Curcumin analogues 2,5-bis-(4’-hidroxy-3’-methoxy)-benzilidinecylopentanone (PGV-0) and 2,5-bis-(4’hidroxy-3’,5’-dimethyl)-benzilidinecylopentanone (PGV-1) have a potency to be developed as cytotoxic agent. The aims of this research are to elucidate the geometric isomer and to study the cytotoxic effect on T47D cells of both compounds. To establish the geometric isomer these compounds, they were elucidated by LC-MS, 1H-NMR, 13C-NMR, HMBC, HMQC, NOESY. Their cytotoxic effect were evaluated by MTT assay method on T47D cells. The results concluded that the geometric isomer of PGV-1 is zusammen-zusammen (Z-Z) and PGV-0 is entgegen-entgegen (EE). The IC50 of both compounds are 1.74 and 9.39 μM respectively.Key words: PGV-0, PGV-1, Cytotoxicit

Da'i, Muhammad

Jenie, Umar Anggara

M., Supardjan A.

Meiyanto, Edy

INDONESIAN JOURNAL OF PHARMACY

English

Toxicity test ofCentella asiaticaLinn. water extract had been carried out. Extract  were  administered  via  oral,  single  and  daily  doses  in  mice  (Mus musculus).  The  effect  of  extract  on  organs  and  tissue  were  evaluated  by observing  the  pathology  anatomy  of  heart,  liver  and  kidney  and  alsoLD50 value. The influences of acute and sub-chronic toxicity on morpho-anatomy organs and histo-pathology tissue were in accordance with LD50experiment. The results of morpho-anatomy showed that all organs were still in normal condition at  low  dose  treatment,  but stain  and  white  spots  appeared  on  liver, black  spot on  heart  and  white  spots  on  kidney  at  higher  dose.  Histo-pathology  indicated that low dose administration of extract resulted that, all of tissue observed were in  good  and  normal  condition,  but  at  higher  dose  resulted  injure  of   muscle fibres  and  also  injure  of  nucleus  cells  of  heart  tissue  also  the  colour  of  liver tissue  was  brick  red  and  white  stain  manifestation  (because  the  damaged  of cells  and  accumulation  of  leucocytes).  Degeneration  and  congestive  sinus occurred  in  liver  tissue,  while  necrosis  and  protein  degeneration  formed  in kidney  because  of  glomerulus  membrane  damaged.  LD50 of Centella extract was  13.6  g/kg.  This  dose  indicated   that  Centella  extract   is   slightly   toxic (5 - 15 g/kg), so that it is suggested that if it was used in phyto-therapy had to be under supervision of an expert or specialist and administered in low dose and short period.Keywords : Centella  asiatica Linn;  extract;  acute  and  subchronic  toxicity;  anatomy patholog

., Praptiwi

Wulansari, Dewi

., Chairul

Indonesian Journal of Pharmacy

Toxicity  effect  of Centella  asiatica Linn.   extract  on  mice  (Mus musculus) organ and tissue

Toxicity test ofCentella asiaticaLinn. water extract had been carried out. Extract  were  administered  via  oral,  single  and  daily  doses  in  mice  (Mus musculus).  The  effect  of  extract  on  organs  and  tissue  were  evaluated  by observing  the  pathology  anatomy  of  heart,  liver  and  kidney  and  also

LD50 value. The influences of acute and sub-chronic toxicity on morpho-anatomy organs and histo-pathology tissue were in accordance with LD50experiment. The results of morpho-anatomy showed that all organs were still in normal condition at  low  dose  treatment,  but stain  and  white  spots  appeared  on  liver, black  spot on  heart  and  white  spots  on  kidney  at  higher  dose.  Histo-pathology  indicated that low dose administration of extract resulted that, all of tissue observed were in  good  and  normal  condition,  but  at  higher  dose  resulted  injure  of   muscle fibres  and  also  injure  of  nucleus  cells  of  heart  tissue  also  the  colour  of  liver tissue  was  brick  red  and  white  stain  manifestation  (because  the  damaged  of cells  and  accumulation  of  leucocytes).  Degeneration  and  congestive  sinus occurred  in  liver  tissue,  while  necrosis  and  protein  degeneration  formed  in kidney  because  of  glomerulus  membrane  damaged.  LD50 of Centella extract was  13.6  g/kg.  This  dose  indicated   that  Centella  extract   is   slightly   toxic (5 - 15 g/kg), so that it is suggested that if it was used in phyto-therapy had to be under supervision of an expert or specialist and administered in low dose and short period

Praptiwi

Dewi Wulansari

Chairul

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Toxicity effect of Centella asiatica Linn. extract on mice (Mus musculus) organ and tissue

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Muhammad Da’i Majalah Farmasi Indonesia, 18(1), 2007 40 Isomers Geometric dan efek  sitotoksik pada sel T47D dari analog kurkumin PGV-0 and PGV-1*  Geometric Isomers and Cytotoxic Effect On T47D Cells of Curcumin Analogues PGV-0 and PGV-1*  Muhammad Da’i 2), Supardjan AM 1),  Edy Meiyanto 1,**)  dan Umar Anggara Jenie 1) 1) Faculty of Pharmacy, Gadjah Mada University  2) Faculty of Pharmacy, Muhammadiyah University of Surakarta  * Part of Thesis for doctoral program at Postgraduate School Gadjah Mada University  Abstrak  Analog kurkumin 2,5-bis-(4’-hidroksi-3’-metoksi)-benzilidinsiklopen-tanon atau PGV-0 dan 2,5-bis-(4’-hidroksi-3’,5’-dimetil)-benzilidinsiklo-pentanon atau PGV-1 memiliki potensi untuk dikembangkan sebagai senyawa yang bersifat sitotoksik. Penelitian ini dilakukan untuk menentukan struktur geometrik kedua senyawa tersebut dan efek sitotoksiknya terhadap sel kanker payudara T47D. Senyawa dielusidasi dengan LC-MS, 1H-NMR dan 13C-NMR, HMBC, HMQC dan NOESY untuk menentukan struktur geometriknya. Senyawa diuji efeksitotoksiknya terhadap sel T47D dengan menggunakan metoda MTT assay. PGV-1 memiliki struktur geometrik engagen-engagen (E-E) dan PGV-0 memiliki struktur geometric Z-Z. Nilai IC50 terhadap sel T47D masing-masing 1,74 dan 9,39 µM. Kata kunci : PGV-0, PGV-1, Sitotoksisitas  Abstract  Curcumin analogues 2,5-bis-(4’-hidroxy-3’-methoxy)-benzilidinecylo-pentanone (PGV-0) and 2,5-bis-(4’-hidroxy-3’,5’-dimethyl)-benzilidinecylo-pentanone (PGV-1) have a potency to be developed as cytotoxic agent. The aims of this research are to elucidate the geometric isomer and to study the cytotoxic effect on T47D cells of both compounds. To establish the geometric isomer these compounds, they were elucidated by LC-MS, 1H-NMR, 13C-NMR, HMBC, HMQC, NOESY. Their cytotoxic effect were evaluated by MTT assay method on T47D cells. The results concluded that the geometric isomer of PGV-1 is zusammen-zusammen (Z-Z) and PGV-0 is entgegen-entgegen (E-E). The IC50 of both compounds are 1.74 and 9.39 µM respectively. Key words: PGV-0, PGV-1, Cytotoxicity  Introduction Curcumin has several biological activities, for example anti-inflammatory, antioxidant, and anticancer. Curcumin is unstable compound at pH above 6.5 and on the presence of light (Tonnesen and Karlsen, 1995; van der Goot, 1997). The instability is attributed to the active methylene group. Omission of this group leads to curcumin analogue stabilization with the same biological properties.   Robinson (2003), divided curcumin pharmacopore in three part (Fig. 1). In a continuing research for potent and stable compound, the geometric isomer of these two curcumin analogues, PGV-0 and PGV-1, have been studied together with their cytotoxic properties on T47D cells. Toxicological studies indicated that both compounds are non toxic even at a high dosage. Biological activities studied for their anti-inflammatory and antioxidant properties showed higher potency than those of the Majalah Farmasi Indonesia, 18(1), 40 – 47, 2007Geometric Isomers and Cytotoxic Effect................ Majalah Farmasi Indonesia, 18(1), 2007 41curcumin (Tim Molnas, 2001). PGV-0 also exhibits cytotoxic effect on Myeloma, HeLa, Raji cell lines better than that of the curcumin (Da’i, 2003). PGV-0 and PGV-1 induce apoptosis on T47D cells that was induced by estrogen hormone (Melannisa, 2005; Nurulita, 2005).  This paper focuses on the elucidation of the geometric isomer of both compounds and their on T47D cells. Structure studies showed that both curcumin analogues have 3 geometric isomers (Fig 2.) These are entgegen-entgegen (E-E), entgegen-zusammen (E-Z) and Z-Z structures. It seems that, this geometrical structure is responsible for the activity of the compound for cytotoxic effect.       Methodology Material PGV-0 and PGV-1 were obtained from the MOLNAS (National Molecule) team UGM, T47D cells were given by Prof. Tatsuo Takeya, NAIST Japan, cells were grown at 37oC in a 5.% CO2 /95% air atmosphere. Cells were cultured in RPMI 1640 medium supplemented with 10.% fetal bovine serum (FBS) contain penicillin-streptomycin antibiotic and fungizon for antifungal, all were purchased from Gibco.    Methods Structure Elucidation The purity of these compounds were determined by HPLC method using methanol and water (70:30). Structure elucidation were carried out by using LC-MS, 1H-NMR and 13C-NMR spectra were recorded  on 400 MHz Jeol Instrument using d6-DMS0 as solvent. The NMR spectra were recorded using HMBC, HMQC, NOESY and COSY patterns.  In vitro cytotoxic effect test   The cells were seeded at 1,5 X 104 cells/ well onto 96 well-plates and than incubated for 24 hours in humidified atmosphere at 37oC and in CO2 5%. At time 0, medium   was  replaced   with  fresh   complete  Figure 1. Curcumin and curcumin analogues (PGV)  pharmacophore division. Part A and C, both are aromatic ring could be heteroaromatic and also symmetric or not symmetric, these part important for reseptor binding. Part B of curcumin, β-diketone group, has –CH2- active mathylene group initiate  curcumin instabilization. (PGV-0: R1 is H, R2 is -OCH3, PGV-1: R1 and R2 are -CH3) (Based on Robinson paper, 2003) Figure 2. The geometric isomer of PGV (A) Entgegen-entgegen (E-E) (B) Entgegen-Zusammen (E-Z) (C) Z-Z structures A C B Muhammad Da’i Majalah Farmasi Indonesia, 18(1), 2007 42 Figure 3.   A. The 400 MHz spectras 1H-NMR of PGV-1 (A) and PGV-0 (B), both of samples are diluted using DMSO-d6. Geometric Isomers and Cytotoxic Effect................ Majalah Farmasi Indonesia, 18(1), 2007 43medium with samples: Curcumin, PGV-0 and PGV-1, each  added at concentrations of 100, 50, 25, 10, 5, 2,5 and 1 µMs. Then, plate incubated for 24 hours at humidified atmosphere 37oC in CO2 5%. At the end of the treatment, medium was replaced and the cells were washed with PBS and added new fresh medium 100 and 15 µL MTT 0,5% in PBS. Plate incubated for 4 hours in humidified atmosphere at 37oC in CO2  5%. The bio-reduction of the MTT dyes was dissolved in SDS 10 % and assessed by measuring the absorbance of each well at λ 550 nm. Viable cells were expressed as a percentage of the absorbance treatment cells divided by absorbance control cells multiply with 100%. Both absorbance are corrected by using medium absorbance only.  % Viable Cell: ((A – B)/ (C – B))X100%   A : Absorbance of treated cells B : Absorbance of medium only C : Absorbance of control cells  The IC50 values are from graphic plot of percentage viable cell versus concentration. The IC50 values express 50 % cell death induced by the concentration and inform about the potency of such compounds.    Result and Discusion The purity of these compounds were determined by HPLC and showed that there is no other signal on PGV-1 (data not showed). However, the PGV-0 still contain vanillin about 0.99% (Martono; 2004, Da’i, 2003). LC-MS chemical ionization method confirm the molecular weight of the compounds with MW 352 and 348 which was belonged to PGV-0 and PGV-1 respectively (data not showed). A series of one and two dimensional NMR spectroscopic experiments using COSY, NOESY, HMBC and HMQC patterns were performed to assign the proton and carbon resonance correlation of the compounds. The experiment resulted that the 1H-NMR and 13C-NMR showed the similarities and differences patterns as indicated each compound (Table 1 and Fig 3). Both compounds were dissolved in DMSO-d6, as expected this solvent give additional peak at δH 2,50 and 3,39 and δC 39,50 ppms (Williams and Fleming, 1995 ).  Assignment of both compound showed similar result for cyclopentanone moeities. The signal pattern of the aromatic ring showed the Table I. Chemical shift (ppm) of PGV-0 and PGV-1  were measured by 1H-NMR and 13C-NMR     OHO OHOCH3H3COH HH H(E)(E)2341'2'4'7'OH H(Z)(Z)OHH3CCH3HOCH3H3C1342'4'6'1'7'8' Atom No δH δC δH δC 1  194.80 194.81 2,5  132.82 132.56 3,4 3.06 25.90 2.99 25.94 1’  127.16 126.72 2’ 7.24 114.50 7.26 131.41 3’  147.69  124.62 4’  148.50  155.14 5’ 6.90 (d/ doublet) 124.76  124.62 6’ 7.14 (d) 115.89 7.26 131.41 7’ 7.37 148.50 7.26 134.54 8’   2.21 16.74 OH 9.70 8.90OCH3 3.84 55.56Muhammad Da’i Majalah Farmasi Indonesia, 18(1), 2007 44 influence of methoxy (-OCH3) and methyl (-CH3) as be shown by the δC of both compounds for C3. The presence of an electron-withdrawing meta (-OCH3) group in meta position of PGV-0 resulted in a downfield shift (147.69) of the adjacent C3. The contrast effect was showed by the presence of an electron-donating methyl (-CH3) group in PGV-1, resulted in an up field shift (124.l62). The presence of both methyl groups in PGV-1 also influence the chemical shift for the proton no 7’, this electron donating group reduces the positive charge of C7’, thus resulted in a up field shift for this proton. The pattern of chemical shift of the aromatic signal and α,β-unsaturated carbonyl of PGV-1 were established by Heteronuclear Multiple Bond Coherence (HMBC) spectra pattern. This spectrum capitalizes on two and or three bond couplings of our compounds (Silverstein and Webste, 1998). Based on the data, at δC 131.41 belongs to C2’(6’) due to coupling H7 to this carbon and  at δC belongs to C2(5) α-carbonyl (Table II).  PGV-0 is not too complex to deduce the pattern of the aromatics protons due to no symmetry of the atom in this compound. Table II. HMBC correlations for PGV-1 Proton Carbon C1 C2(5) C3(4) C1’ C2’(6’) C3’(5’) C4’ C7’ C8’ ppm 194.81 132.56 25.94 126.72 131.41 124.62 155.14 134.54 16.74 H3(4) 2.99 β α(β) Bond     β  H2’(6’) 7.26     Bond     H7’ 7.26 β α α β  Bond H8’ 2.21  β α β  BondH(-OH) 8.90  β α  Figure 4.  Selected COSY spectra of PGV-1 (A) and PGV-0 (B). Dashes line indicate coupling between H71 and H-3(4) of pentanone group. The spectra of PGV-1 also indicated cross peak between H-21(61) and H of dimethyl group (H-81)    Geometric Isomers and Cytotoxic Effect................ Majalah Farmasi Indonesia, 18(1), 2007 45For convenience of interpretation it is extremely helpful to use two dimensional spectrum i.e. COSY, NOESY and HMBC. 1H-1H COSY spectrum indicates all the spin-spin coupled proton. COSY spectra can be obtained to emphasize long-range couplings (Williams and Fleming, 1995). NOESY spectrum which indicate protons that are close in space, provides crucial information about the geometry of molecule (Williams and Fleming, 1995).  Selected COSY spectrum shown H7’ of PGV-0 and PGV-1 couple with H3(4) (Fig 4).  Selected NOESY spectrum shown H-71 of PGV-0 has no cross peak with H3(4) of cyclopentanone. This data sugest that H71 of PGV-0 is not in the same space with H3(4) proton. The NOESY spectra of PGV-1 showed difference phenomena. The H71 proton of PGV-1 showed cross peak with H3(4) of cyclopentanone. Based on this result, the proton H-71 may be is in the same space with H-3(4) proton of cyclopentanone group (Fig 5). This result indicated the possibilities that PGV-1 has Z-Z isomer. Pentagamavunon-0 can be concluded exactly as E-E isomer. Additional cross peak of PGV-1 showed the same space of H-21(61) with H-81 of dimethyl group. Both compounds were evaluated the cytotoxic effect on T47D cells line. The IC50 values of PGV-0 and PGV-1 are 11 µM and 5 µM respectively (Fig 6). As comparison the IC50 values of curcumin on T47D cells is 22 µM Based on this result, PGV-1 was the most potent compound as cytotoxic agent on T47D.  The activity of PGV-0 and PGV-1 should follow the curcumin activity. Curcumin change the proteins level that involve in the cell cycle and apoptosis process. Incubation of breast cancer cells with 20 and 40 microM Figure 5.  Selected NOESY spectra of PGV-1 (A) and PGV-0 (B) dashed line showed cross peak between H71 of PGV-1 and H3(4) but there is no cross peak in PGV-0 Figure 6.  The result of citotoxicity test of PGV-0 and PGV-1 on T47D cell lines showed by IC50 values compared with curcumin  (n=5). Pentagamavunon-1 (PGV-1) has better potency as cytotoxic agent on T47D cells compare with PGV-0 and curcumin. Muhammad Da’i Majalah Farmasi Indonesia, 18(1), 2007 46 curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively  (Ramachandran and You, 1999).  It is suggest that antiproliferative effect of curcumin modulated cell cycle related gene expression. Curcumin down-regulates cyclin D1 expression through activation of both transcriptional and post-transcriptional mechanisms, and this may contribute to the antiproliferative effects of curcumin against various cell types (Mukhopadhyay et al., 2002). Curcumin also was found to induce G0/G1 and/ or G2/M phase cell cycle arrest, up regulates CDKIs, p21, p27 and p53 and slighly down-regulate cyclin B1 in umblical vein endothelial cells (Park et al., 2002). Curcumin induces apoptosis accompanied by an increase in p53 level as well as its DNA-binding activity followed by Bax expression at the protein level. The research also proved that curcumin increase the precentage of cell with G0/G1 DNA content (Choudhuri et al., 2002). It must be elucidated the mechanism of PGV-1 and PGV-0 to inhibit the cancer cell. The PGV-1 structure must be clarified by using x-ray crystallography to confirm the geometric isomer of PGV-1.    Conclusion The result concluded that the geometric isomer of PGV-0 is E-E. Pentagamavunon-1 has possibility for Z-Z isomer. The IC50 values of PGV-0 and PGV-1 are 9.39 and 1.74 µM. Pentagmavunon-1 is more potent than the PGV-0 to inhibit the T47D cell growth.  Acknowledgment This experiment was sponsored by Hibah Bersaing project, DIRJEN DIKTI RI. We also thank to Mrs. Sachiko Iida and Prof. Masashi Kawaichi (Nara Institute of Science and Technology - Japan) for their support to continue this experiment in molecular targets mechanism of PGV-0 and PGV-0.  References Choudhuri, T., Pala, S., Munna L. Aggarwal, B. B.,  Dasa, T., and Saa, G., 2002, Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction, FEBS Letters, 512, 334-340. Da’i, M., 2003, Aktivitas antiproliferatif Pentagamavunon-0 terhadap sel Raji, Sel HeLa dan sel Myeloma, Thesis, Program Pascasarjana UGM, Yogyakarta Martono, S., 2004, Pengembangan Metoda Analisis Pentagamavunon-0 dalam Cairan Biologis Secara Kromatografi Cair Kinerja Tinggi, Laporan Penelitian Proyek Molekul Nasional, Fakultas Farmasi Universitas Gadjah Mada, Jogjakarta Melannisa, R., 2004, Pengaruh PGV-1 Pada Sel Kanker Payudara T47D Yang Diinduksi 17β-estradiol: Kajian Antiproliferasi, Pemacuan Apoptosis, dan Antiangiogenesis, Tesis, Sekolah Pascasarjana, Universitas Gadjah Mada Mukhopadhyay, A., Banerjee, S., Stafford, L. J., Xia, C., Liu, M., and Aggarwal, B. B., 2001, Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDJ4-mediated retinoblastoma protein phosphorylation, Oncogen, 21(57), 8852-8861 Park, M. J., Kim, E. H., Park, I. C., Lee, H. C., Woo, S. H., Lee, J. Y., Hong, Y. J., Rhee, C. H., Choi, S. H., Shim, B. S., Lee, S. H., Hong, S. I., 1998, Curcumin inhibits cell cycle progression of immortalized human umbelical vein endhotelial (ECV304) cells by up regulating cyclin dependent kinase inhibitor, p21WAF1/CIP1, p27Kip1 and p53, International Journal of Oncology, 21: 379-383 Ramachandran, C. and You, W., 1999, Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin, Breast Cancer Res. Treat., 54(3), 269-278. Robinson, T. P., Ehler, T., Hubbard, R.B., IV, Bai, X., Arbiser J.L., Goldsmith D.J., and Bowen J.P., 2003, Design, synthesis amd biological evaluation of angiogenesis inhibitors: Aromatic enone and dienone analogues of curcumin, Bioor. & Med. Chem. Lett., 13, 115-117. Geometric Isomers and Cytotoxic Effect................ Majalah Farmasi Indonesia, 18(1), 2007 47Silverstein, R. M., and Webster, F. X., 1998, Spectrometric Identification of Organic compound, 6th edition, Robert M. Silverstein, Francis X. Webster, John Wiley & Sons Inc., New York Tonnesenn H. H., and Karlsen, J., 1985, Studies on curcumin and curcuminoids, VI: Kinetics of Curcumin Degradation in Aqueous Solution, Original Paper, Z. Lebensm. Unters. Fosch. 402-404. Van der Goot H, 1997, The chemistry and qualitative structure-activity relationships of curcumin, in Recent Development in Curcumin Pharmacochemistry, Procedings of The Internastional Symposium on Curcumin Pharmacochemistry (ISCP), August 29-31, 1995, edited by Suwijyo Pramono, Aditya Media, Yogyakarta Indonesia. Williams, D. H., and Fleming, I., 1995, Spectroscopic methods in organic chemistry 5th edition, MacGraw-Hill Companies, London, p162     **Korespondensi: Dr. Edy Meiyanto, M.Si., Apt.     Fakultas Farmasi UGM, Sekip Utara, Yogyakarta 55281,         Telephone/Fax: +62 (0274) 543120,      E-mail: edy.meiyanto@gmail.com 