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Cytotoxicity Comparation of Ethyl Acetate Extract and Their Isolate From SKY III.2.

1, An Endophytic Fungi from Srikaya Plants (Annona squamosa) .


Prasetyawan Yunianto1,3*), Fery Azis Wijaya1), Nurhadi 1), Syofi Rosmalawati2), Wahyudi Priyono Suwarso3), Wahono Sumaryono1) 1) Pusat Teknologi Farmasi dan Medika BPPT, Jakarta, Indonesia 2) Balai Pengkajian Bioteknologi-BPPT, Serpong, Indonesia
3) Depatemen Kimia FMIPA UI, Depok, Indonesia

ABSTRACT

SKY III.2.1 was an endophytic fungi from twig of srikaya (Anonna squamosa) that was isolated and selected by microscopic and macroscopic analysis. SKY III.2.1 fungi was fermented in liquid media for three weeks. The liquid and mycelium were extracted using ethyl acetate for 24 hours and sonicated for 15 minutes three times. Whole extraction of SKY III..2.1 was partitioned and evaporated to obtain each ethyl acetate extract. Ethyl acetate extract was isolated by chromatography column with silica as stationery phase and mixture of 98 % ethyl acetate and 2 % metanol as mobile phase. From this isolation method, 14 isolates were obtained. Cytotoxicity assay of ethyl acetate extract and 9 from 14 isolate was carried out at level 100 ppm by MTT viability test towards MCF-7 a breast cancer cell. The result indicated that activity of 9 fractions from the isolation were lower than that of the initial extract (ethyl acetate extract of SKY III.2.1). It was concluded that the ethyl acetate extract of SKY III.2.1 had synergistic anti cancer activity.
Key word : endophytic fungi, srikaya (Annona squamosa), MTT Viability test.

Introduction Until now, a quarter of modern medicines circulating in the world comes from the active ingredients are isolated and developed from plants. According to Cragg et al. (1999) more than 50% of new chemical compounds are registered with the FDA as a medicinal raw material compounds are metabolites of plants and their derivatives. Phytochemical compounds are very heterogeneous mixtures of single substances acting in a synergistic or antagonistic manner. Mixtures of active constituents show a broad spectrum of biological and pharmacological activity (Robinson, 1967; Coelho-de-Souza et al., 1998; Atindehou et al., 2002). Endophytic microbes are bacteria or fungi that live in plant tissue in a certain period and can live with to form colonies in plant tissue without harming its host. Results of secondary metabolites of endophytic microbes have been applied in the fields of health, agriculture and industry (Strobel, 2002). Millon dollars have been generated after the first discovery of Taxol (paclitaxel) as an anti-cancer drug that endophytic fungi isolated from the plant Taxus brevifolia (Wani, et.al, 1971). Annonase asetogenin , compounds were derived from Annona sp that was a natural material that has potential as antitumor (Mclaughin, 1998, 1993, 2001), pesticides (Mikolajczak, 1988) and other bioactive as a barrier to electron transport in mitochondria (Azucena Gonzalez C, 2002). Chih, C, L et al (2008) found a new compound derived mono-tetrahydrofuran (THF) annonase type asetoginin extracted from sugar apple seeds have a cytotoxic potential against cancer cells. Endophytic from Annonaceae expected to get the same metabolites was extracted from the host, so it is more economical than extraction from the seeds of its host fruit.
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Correspondence : Prasetyawan Yunianto, Laboratorium of Pharmacy and Medika BPPT, LAPTIAB Gedung 610 PUSPITEK-TANGERANG SELATAN, Tlp 021-7560536, Fax 021-7560536 ext. 122. Email : pyunianto@gmail.com

In this study, we fermented the metabolite from endophytic fungi of annnona squamosa SKY III.2.1 by liquid fermentation and extrated to form ethyl acetat extract and then isolation the extract to some fraction. Ethyl acetat exctract and their fraction was tested as anti-cancer potential based on the viability of breast cancer cells (MCF-7) using in vitro with MTT method Material and Methods Material : Endophytic fungus derived from twigs of Annona squamosa.in Tangerang. Indonesia. Fungal isolation media: Agar (DIFCO), Dried Plant Materials (leafs), Chloramphenicol (Kimia Farma). Fermentation Media: Yeast extract (DIFCO), Malt extract (DIFCO), Peptone (DIFCO), Glucose monohydrate (E-Merck). Solvent for ectraxtion: ethyl acetate, methanol destiled were the resource persons from technical grade Methanol for HPLC (E-Merck), Silica gel 60 (he is from 0.040 to 0.063) (E-Merck), Sphadex LH 20 (Sigma), TLC Plate Silica Gel 60 F254 (E-merck). Cytotoxicity assay using MTT methods. Methods

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Correspondence : Prasetyawan Yunianto, Laboratorium of Pharmacy and Medika BPPT, LAPTIAB Gedung 610 PUSPITEK-TANGERANG SELATAN, Tlp 021-7560536, Fax 021-7560536 ext. 122. Email : pyunianto@gmail.com

Fungal SKY III.2.1 cultures in 500 mL erlenmeyer flask, Winchkerham media


addition of 200 mL EtOac., stand Cell over night ethylacetate disruption/.sonicator FiltratioUltraturrax using n Separatio n

Cell debris

Supernatan t

Medium / Extracti Water phase on with

100 mL phase watern-BuOH saturate phase d nEvaporatio BuOH n

Water

Evaporation Extraction using 100 mL 90 % MeOH and 100 mL n-Hexane

Ethylacet ate phase Washing with 100 mL water

n-BuOH extract

n-Hexane phase
Evaporation

n-Hexane extract

MeOH extract/ EtOAc extract

Evaporatio n

Methanol phase

Silica column (mobile phase = 98 % EtOAc : 2% CH3OH) F.1 22 F.2 F.1 0 F. 9 F. 7 F. 5 F. 4 F. 3 F. 6

Viability test to breast cancer cells (T47-D) using in vitro with MTT methods methmethodsmethod

Result and Discussion

Viability cell (%) Ethyl acetat extract of SKY III.2.1 and their isolated From Figure 1 shows that the activity of all fractions of the separation is smaller than the initial fraction

extract, so it can be concluded that the ethyl acetate extract of synergistic III.2.1. This is confirmed
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Correspondence : Prasetyawan Yunianto, Laboratorium of Pharmacy and Medika BPPT, LAPTIAB Gedung 610 PUSPITEK-TANGERANG SELATAN, Tlp 021-7560536, Fax 021-7560536 ext. 122. Email : pyunianto@gmail.com

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mAU
mAU 10 00 10 20 10 40
10 0 15 2 10 5 15 7 20 0

mAU

20 0
-0 5 -5 2

40 0
10 0 15 2 10 5 15 7 -0 5 -5 2
2 5 5 0 7 5

60 0
2 5 5 0 7 5

80 0

0
0
0

0
0

0.4 483

1.4 908171 1.81397753 1.4482120 1.7 456381 1.91019073 2.2 905149 R te tio T e e n n im Ae ra
3.1 3242697
K2 0 [ ] - 8 01

1.4 840736 1.9 2086121

Rte tio T e e n n im Ae ra

R te tio T e e n n im Ae ra 3685650 3.33.0 5689384 4.7 2004690


5

K2 0 [ ] - 8 01

K 80 ] -2 0 [1

3.416226796
5

4.0 1350214 4.2 876507 4.6 1002263 4.9 633744 5.2 956575 5.9 3625581

6.05893686

6.09796655

8.31649281 8.6 4409149


9.3 3030471
1 0

7.9 6403615

1 0
1 0

9.1 2369144 9.51538805 10.0 881278 10.7 1951583 11.3 1030841 9.41435167 10.0 1832222 10.6 944642 11.3 1676383 12.2 43626680 13.4 5386253 13.9 6238951
1 5

10.1 1693713 11.2 2048367 11.6 500597

12.2 3760338
13.3 4392020

1 5
1 5

14.2 5107705 14.9 1621464 15.3 4304982 15.1 10499087 16.5 4711384 16.8 8215329 17.9 4795955 19.6 4048506 20.9 1617766 21.7 2983072 23.2 1424715
2 5

15.0 12279957 16.1 9660049 17.5 4706514

16.4 3978547

17.9 2797148

19.1 1726225 19.6 1353011


2 0

19.3 6182960
2 0

2 0

20.9 1653828

21.8 4582099

22.6 1882898

23.2 986774

2 5

2 5

24.6 2419271 25.2 887384

23.5 299146 23.9 582600 24.2 298529 24.8 1028079 25.2 706089

26.0 1867207

24.6 1627064 24.9 365601 25.2 1064815 25.9 626001 26.3 1195385 27.3 972115 28.3 421345 28.8 565297 29.3 1058667

Mu s i te n

Mu s in te

Chromatogram HPLC of Fraction 4

Chromatogram HPLC of Fraction 6

Mu s in te
3 0

27.0 1635013

29.0 644483 29.7 1067167 31.3 2183743 32.2 382961 32.9 610237

26.1 834242 26.4 553023 26.9 600818 27.5 570856 28.2 402057 28.7 568252 29.3 513054 29.8 785947
3 0

3 0
3 5

30.8 473448

31.6 1810240

30.6 212646 31.1 1639100 31.8 185985 32.4 732941 32.9 1064248

3 5

3 5

32.9 524839 33.5 213362 33.9 252557 34.5 442155 35.0 300347 35.4 648331

34.4 837964 34.9 344192 35.3 568960 36.3 224276

33.7 320856 34.2 767922 34.8 327880 35.2 510659 35.5 1252282

Chromatogram HPLC of Fraction 7


4 0

36.3 159341 36.9 261921 37.6 178968

38.6 1179683
4 0

4 0
4 5 5 0 5 5

39.5 1057974 40.0 512525 40.4 433565 41.0 555896

41.9 333674 42.5 361496 43.0 244568

39.5 754052 39.9 244457 40.5 492621 41.0 197618 41.3 115724 41.5 141616 41.8 355515 42.5 472875 42.9 215957 43.7 328594

39.5 1096448 40.1 1053608 40.4 969807 41.0 829284 41.5 381585 41.8 794250 42.4 1165016 42.9 745894 43.4 1628548
4 5

43.7 575053

4 5

45.0 20812

46.8 34828

46.1 252147 46.6 359612 48.3 412990 48.6 337616 49.0 165244 49.2 778252
5 0

5 0

49.6 741073 50.0 309632

51.2 676495 51.8 336617

49.3 1114571 49.4 150920 49.6 174132 49.9 227490 50.1 234531 50.5 206121 50.6 224104 51.1 560016 51.8 638196 53.0 189348 53.7 132408 54.2 96725

49.9 472960 50.5 244016 50.9 667689

52.7 54636 54.3 165199


5 5

53.4 23155

Correspondence : Prasetyawan Yunianto, Laboratorium of Pharmacy and Medika BPPT, LAPTIAB Gedung 610 PUSPITEK-TANGERANG SELATAN, Tlp 021-7560536, Fax 021-7560536 ext. 122. Email : pyunianto@gmail.com

also by differences in activity between fractions 4 and 6 greater than the fraction 7 and 9, where the HPLC chromatogram of fraction F7 and F9 relatively more single than F4 and F6.

54.2 12727 54.8 3972

5 5

Conclusion Activity of 9 fractions from the isolation were lower than that of the initial extract (ethyl acetate extract of SKY III.2.1), so that the ethyl acetate extract of SKY III.2.1 had synergistic anti cancer activity. References Atindehou, K.K., M. Kon, C. Tenneaux, D. Traore, K. Hostettman and M. Dosso, 2002. Evaluation of the antimicrobial potential of medicinal plants from the Ivory Coast. Phytother. Res., 16: 497-502 Coelho-de-Souza, A.N., D.N. Criddle and J.H. Leal-Cardoso, 1998. Selective modulatory effects of the essential oil of Croton zehntneri on isolated smooth muscle preparations of the guinea pig. Phytother. Res., 12: 184-194. Cragg, GM., MR Boyd, R Khanna, R Kneller, TD Mays, KD Mazan, DJ Newman, EA Sausville. 1999. International collaboration in drug discovery and development: the NCI experience. Pure Appl. Chem. 71 (9): 1619-1633 Robinson, T., 1967. The Organic Constituents of Higher Plants, Their Chemistry and Interrelationships. 1st Edn., Burgress Publishers Company, USA Strobel,GA. (2002). Microbial gifts from rain forests. Can.J.Plant Pathol. 24: 14-20. Strobel GA., and B. Daisy (2003),Bioprospecting for Microbial Endophytes and Their Natural Products. Microbiol. and Mol. BiologyRev. 67(4):491-502 Wani,M.C,Taylor,H.L,Aal, M.E,Goggon,P,McPhail,A.T, 1971,Plant Antitumor-agent IV,The Isolation and structure of taxol, a novel antileukemic and antitumor,J.Am.Chem.Soc,93,2325 2327.

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Correspondence : Prasetyawan Yunianto, Laboratorium of Pharmacy and Medika BPPT, LAPTIAB Gedung 610 PUSPITEK-TANGERANG SELATAN, Tlp 021-7560536, Fax 021-7560536 ext. 122. Email : pyunianto@gmail.com

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