ANALYSIS OF SECONDARY METABOLITES PROFILE OF LEMPUYANG GAJAH (Zingiber zerumbet Smith) ETHANOL EXTRACT USING GAS CHROMATOGRAPHY MASS SPECTROSCOPY WITH DERIVATIZATION Analisis Profil Metabolit Sekunder Ekstrak Etanol Lempuyang Gajah (Zingiber zerumbet Smit

ANALYSIS OF SECONDARY METABOLITES PROFILE OF LEMPUYANG GAJAH

  

(Zingiber zerumbet Smith) ETHANOL EXTRACT USING GAS CHROMATOGRAPHY

MASS SPECTROSCOPY WITH DERIVATIZATION

  

Analisis Profil Metabolit Sekunder Ekstrak Etanol Lempuyang Gajah (Zingiber

zerumbet Smith) Menggunakan Kromatografi Spektroskopi Massa

Terderivatisasi

Dedi Hanwar, Mutia Sari Dewi, Andi Suhendi, Ika Trisharyanti D.K.

  

Fakultas Farmasi, Universitas Muhammadiyah Surakarta

e-mail: dedi.hanwar@ums.ac.id

ABSTRAK

  

Lempuyang Gajah (Zingiber zerumbet Smith) adalah salah satu spesies tanaman yang mengandung metabolit

sekunder yang penting dalam pengobatan penyakit. Penelitian ini dilakukan untuk mengetahui metabolit sekunder

yang terkandung dalam ekstrak etanol lempuyang gajah dari dua daerah (Solo dan Yogyakarta) setelah dideriva-

tisasi dan menentukan tingkat zerumbon nya. Analisis profil metabolit dilakukan dengan kromatografi gas dengan

detektor massa spektroskopi, sistem injeksi split, dan helium sebagai fase gerak dengan kecepatan konstan 3,0 mL/

menit dan diderivatisasi dengan BSTFA. Sementara zerumbon tingkat ditentukan dengan metode yang sama tetapi

tanpa derivatisasi. Hasil penelitian menunjukkan bahwa terdapat perbedaan profil metabolit sekunder ekstrak

etanol lempuyang gajah dari Solo dan Yogyakarta, namun tingkat zerumbon yang tidak berbeda dalam dua ekstrak

adalah 13,00% (Solo) dan 13,35% (Yogyakarta).

  Kata kunci: profil metabolit sekunder, GCMS, Zingiber zerumbet Smith, zerumbon, BSTFA

_ABSTRACT

_{tained in the ethanol extract of lempuyang gajah from two regions (Solo and Yogyakarta) after derivatized and}

_{are important in diseases treatment. This study was conducted to determine the secondary metabolites con-}

Lempuyang gajah (Zingiber zerumbet Smith) is one of the plants species that contain secondary metabolites that

_{derivatized with BSTFA. While zerumbone levels determined by the same method but without derivatization.}

_{troscopy detector, split injection system, and helium as the mobile phase at a constant rate of 3.0 mL/min and}

determine its zerumbon level. Metabolite profile analysis performed by gas chromatography with mass spec-

The results showed that there were differences in secondary metabolite profiles of ethanol extract of lempuyang

gajah from Solo and Yogyakarta, but its zerumbon levels did not differ in the two extracts were 13.00% (Solo) and

_Keywords: 13.35% (Yogyakarta). profile of secondary metabolites, GCMS, Zingiber zerumbet Smith, zerumbone, BSTFA

  INTRODUCTION Zingiber zerumbet has been widely use in the preparation of traditional medicines.

  Materials used in this research were Lempuyang gajah and methanol. The instru- ment used were a set of Gas Chromatography Shimadzu GC-2010 equipped with a Shimadzu GC-Mass Selective Detector 2010s with Rxi TM-1MS column, micropipette, refrigerators.

  ture

  10:1), the injector temperature of 280 °C, column temperature of 70 °C (5 min) - 270 °C (15 min) with a temperature rise of 10 °C/min. While the condition of MS, ion source tempera-

  tio

  and RxiTM-1ms capillary column (30m x 0.25 mm, film thickness 0.25 lm). Used helium carrier gas with a constant rate of 3.0 mL/min, injected as much as 1 mL (split ra-

  detector

  Solution B in eppendorf tubes ready to be injected into GCMS. Analyses were per- formed using a Shimadzu GC-2010 equipped with a Shimadzu GCMS-2010s mass selective

  Analysis of Secondary Metabolites Profiles Us- ing GCMS

  A 100 µL solution was taken and evap- orated to dryness under nitrogen gas. Then added 100 μl BSTFA and heated at 70 oC for 10 min and then injected at GCMS (Solution B).

  Derivatization Process

  A total of 10.0 mg of lempuyang gajah extract weighed accurately and dissolved with methanol and then inserted into the measur- ing flask and 5 mL of methanol was added to the limit (Solution A).

  Methods Sample Preparation

  MATERIALS AND METHOD Materials

  Its rhizome used as a slimming drug, appe- tite enhancer, body warmers, headache medi- cine, remedy dysentery, and helps remove gas (carminative) abdominal bloating. Its ethanol extract of the rhizome has analgesic and an- tipyretic activity capable of inhibiting inflam- mation caused by induction of prostaglandin (Somchit et al

  Smith from different regions.

  GCMS for separation and detection, re- quiring organic compounds volatile gases and inorganic compounds in a mixture (Settle, 1997) and is stable at temperatures of testing, mainly of 50-300°C. If the compound is not volatile or unstable at the test temperature, the compound must be derivatized in order to be analyzed by GCMS (Mardoni et al., 2007). Therefore Zingiber zerumbet raw material har- vested from different areas of each unknown secondary metabolite profiles were analyzed using GCMS with derivatization, this study is to provide an overview of each of the chromatographic profiles of secondary metab- olites Zingiber zerumbet

  ., 2008). Metabolite profiles are used for relative quantitation of metabolites of a num- ber of samples. GCMS can yield more accurate data on the identification of compounds that are equipped with molecular structure (Pavia, 2006).

  al

  ., 2000; Roessner et al., 2000; cit. Cha et

  et al

  ., 2005; Fienh

  Zerumbone assay can be performed with gas chromatography mass spectrometry (GCMS) method for qualitative and quantita- tive analysis. In addition, GCMS was also one of the technology for metabolite profiling (Ko- pka, 2006), because it has a good reproducibil- ity and wide applications for various types of classes of metabolites (Dunn et al

  et al ., 2000; Bhuiyan et al., 2009).

  ., 2010; Rout et al., 2011). Zerum- bone and α-caryophyllene contained in the leaves and rhizomes, and these compounds at high concentrations showed anti-inflammato- ry activity, antiulcus, antioxidant and antimi- crobial (Jaganath and Ng, 2000; Somchit and Shukriyah, 2003; Mascolo et al., 1989; Agrawal

  ., 2005), and antioxidants activity (Stanly et al

  250 °C, interface temperature 300 °C, Dedi Hanwar, Mutia Sari Dewi, Andi Suhendi, Ika Trisharyanti D.K. and the solvent cut time 3 minutes. Ionization energy of 70 eV with a scan range of 0.5 sec- onds with the weight of fragment 35-550 m/z. Components were identified by comparing the mass spectra of samples with internal Willey

  Library. Quantitative Test

  a. Preparation of Standard Curve Taken 100 mL, 150 mL, 200 mL, 400 mL, and 800 mL of stock solution isolates zerum- bon, then inserted into the Eppendorf tube and added to 1.0 mL of methanol, to obtain a concentration of 0.02%; 0.03%; 0.04%; 0.06%; 0.08%. Then each concentration was analyzed by GCMS system. The area acquired is made proportional to the concentration equation as X-axis and Y-axis area as.

  b. Sample Preparation A total of 10.0 mg of lempuyang gajah extract weighed carefully and dissolved with methanol and then inserted into the measur- ing flask and 5 mL of methanol was added to the limit.

  Metabolite profile analysis using GCMS

  Rhizomes were taken from lempuyang gajah (Zingiber zerumbet Smith) from two dif- ferent regions, namely Solo and Yogyakarta. Analysis using gas chromatography for non- volatile polar compounds, such as phenolic and acidic compounds, the assay is less sensi- tive and there is peak tailing, the derivatization method used to improve the accuracy of gas chromatography, reproducibility, and sensitiv- ity (Nakashima et a l., 2004).

  After derivatization and analyzed us- ing GCMS, there are significant differences be- tween the sample and the sample of non de- rivatization with derivatization, as seen that many peaks appear (Figure 1). In the non-po- lar volatile components such as phenolic com- pounds and acidic compounds detected after many derivatized, for example, lactic acid, acetic acid, formic acid, citric acid and oxalic acid.

  Derivatization will differ among plant species and depends on the genetic composi- tion (endogenous enzyme activity) (Pierce, 1968). According to Kaufman et al. (1999), the environment is one factor that is important in the biosynthesis of metabolites in plants. Plants can regulate metabolite production in accordance with changing factors that exist in the environment.

  According to Gupta (1994), a medicinal plant that grows in the soil and different sea- sons will produce chemical compounds and different therapeutic effects. This suggests that the environment influence the chemical constituents of plants.

  Derivatization method for gas chroma- tography mass spectroscopy is the method of Silylation (BSTFA + 1% TMCS) because it cov- ers a broad range in variety of applications, excellent thermal stability, and good chro- matographic characteristics (Pierce, 1968). In addition, because it is easy to prepare and use, time efficient, and a variety of reagents are available.

  Silylation reagents are generally sensi- tive to moisture. It can be controlled by drying using nitrogen to prevent deactivation. BSTFA reagent was used because of its availability, easy to use, faster reaction, and efficient time (Kuo and Ding, 2004) (Shin et al., 2001).

  Figure 1. GCMS Profiles of Lempuyang Gajah (Zingiber zerum _{bet Smith) Extract. (A) non derivatization and (B) with derivatization}

  _{(Zingiber zerumbet} Smith) ETHANOL EXTRACT USING GAS CHROMATOGRAPHY MASS SPECTROSCOPY WITH DERIVATIZATION

ANALYSIS OF SECONDARY METABOLITES PROFILE OF LEMPUYANG GAJAH

  Analisis Profil Metabolit Sekunder Ekstrak Etanol Lempuyang Gajah (Zingiber zerumbet Smith) Menggunakan Kromatografi Spektroskopi Massa Terderivatisasi

RESULTS AND DISCUSSION

  Silylation reagents are generally sensi- tive to moisture. It can be controlled by drying using nitrogen to prevent deactivation. BSTFA reagent was used because of its availability, easy to use, faster reaction, and efficient time (Kuo and Ding, 2004) (Shin et al., 2001).

  7.74 Glyoxalite Hydrate 74.40 0.60 + +

  21.31 Stearic Acid 284.48 4.38 + +

  21.04 Octadecanoic acid 284.48 2.35 + -

  20.97 Linoleic acid 278.43 6.75 + -

  19.47 Palmitic Acid 256.43 3.08 + +

  18.99 D-sorbite 182.17 1.56 + -

  18.44 Xylose 150.13 1.28 - -

  17.64 Acid Manonic 104.06 2.95 - +

  17.44 Citric Acid 126.07 1.32 - +

  15.82 Zerumbone 218.340 13.22 + +

  15.50 Acid Arabinonic 166.130 4.62 + -

  13.42 Malic Acid 134.09 1.23 + +

  92.00 5.41 + +

  10.42 Glycerol

  90.08 5.92 + +

  The compounds can be derivatized us- ing BSTFA were alcohol, phenol, carboxylic acids, amines, and amides (Evershed, 1993). TMCS is silylation catalysts, to increase the re- activity of the BSTFA reagent.

  5.46 Lactic Acid

  5.27 Oxalic Acid 126.07 1.92 + +

  3.06 Succinic Acid 118.09 0.93 + -

  Solo Yogyakarta

  

Table 1. Comparison of qualitative secondary metabolite lempuyang elephants from areas Solo and Yogyakarta with a

_{minimum area of 200,000}

RT (min) (N = 3) Name BM % Area Area Metabolites

  version 7 so it can be ex- pected that the compounds contained in the extract (Table 1). The main criteria for the se- lection of a suitable ion for compound identifi- cation should have a high peak areas (> 0.05%) (Jiang et al., 2006). (Solo and Yogyakarta) but both areas the dis- tance is not too far away so that differences in location, climate, rainfall, and intensity of sun light may not vary much.

  ley Library database

  The analysis resulted in several subse- quent peaks of each peak compared with Wil-

  Figure 2. C GCMS Profiles of Lempuyang Gajah (Zingiber _{zerumbet Smith) Extract. (A) Yogyakarta and (B) Solo}

  50000. Replica- tion 3 times produce chromatograms are al- most similar to each other (Fig. 2). Although the plants from different regions,

  GCMS can yield more accurate data in the identification of compounds that are equipped with molecular structure (Pavia, 2006). Both samples were analyzed using gas chromatog- raphy with a integration area

  ., 2005; Fienh et al., 2000; Roessner et al., 2000; cit. Cha et al., 2008). GCMS shown to increase the sensitivity and ideal for many applications in the field of health.

  Gas chromatography is used because it has good reproducibility and wide applica- tions for various types of classes of metabolites (Dunn et al

  (+): Refer to present of compounds; (-): Refer to absence of compounds Dedi Hanwar, Mutia Sari Dewi, Andi Suhendi, Ika Trisharyanti D.K.

ANALYSIS OF SECONDARY METABOLITES PROFILE OF LEMPUYANG GAJAH

  From the above-mentioned standard curve, equation Y = 20000000 X - 355 625 with a correlation coefficient (r ² ) of 0.9967. The equation used to determine the estimated value of Y at a particular level of X, where Y is the area and X is the concentration. Based on the linear regression equation obtained an av- erage grade of zerumbon Solo area by 13% w / w, and from the Yogyakarta at 13.35% w / w.

  Analisis Profil Metabolit Sekunder Ekstrak Etanol Lempuyang Gajah (Zingiber zerumbet Smith) Menggunakan Kromatografi Spektroskopi Massa Terderivatisasi

  _{(Zingiber zerumbet} Smith) ETHANOL EXTRACT USING GAS CHROMATOGRAPHY MASS SPECTROSCOPY WITH DERIVATIZATION

  CONCLUSIONS There were significant differences in the chromatographic profiles of secondary metabo- lites between the Solo and Yogyakarta after deriva- tized.

  around 13.00% w/w and these result showed that zerumbone is a major compounds on lem- puyang gajah extract.

  13.35 Zerumbone levels using GCMS were

  13.00 Yogyakarta

  Table 2. Zerumbone Level of Lempuyang Gajah Extract Area Levels of Zerumbone (% w/w) Solo

  The results of each of the extracts showed peak differences and differences in the amount of metabolites contained. The re- sults of gas chromatography mass spectrosco- py analysis with a minimum area of 200,000, the extract from the Solo and Yogyakarta, there were 13 and 12 compounds were identified re- spectively. While the compounds owned by the both area were 8 compounds.

  Secondary metabolites contained were xylose, palmitic acid, stearic acid, zerumbone, octadecanoic acid, oxalic acid, and malic acid. The results obtained from these two regions indicates that zerumbone is a major compo- nent contained in lempuyang gajah rhizome. Average % area of zerumbone of the Solo and Yogyakarta were 13.44.

  This was done with the assumption that at a given retention time, certain ions are charac- teristic of a particular compound. This is a fast and efficient analysis, especially if the analyst has previous information about a sample or just looking for some specific substances.

  ) GCMS method. Usefulness of the SIM method is to monitor the selected peaks asso- ciated with a particular substance.

  itoring

  GCMS method has been applied to the analysis of lempuyang gajah plants. Based on research Chane-Ming et al. (2003), the larg- est component in lempuyang gajah rhizome is zerumbone, which is the pharmacologically active compounds of the plant. Zerumbon as- say was conducted to determine the levels of compounds responsible for pharmacological activity and can be used as a quality control. Zerumbone assay using SIM (Selected Ion Mon-

  Determination of Levels of Zerumbone Using GCMS

  Each region has differences on nutrient content resulting differences in the results of the metabolic processes of plants, so that the secondary metabolites produced was also dif- ferent. In addition to the differences in location, climate, rainfall, and sunlight intensity can also affect secondary metabolite produced.

  The differences content on the two dif- ferent regions of lempuyang gajah can be in- fluenced by genetic factors and environmental factors, such as regional differences. The re- gional differences may cause the availability of difference nutrients in the soil for theplant.

  Figure 3. Standard curve of zerumbone Zerumbone level of the Solo was 13.00% w/w, and from the Yogyakarta was 13.35% w/w.

  REFERENCES

  metabolomics, in Methods in Pharmacol- ogy and Toxicology: Biomarker Methods in Drug Discovery and Development. Hu- mana Press. Totowa, pp. 317-340.

  Knapp DR. 1979. Derivatisation Handbook of

  Analytical Reactions. John Wiley & Sons,

  New York Ellis DI., Dunn WB., Griffin JL., Allwood JW. and Goodacre R. 2007. Metabolic Fin- gerprinting as a Diagnostic Tool. Phar-

  macogenomic Review, 8(9): 1243-1266.

  Evershed RP. 1993. Advances in Silylation, in: Blau, K., & Halket, JM (Ed.), Handbook

  for Derivatives for Chromatography, 2nd ed

  . John Wiley & Sons, Chichester. Fancy SA., and Rumpel K. 2008. GC-MS-Based

  Fiehn O., Kopka J., Dormann P., Altmann T., Trethewey RN. and Willmitzer L. 2000.

  Dunn WB., Bailey NJC. & Johnson HE. 2005.

  Metabolite profiling for plant function- al genomics. Natural Biotechnology , 18: 1157-1161.

  Fowlin IA. 1995. Gas Chromatography. 2nd ed.

  Chichester: John Wiley & Sons. Gohlke RH and McLafferty FW. 1993. Early gas chromatography/mass spectrom- etry.

  J Am Soc Mass Spectrom., 4:367- 371.

  Hanafi M., Sutedja L., Mulder R. and Braybook

  C. 2001. Isolation and identification of chemical Constituents of Zingiber

  zerumbet the rhizome. Proceedings of the International Seminar on Natural Products Chemistry and Utilization of Natural Resources. VI-Depok: 173-177.

  Measuring the metabolome: current analytical technologies. Analyst , 130: 606-625.

  Max-Planck-Institute of Molecular Plant Physiology, Plant Nutrition Group, Ger- many. p. 166

  Pierce AE. 1968. Silylation of Organic

  Boris A.2012. Toward the Complete Character

  Compounds (a technique for gas-phase analysis)

  , Pierce Chemical Co. Rockford,

  III Agrawal AK.,Rao CV.,Sairam K.,dan Joshi V.K. 2000. Antipyretic dan Analgesic Activi- ties of Zingiber zerumbet Extracts. In-

  dian J Exp Biol., 38: 994-998.

  Bhuiyan NI., Chowdury JU. and Begum J. 2009.

  Chemical Investigation of the Leaf and Rhizome Essential Oil of Zingiber zerumbet (L.) Smith from Bangladesh.

  Bangladesh J Pharmacol , 4: 9-12.

  iztion of Atmospheric Organic Particulate Matter: Derivatization and Comprehen- sive Two-Dimensional Gas Chromatog- raphy/ Time of Flight Mass Spectrom- etry as a Method for the Determination of Carboxylic Acids, theses, Master of Sci-

  Desbrosses G., Steinhauser D., Kopka J. & Udvardi M. 2005. Metabolome Analysis Using GC-MS, Lotus japonicus Handbook.

  ence in Chemistry Portland State Uni- versity Chane-Ming J., Vera R. and Chalchat JC. 2003.

  Chemical composition of the essential oils from rhizomes, leaves and flowers of Zingiber zerumbet Smith from Re- union Island.

  J Essent Oil Res, 15: 202- 05.

  Cronquist A. 1981. An Integrated System of

  Classfication of Flowering Plants. Colum-

  bia University Press: New York. p 447 Dai JR.,Cardellina JHII.,Mc Mahon JB, Boyd MR. 1997. Zerumbone, an HIV-inhibitory and cytotoxic sesquiterpene of Zingiber

  aromaticum and Z. zerumbet. Nat.Prod. Lett ., 10:115-118.

  Dedi Hanwar, Mutia Sari Dewi, Andi Suhendi, Ika Trisharyanti D.K.

ANALYSIS OF SECONDARY METABOLITES PROFILE OF LEMPUYANG GAJAH

  ogy

  Acta Chromatographi- ca

  , 443-452. Settle F. (Editor). 1997. Handbook of Instru

  mental Techniques for Analytical Chem- istry

  . Prentice Hall PTR, New Jersey, USA. Somchit N., Hareet M., Nur Shukriyah MH.

  2003. Anti-inflammatory property of ethanol and water extracts of Zingiber

  zerumbet. Indian J Pharmacol

  , 35: 181- 82. Stanly C., Bhatt S. and Keng CL. 2010. A comparative study of

  Curcuma zedoaria

  and Zingiber zerumbet plantlet produc- tion using different micropropagation systems. African Journal of Biotechnol-

  , 9(28): 4326-4333 Sakinah SSA., Handy ST. and Hawariah LPA. 2007. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/ Bcl-2 ratio.

  Development and Validation of an HPTLC Method for Analysis of Zerum- bone, the Anticancer Marker from Zin-

  Cancer Cell Int., 7: 4.

  Basak ST., Sarma GC. and Rangan L. 2010.

  Ethnomedical Zingiberaceous the uses of plants of Northeast India.

  J. Ethnop- harmacol

  ., 132: 286-296. Villas-Boas SG., Mas S.,Åkesson M.,Smedsgaars

  J. & Nielsen J. 2005. Mass. Spectrofo- tometry in metabolome Analysis.

  Mass. Spectrofotometry Review, 24: 613-646.

  _{(Zingiber zerumbet} Smith) ETHANOL EXTRACT USING GAS CHROMATOGRAPHY MASS SPECTROSCOPY WITH DERIVATIZATION

  Analisis Profil Metabolit Sekunder Ekstrak Etanol Lempuyang Gajah (Zingiber zerumbet Smith) Menggunakan Kromatografi Spektroskopi Massa Terderivatisasi

  giber zerumbet.

  Harrigan GG. and Goodacre R. 2003. Introdu tion. Di in: Harrigan GG, Goodacre R, editors.

  Metabolic Profiling: Its Role inBiomarker Discovery and Gene Func- tion Analysis

  nopharmacology , 27: 129.

  . Dordrecht: Kluwer Aca- demicPb. p. 1-8. Hasnah MS. 1991. Chemical Constituents of some medicinal plants of Zingiberace- ae. Proceedings of the conference on the

  medicinal products from tropical rain forest

  ; Forest Research Institute of Ma- laysia, Kuala Lumpur; 2: 299-304. Jaganath IB. dan Ng LT. 2000. Herbs: The

  Green pharmacy of Malaysia. Vinpress

  Sdn. Bhd. and Malaysia Agricultural Re- search and Development Institute, pp 95-99. Kopka J. 2006. Current Challenges and Devel opments in GC-MS metabolite profiling

  Based Technology. Journal of Biotech-

  nology , 124: 312-322.

  Mascolo N., Jain R., Jain SC. dan Capasso FJ.

  1989. Ethnopharmacologic Investiga- tion of Ginger (Zingiber officinale). Eth-

  Murakami A., Takahashi M., Jiwajinda S., Koshimizu K., Ohigashi H. 1999. Iden- tification of Zingiber zerumbet zerum- bone in Smith as a potent inhibitor of 12-Otetradecanoylphorbol-13-acetate- induced Epstein-Barr virus activation.

  Nat Rev Mol Cell Biol, 8(1) :49- 62.

  Biosci Biotechnol Biochem

  , 63:1811- 1812. Murakami A., Takahashi D., Kinoshita T.,

  Koshimizu K., Kim HW., Yoshihiro A., Nakamura Y., Jiwajinda S., Terao J., Ohi- gashi H 2002. Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompa- nied by apoptosis: the alpha, beta-un- saturated carbonyl group is a prerequi- site.

  Carcinogenesis, 23(5) :795-802.

  Pavia DL., Lampman GM., Kritz GS., Engel RG.

  2006.’s Introduction to Organic Labo-

  ratory Techniques (4th Ed.). Thomson Brooks / Cole. pp. 797-817.

  Perry LM. 1980. Medicinal plants of East and

  South East Asia: Attributed properties and uses

  . MIT Press, Cambridge. Perkins ND. 2007. Integrating cell-signaling pathways with NF-kappaB and IKK function.

  Rout KK., Mishra SK., and Sherma J. 2009. Sun Y., Irie M., Kishikawa N., Wada M., Kuroda N. and NakashimaK. 2004.Biomed.Chro-

  matogr ., 18; 501.

  Dedi Hanwar, Mutia Sari Dewi, Andi Suhendi, Ika Trisharyanti D.K.