraksi Aktif Ekstrak Etanol Akar Pasak Bumi

(Eurycoma longifolia Jack) Sebagai Antioksidan

  

Vera Nurviana, Nurkhasanah, Laela Hayu Nurani

Faculty of Pharmacy, Ahmad Dahlan University

Jl. Prof Soepomo, Janturan, Yogyakarta,

e-mail: vevey06farm@gmail.com

  

ABSTRAK

Eurycoma longifolia Jack telah digunakan sebagai obat tradisional untuk menyembuhkan berbagai jenis penya-

kit. Tumbuhan tersebut dilaporkan mempunyai aktivitas sebagai antioksidan, antimalaria, antikanker, penambah

stamina, dan antiangiogenesis pada beberapa penelitian. Tujuan dari penelitian ini adalah skrining fraksi aktif

sebagai antioksidan dari ekstrak etanol Eurycoma longifolia Jack. Ekstrak etanol masing-masing difraksinasi meng-

gunakan n-heksan, etil asetat, dan metanol. Aktivitas antioksidan yang diamati menggunakan metode DPPH. Hasil

penelitian menunjukkan bahwa semua perlakuan memiliki aktivitas sebagai penangkap radikal bebas, adalah ek-

strak etanol dengan nilai ES50 (60.796 ± 0.051 µg/ml) fraksi n-heksana (237.621 ± 1,662 µg/ml) fraksi etil asetat

(53.597 ± 0.028 µg/ml), dan fraksi metanol (109760 ± 0,34 µg/ml). Aktivitas antioksidan tertinggi E. longifolia

menggunakan metode DPPH adalah fraksi etil asetat.

  Kata kunci: Eurycoma longifolia Jack., antioksidan, radikal bebas, DPPH.

  

ABSTRACT

Eurycoma longifolia Jack has been used as traditional medicines to cure many kinds of diseases. Previous re-

searches reported to have antioxidant, antimalaria, anticancer, stamina enhancer, and antiangiogenesis activities.

  

The objective of this research was screen active fraction as antioxidant from ethanolic extract of Eurycoma longi-

folia Jack. The ethanolic ectract was fractionated using n-hexan, ethyl acetate, and methanol. Antioxidant cctivity

were observed using DPPH method. The results showed that all the treatments had the activity as scavenging the

free radical, with the value ES50 ethanolic extract was (60,796 ± 0,051 µg/ml) fraction of n-hexan (237,621 ±

1,662 µg/ml) ethyl acetate fraction was (53,597±0,028 µg/ml), and methanolic fraction was (109,760±0,34 µg/

ml). The best activity radical scavenging using DPPH method is ethyl acetate fraction.

  Key words: Eurycoma longifolia Jack., antioxidant, free radical, DPPH.

  INTRODUCTION

  Antioxidant is a compound that is very important in maintaining good health. Some diseases such as cancer, heart disease, arthritis, diabetes, liver and other degenerative diseases are increasingly being suffered by the people of Indonesia. Such diseases can be caused due to antioxidants present in the body is not able to neutralize the increase of the concentration of free radicals.

  If free radicals are not inactivated, their chemical reactivity can damage cellular macro- molecules including proteins, carbohydrates, lipids and nucleic acids. Their destructive effect on protein may play a role in the development of diseases, like cataracts. Free radical damage to DNA is also implicated in the development of cancer and its effect on LDL cholesterol is verylikely responsible for heart disease. Free radicals are also responsible for ageing (Bagchi and Puri, 1998).

  One way to reduce or prevent oxidative damage to cellular and molecular level is con- about the possible side effects of synthetic an- tioxidants, it is suggested to use natural alter- native antioxidants instead (Sunarni, 2005). Natural antioxidants can be obtained from natural ingredients derived from plants. Indo- nesia as a tropical country has a high biodiver- sity. Based on the survey, Indonesia has 13-15 % of all plant species in the world, or about 35.000-40.000 plants (LIPI, 2011).

  One such plant is pasak bumi (Eurycoma

  longifolia

  Jack), that is one of the 13 seed plant set by the government (Budianto dkk., 2004). Eurycoma longifolia Jack has been used as an antioxidant, malaria drugs, increase stamina, anticancer, and aphrodisiac (Nurani, 2013). Eurycomanone was cytotoxic on HeLa cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2 (Mahfudh and Pihie, 2008). Additionally eury- comanone of the E. longifolia Jack can inhibit angiogenesis (Salamah et al., 2009).

  Compounds contained in E. longifolia Jack is quasinoid (Miyake, 2009), and alkaloid 9-metoksisantin-6-one (Rosli et al., 2009).

  From the roots, several classes of compounds have been identified and they included quassi- noids, cathin-6-one alkaloids, β-carboline al- kaloids, tirucallane-type triterpenes, squalene derivatives, and biphenylneolignans (Lin et al., 2001), and coumarin (7-Hydroxy-6-methoxy- 2H-1- benzopyran-2-one) (Nurzaini, 2010).

  Previous studies suggested that the methanol extract of the roots of the E. longifolia Jack has antioxidant activity that is expressed in ES 50 value 423.5135 µg/ml (Filza dkk., 2006). In addition, the ethyl acetate fraction of ethanol extract, as well as the roots of the earth peg 1 isolates of E. longifolia Jack have antioxidant activity as DPPH free radical catcher with ES50 price of ethanol extract of E. longifolia

  Jack is 15.636 µg /ml and ES50 ethyl acetate fraction of ethanol

  Jack is 13.948 µg/ml and 1 isolate of 3.961 µg/ml (Nurani, 2013). The aim of the present study was to evaluate the antioxidant activity of ethanol ex- tract and its derived fraction from E. longifolia Jack.

  MATERIALS AND METHOD Materials

  The plant material examined in this study is the root of Eurycoma longifolia Jack obtained from the center of herbal raw mate- rials supplier, Pasar Gede, Solo. Plant material was determined in the Laboratory of Pharma- cognosy, Pharmaceutical Biology Section.

  The chemical material used are aquades- tilata, ethanol 96%, n-hexan, ethyl acetate, methanol pa, aquades, DPPH solutions. The tools used for the extraction and fractionation of the roots of E. longifolia Jack are glassware, balance sheets, ^{Vera Nurviana, Nurkhasanah, Laela Hayu Nurani} electric stirrer, rotary evaporators, separating funnel, Buchner funnel, vacuum, shacker orbit- al, waterbath, thin layer chromatografi plates. Tool for testing the antioxidant is UV-Vis spec- trophotometer.

  Preparation Of The Sample

  Determination of wavelength (ʎ) of maximum absorbance of DPPH solution per- formed as follows: l,0 ml 0.2 mM DPPH solu- tion added 1.0 ml methanol pa, whipped ho- mogeneous, absorbance was measured over a range of wavelength of 400-600 nm.

  (Eurycoma longifolia Jack) ROOT AS ANTIOXIDANT _{Skrining Fraksi Aktif Ekstrak Etanol Akar Pasak Bumi (Eurycoma longifolia Jack) Sebagai Antioksidan}

  ,. 2008). DPPH is characterized as stable free radical by virtue of the delocalization of the spare electron, where the molecule as a whole, so that the molecule do not dimerise, as would be the case with most other free radi- cals. The delocalization gives rise to the deep violet colour, characterized by an absorption band (517 nm) (S. Arokiyaraj et al,. 2008). ^{SCREENING ACTIVE FRACTION OF ETHANOLIC EXTRACT OF PASAK BUMI}

  DPPH radical scavenging model is wide- ly used method to evaluate antioxidant activity of natural compound and plant extracts. The degree of discoloration indicates the scaveng- ing potential of the antioxidant extract, which is due to the hydrogen donating ability (Barrei- ra et al

  DPPH scavenging = x 100% activity (%) Absorbance of negative control Data obtained in the form ES50 analyzed statistically with confidence 95% using the t test.

  The scavenging ability of the plant ex- tract was calculated using this equation: _{Absorbance of negative control – abso bance of the sample}

  Data Analysis

  Each 1.0 ml of the extract and fractions of E. longifolia Jack added with a solution of DPPH 0.2 mM. The mixture solution was stored dark place during operating time. Then the ab- sorbance was measured The reaction mixture was vortexed thoroughly and left in dark at room temperature for 60 min. The absorbance was measured spectrophotometrically at 516 nm.

  Absorbance measurements catcher of free radi- cals by DPPH method

  Determination of Wavelength maximum ab- sorption

  Extraction

  Each 1.0 ml of the sample solution is shaken with 1.0 ml of 0.2 mM DPPH solution, then observed its absorbance for 120 minutes at 516 nm wave lenght.

  Determination of operating time

  Three fractions and ethanol extract were characterized by TLC (silica gel 60 F254, Merk). The extracts were spotted into TLC plate, which then were developed with solu- tion of ethyl acetate:ethanol:water (100:10:1), and then evaluate under uv light at 254 and 360 nm. 2,2- Diphenyl- 1- Picrylhydrazyl (DPPH) Assay

  Thin-layer chromatography (TLC) analysis

  Ethanol extract then successively frac- tionated using solvents with different levels of polarity, namely n-hexane, ethyl acetate and methanol. Compounds were dissolved in n- hexane separated called the n-hexane fraction dissolved in ethyl acetate, ethyl acetate soluble fraction called ethyl acetate fraction. Further more insoluble fraction of ethyl acetate then dissolved with methanol (methanol fraction). All fractions obtained and ethanol 96% ex- tracts were analyzed by thin layer chromatog- raphy (TLC).

  Fractionation

  Maserat II was evaporated using a rotary evap- orator to get thick extract.

  Two kilograms of E. longifolia Jack macerated using 10 L ethanol 96 %. Macera- tion process is optimized with stirring using an electric stirrer for 1 hour, then save for 24 hours at room temperature. The filtrate ob- tained by filtering using a funnel Buchner, in order to obtain maserat. Dregs of the first filter was macerated again using 10 L ethanol 96 %.

RESULTS AND DISCUSSION

  Vera Nurviana, Nurkhasanah, Laela Hayu Nurani

  The violet colour disappears or decrease when is their cytotoxicity (Nobory, 1994) with pos- an antioxidant is present in the medium. Thus, sible interaction with cell wall and DNA. In the case of indole alkaloids, many studies have antioxidant molecules can quench DPPH free shown to have biological activity such as anti- radicals and convert them to a colourless prod- uct, resulting in a decrease in absorbance at tumoral, anti-inflammatory, analgesic, antioxi- dant, and antimycobacterial effects (Shi et al,. 517 nm. In this quantitative assay the extract exhibited a notable dose dependent scaveng-

  2011). Flavonoid also inhibit enzymes such as aldose reductase and xanthine oxidase. They ing of the DPPH activity. are potent antioxidants and have free radi-

  Absorbance obtained from these mea- surements are used to calculate the percent cal scavenging abilities (Daniel et al,. 2012).

  Therefore it can be assumed that the presence capture of free radicals, that is the reduction of these components may have contributed to in absorbance of the solution with the negative these antioxidant activities of the ethyl acetate control absorbance of the solution of test mate- fraction of ethanolic extract. The highest anti- rial divided by the absorbance of negative con- trol. The absorbance of the negative controls oxidant activity of ethyl acetate fractions (Table

  1) can be attributed to the fact that there are showed the total amount of DPPH in solution. more antioxidant components present in these

  Percent capture of free radicals that illustrate extracts which could react rapidly with DPPH the many DPPH free radicals were arrested or reduced by compounds in the test solution. radicals and reduce most of DPPH radical mol- ecules (Canadanovic-Brunet et al. 2005).

  The results of the calculation of percent DPPH radical arrest by ethanol extract, ethyl acetate fraction of the ethanol extract of E. longifolia Jack, the fraction of n-hexan, ethyl acetate and methanol fractions can be seen in Tables I and

  The interaction of a potential antioxi- dant with DPPH depends on its structural con- formation. The number of DPPH molecules that are reduced is correlated with the number of available hydroxyl groups (Brand-Williams

  et al

  ., 1995). It is strongly suggested that the DPPH free radical abstracts the phenolic hydro- gen of the electron-donating molecule and this could be the general mechanism of the scav- enging action of antiperoxidative flavonols, for example.

  Medicinal plants antioxidant activity is mainly due to the presence of secondary me- tabolites (Sayed, A et al,. 2011). Ethyl acetate is The ethyl acetate fraction of eurycoma longi-

  folia. Jack showed the presence of secondary

  metabolites like alkaloids, flavonoid, and sterid. Alkaloids have been associated with medicinal uses for centuries and one of their common biological properties

SCREENING ACTIVE FRACTION OF ETHANOLIC EXTRACT OF PASAK BUMI

  _{Skrining Fraksi Aktif Ekstrak Etanol Akar Pasak Bumi (Eurycoma longifolia Jack) Sebagai Antioksidan} (Eurycoma longifolia Jack) ROOT AS ANTIOXIDANT

  

Table 1. Percent DPPH radical arrest by ethanol extract n-hexan fraction, ethyl acetate fraction and methanol fraction of

ethanol extract of E. longifolia Jack

Ethanolic n-hexan Ethyl acetate Methanol extract fraction fraction fraction Mean ± SD

  60.796 ± 237.621 ± 53.597 ± 109.760 ± 0.340 0,051 1.662 0.028 CV

  

0.0832% 0.6996% 0.0514% 0.3097%

Table 2. Linear regression equation between radical concentration vs. percent arrest

  Materials test Linear equation Correlation R table ES50 ₂₎ (r ) Ethanol extract y = 0.004x + 0.207 0.995 0.95000 60.796 n-hexan fraction y = 0.001x + 0.078 0.955 0.95000 237.621

  Ethyl acetate fraction y = 0.008x + 0.059 0.988 0.95000 53.597

  Methanol fraction y = 0.002x + 0.229 0.985 0.95000 109.760

  Based on results obtained from data in The spots seein in Figure I viewed un- Tables I and II as well as those of statistical der UV light at 254 and 366 nm suggested analysis, we can say that ethanol extracts of E. that there are antioxidant cmpound contain in

  longifolia ethanolic extract and solvent fractions from E.

  Jack showed lower ES50 value than n-hexan fraction and methanol fraction but longifolia Jack root. higher than ethyl acetate fraction.

  CONCLUSION

  The result of thin layer chromatography The best activity of radical of Eurycoma

  longifolia

  (TLC)analysis on figure I. Jack extract using DPPH method found in ethyl acetate fraction.

  Figure I. TLC of Profil Sample (A= water fraction, B=methanol fraction, C= ethanol 96% ex- tract, D= ethyl acetate fraction, E= n-hexan fraction).

  Plants Research Vol. 5(3), pp. 300-308 Mahfudh N. and Pihie AHL., 2008. Euryc- manone induces apoptosis through the up regulation of P53 in human cervical carcinoma cells. Journal of Cancer Mol-

  Keanekaragaman Hayati Indonesia. Bo- gor: LIPI Press. p 7.

  Lu-E Shi,Zhi-Liang Zhang,Liang-Ying Xing, Dan Dan Yang, Yu-Peng Guo, Xiao-Feng Guo, Li-Ming Zhaoand Zhen-Xing Tang., 2011.

  Chin Pharmaceutical J , 53: 97-106.

  2001. Reinvestigation of the chemical constituents of Eurycoma longifolia.

  Lambung Mangkurat. Lin LC.,Peng CY.,Wang HS.,Lee KW.,Paulus WS.,

  lia Jack) sebagai Model Antipenuaan In Vitro. Fakultas Kedokteran, Universitas

  Filza MR., Kristanto A., Mayasari DI., Sari NI., P tri DPS., 2006. Pemanfaatan Ekstrak Akar Pasak Bumi (Eurycoma longifo-

  Abay, Medicinal Plants as Antioxidant Agents: Understanding Their Mecha- nism of Action and Therapeutic Effi- cacy, 2012: 97-145 ISBN: 978-81-308- 0509-2 Editor: Anna Capasso

  nya sebagai inhibitor kerusakan pro- tein akibat reaksi glikosilasi. Chem. Rev., 7(2): 89-97. Daniel Seifu, Freshet Assefa and Solomon M.

  rycoma longifolia Jack.) serta peranan-

  Budianto R.,Firdaus RT.,ParamitaD.,Vianty TA., Damayanti ED., Suhartono E., 2004. Uji antioksidan tumbuhan pasak bumi (Eu-

  Wiss . Technol., 28: 25-30.

  Brand-Williams W., Cuvelier ME. and Berset C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensm.-

  Pereira JA., 2008. Antioxidant activities of the extracts from chestnut flower, leaf, skins and fruit. Food Chem, 107:1106– 1113. Biology Research Center–LIPI, 2011. Status

  ecules , 4(4): 109-115.

  4(2): 350-360. Barreira JCM, Ferreira ICFR, Oliveira MBPP,

  ern Mediterranean Health Journal

  Bagchi K and Puri S, 1998. Free radicals and antioxidants in health and disease. East-

  REFERENCES

  Farmasi Indonesia , 20(3): 118-126.

  1, pp. 1–3. Salamah N., Sugiyanto, Hartati MS., Hayati F., J mariyatno F., 2009. Isolation and Iden- tification Of Eurycomanone From Akar Pasak Bumi (Eurycoma Longifolia, Jack) and Its Antiangiogenic Activity. Majalah

  “Free radical scavenging activity and HPTLC finger print of Pterocarpus san- talinus L, an in vitro study,” Indian Jour- nal of Science and Technology, vol. 7, no.

  S., 2009. Factors affecting the accumu- lation of 9-methoxycanthin-6- one in callus cultures of Eurycoma longifolia. J. Forest. Res. 20: 54-58. S. Arokiyaraj, S.Martin, K. Perinbam, P.Marie Arockianathan, and V. Beatrice., 2008.

  (Eurycoma longifolia Jack.) origin Sin- tang, West Kalimantan. School of Life Sciences and Technology ITB. Rosli N., Maziah M., Chan KL.and Sreeramanan

  Nurani LH., 2013. Isolation and free radicals scavenging activity of isolate-1, ethyl acetate fraction, and ethanolic extract of Pasak Bumi (Eurycoma longifolia Jack) root. Faculty of Pharmacy Ahmad Dahlan University, Yogyakarta. Nurzaini RR., 2010. Content analysis of polar fractions roots and stem Pasak Bumi

  Nobori T, Miurak K, Wu DJ, Takabayashik LA, Carson D A. Deletion of the cyclin-de- pendent kinase- 4 inhibitor gene in multiple human cancers. Nature 1994; 368 (6473): 753-75

  longifolia. Journal of Natural Products, 72(12): 2135-2140.

  Miyake K., Tezuka Y., Awale S., Li F. and Kadota S., 2009. Quassinoids from Eurycoma

  Antioxidants extraction by supercritical CO2 Journal of Medicinal ^{Vera Nurviana, Nurkhasanah, Laela Hayu Nurani}

SCREENING ACTIVE FRACTION OF ETHANOLIC EXTRACT OF PASAK BUMI

  _{Skrining Fraksi Aktif Ekstrak Etanol Akar Pasak Bumi (Eurycoma longifolia Jack) Sebagai Antioksidan} (Eurycoma longifolia Jack) ROOT AS ANTIOXIDANT

  Sunarni T., 2005. Aktivitas antioksidan penan kap radikal bebas beberapa kecambah dari biji tanaman familia Papilionaceae.

  Jurnal Farmasi Indonesia,

  4: 34-39