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Title:
ISOLATE COMPOUNDS FROM PHALERIA MACROCARPA AS ANTI CANCER
Document Type and Number:
WIPO Patent Application WO/2010/064172
Kind Code:
A2
Abstract:
A pharmaceutical dosage form which is isolated and identified as DLBS1425F1 and DLBS1425E2.2 compound derived from Phaleria macrocarpa (Scheff.) Boerl. plant. Such two compounds, as single active compound or in combination, function as anti proliferation of MDA-MB-231 cancer cells, femalerelated diseases or other gynecologic diseases such as cancer, adenoma, and cyst related to breast, uterus, cervical, and ovary. DLBS1425E2.2 pure compound has a basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 3 hydroxyl groups (-OH) substituted at carbon 2, 6, and 4'; methoxy group (-OCH 3 ) substituted at carbon 4. DLBS1425F1 pure compound has basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 2 hydroxyl groups (-OH) substituted at carbon 4' and 6; a methoxy group (-OCH 3 ) substituted at carbon 4, and β-D-glycopyranoside substituted at carbon 2.

Inventors:
ARIPIN, Asep (Perum Graha Puspa, RT05/RW03 Kelurahan,Karangpawitan, Kecamatan Karawang Bara, Karawang ., 41315, ID)
ARIFIN, Poppy Firzani (Gg. Damai no.51, Kelurahan SrengsengSawah, Kecamatan Jagakars, Jakarta Selatan ., 12640, ID)
TJANDRAWINATA, Raymond R. (Jl. Bangka VIII A/22, RT001/RW012,Kelurahan Pela Mampang, Kecamatan Mampang Prapata, Jakarta Selatan ., 12720, ID)
Application Number:
IB2009/055353
Publication Date:
June 10, 2010
Filing Date:
November 26, 2009
Export Citation:
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Assignee:
PT. DEXA MEDICA (Jl. Letjen Bambang Utoyo No.138, Kelurahan 8 Ilir Kecamatan Ilir Timur I, Palembang ., 30115, ID)
ARIPIN, Asep (Perum Graha Puspa, RT05/RW03 Kelurahan,Karangpawitan, Kecamatan Karawang Bara, Karawang ., 41315, ID)
ARIFIN, Poppy Firzani (Gg. Damai no.51, Kelurahan SrengsengSawah, Kecamatan Jagakars, Jakarta Selatan ., 12640, ID)
TJANDRAWINATA, Raymond R. (Jl. Bangka VIII A/22, RT001/RW012,Kelurahan Pela Mampang, Kecamatan Mampang Prapata, Jakarta Selatan ., 12720, ID)
Domestic Patent References:
2009-12-23
Other References:
OSHIMI S. ET AL: 'Studies on the constituents from the fruits of Phaleria macrocarpa' JOURNAL OF NATURAL MEDICINE vol. 62, 2008, pages 207 - 210, XP008137409
TSUKAMOTO S. ET AL: '7-Hydroxy-3-(4-hydroxybenzyl)chroman and Broussonin B: Neurotrophic Compounds, Isolated from Anemarrhena asphodeloides BUNGE, Function as Proteasome Inhibitors' BIOLOGICAL & PHARMACEUTICAL BULLETIN vol. 28, no. 9, 2005, pages 1798 - 1800, XP007905864
MATSUDA H. ET AL: 'Testosterone 5a-Reductase Inhibitory Active Constituents from Anemarrhenae Rhizoma' BIOLOGICAL & PHARMACEUTICAL BULLETIN vol. 24, no. 5, 2001, pages 586 - 587, XP008158463
FERRARI J. ET AL: 'Benzophenone glycosides from Gnidia involucrate' PHYTOCHEMISTRY vol. 54, 2000, pages 883 - 889, XP027253762
DATABASE CA XP008158471 Database accession no. 1996:345028
NIKAIDO T. ET AL: 'Inhibitors of Cyclic AMP Phosphodiesterase in Medicinal Plants' PLANTA MEDICA vol. 43, 1981, pages 18 - 23, XP009164100
CORREA D. ET AL: 'Duckein, an Alkaloid from Aniba duckei' PHYTOCHEMISTRY vol. 14, 1975, pages 271 - 272, XP008158464
FARIED A. ET AL: 'Anticancer effects of gallic acid isolated from Indonesian herbal medicine, Phaleria macrocarpa (Scheff.) Boerl, on human cancer cell lines' INTERNATIONAL JOURNAL OF ONCOLOGY vol. 30, 2007, pages 605 - 613, XP008137412
MAE S.H.W. ET AL: 'Phalerin, a new benzophenoic glucoside isolated from the methanolic extract of Mahkota Dewa [Phaleria macrocarpa (scheff). Boerl.] leaves' MAJALAH FARMASI INDONESIA vol. 16, no. 1, 2005, pages 51 - 57, XP008158467
TAMBUNAN R.M. ET AL: 'Determination of chemical structure of antioxidant compound benzophenon glycoside from n-butanolextract of the fruits of Mahkota Dewa [Phaleria macrocarpa (Scheff) Boerl.]' MAJALAH FARMASI INDONESIA vol. 17, no. 4, 2006, pages 184 - 189, XP008158469
HENDRA P. ET AL: 'Synthesis of Benzophenone Glucopyranosides from Phaleria macrocarpa and Related Benzophenone Glucopyranosides' BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY vol. 73, no. 10, 2009, pages 2172 - 2182, XP008158465
YOUN U.J. ET AL: 'Identification of Antiadipogenic Constituents of the Rhizomes of Anemarrhena asphodeloides' JOURNAL OF NATURAL PRODUCTS vol. 72, 2009, pages 1895 - 1898, XP008158466
See also references of EP 2370390A2
Attorney, Agent or Firm:
ROSALINA, Belinda (Jl. Permata Hijau Raya B-29 Senayan, Jakarta Selatan, 12210, ID)
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Claims:
Claims

1. A pure compound having a basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 3 hydroxyl groups (-0H) substituted at carbon 2, 6, and 4'; methoxy group (-OCH3) substituted at carbon 4.

2. The pure compound according to claim 1, wherein such compound has a relative molecular mass of 260.88 g/mol.

3. The pure compound according to claim 1, wherein such compound has hydroxyl group, carbonyl group, 0-ether group, conjugated double carbon bond, and hydrogen-carbon aliphatic bond.

4. The pure compound according to claim 1, wherein NMR experiment, hydrogen atoms of such compound show chemical shift at 5.9 (s) ; 7.52 (d, JHz =8.55); 6.68 (d, JHz =8.55) ; 3.68 (s) .

5. The pure compound according to claim 1, wherein NMR experiment, its carbon atoms of such compound show chemical shift at 198.5; 165.6; 161.2; 161; 133; 132.8; 115.5; 108; 94.3; 55.5.

6. 1 A pure compound having a basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 2 hydroyl groups (-0H) substituted at carbon 4' and 6; a methoxy group (-OCH3) substituted at carbon 4, and β-D-glycopyranoside substituted at carbon 2.

7. The pure compound according to claim 6, wherein such compound has relative molecular mass of 422.6 g/mol.

8. The pure compound according to claim 6, wherein such compound has hydroxyl group (binding to C aromatic) , hydroxyl group (binding to C aliphatic) , carbonyl group, O-ether group, conjugated carbon bond, and hydrogen- carbon aliphatic single bond, respectively.

9. The pure compound according to claim 6, wherein NMR experiment, its hydrogen atoms of such compound show chemical shift at (δ) : 6.2; 6.4; 6.8; 7.7; δ 6.2 (IH, d, JHz = 1.85); δ 6.4 (IH, d, JHz = 1.85); δ 6.8 (2H, d, JHz = 8.5) dan δ 1.1 (2H, d, JHz = 8.5); δ 3.8 (IH, s) , δ 4.8 (2H, d, JHz= 7.9) .

10. The pure compound according to claim 6; wherein NMR experiment, its carbon atoms of such compound show chemical shift at δ = 164.3; 164.10; 159.03; 158.51; 133.69; 131.84; 116.04; 111.77; 102.55; 96.80; 94.90; 78.41; 77.91; 4.83; 71.31; 62.65; 56.01; and δ 192.5 (C=O) . 11. The use of Phaleria macrocarpa

(Scheff.) Boerl . extract including E2.2 and/or Fl compound (s) which derived there from to make pharmaceutical dosage forms as anti proliferation of cancer cells, apoptosis inducer, anti cancer, also diseases related to neoplasia and treatment for female related diseases or other gynecologic diseases such as cancer, adenoma, cyst related to breast, uterus, cervical, and ovary.

Description:
Description

ISOLATE COMPOUNDS FROM Phaler±a macrocarpa AS ANTI CANCER

Field of the Invention

The present invention relates to compounds from Phaleria macrocarpa (Scheff.) Boerl. plant including preparation process of compound isolation and biological activities of such compounds. Two compounds have been isolated from Phaleria macrocarpa named DLBS1425E2.2 and DLBS1425F1. Both compounds show anti proliferation activity against MDA-MB-231 cancer cells. DLBS1425E2.2 compound also shows apoptosis inducer activity. Both compounds in accordance with the present invention can be applied in treatment of female related diseases or other gynecological diseases such as cancer, adenoma, cyst, related to breast, uterus, cervical, and ovary.

Background of the Invention

Cancer is one of the most leading death causing diseases in Indonesia, since until now no effective treatment has been found, especially for late stage of cancer. Cancer is caused by neoplasia which is an abnormal proliferation of cells within a tissue or an organ, resulting in a mass known as a neoplasm. Tumor is a neoplasm that has formed a lump; while other type of neoplasm may not form a lump, for example cervical intraepithelial neoplasia, anal intraepithelial neoplasia, and leukemia. Neoplasm may be benign; however it can also be malignant. A benign neoplasm includes, for example, leiomyoma or uterine fibroids and melanocytic nevi or moles. A malignant neoplasm includes, for example, teratoma, also includes various kinds of cancer, including breast cancer .

One of the prevention and treatment method against cancer is by using herbal medicine. One of the plants that possess anti cancer properties is Phaleria macrocarpa (Scheff.) Boerl . , a native plant of Papua that is widely known in

Indonesia as "mahkota dewa".

In Indonesian patent application POO 2005 00077, it has been taught that the flavonoid extract of mahkota dewa was useful as anticancer based on its ability to reduce tyrosine kinase activity, its capacity as antioxidant, and its activity against HeLa cancer cells. Another Indonesian patent application POO 2008 00334 has taught that mahkota dewa extract was useful as an antineoplastic, antiinflammatory, and antiangiogenic agent. But in those patent applications, it has not been explained which compound is useful in such plant extract .

In the present invention, it will be learned about benzophenone compounds as major components which are isolated and identified from the fruits of mahkota dewa also their effects as anti proliferating agent shows by their potential in inhibiting cell growth and stimulating apoptosis which may induce cancer cell death.

Brief Description of the Invention

The objects and/or solutions which are taught from the present invention will be set forth in the preferred embodiments. The embodiments illustrated to serve the purpose of understanding the present invention, without limiting the possibilities of other embodiments which can be learned from the practice of the present invention. The objects and/or solutions which are taught in the present invention will be realized from the elements and combinations detailed in the claims herein.

To attain the object and/or solutions of the present invention, as explained in the embodiments and broadly described in this application, the first aspect of the present invention is directed to a pharmaceutical preparation which comprises: (1) Phaleria macrocarpa (Scheff.) Boerl . compound, for the purpose of the present invention it will be used herein namely DLBS1425E2.2 hereinafter abbreviated as E2.2, and (2) Phaleria macrocarpa (Scheff.) Boerl. compound, for the purpose of the present invention it will be used herein namely DLBS1425F1 hereinafter abbreviated as Fl. Such E2.2 and Fl compounds as single active compound or in combination, in effective amount or dose are useful for cancer treatment or therapy. Pharmaceutical dosage forms according to the present invention may also contain excipients that are pharmaceutically acceptable.

The second aspect of the present invention is directed to a pharmaceutical preparation which contains E2.2 and Fl compounds which function as anti proliferation of cancer cells also female related diseases or other gynecological diseases such as cancer, adenoma, and cyst related to breast, uterus, cervical, and ovary.

The third aspect of the present invention is directed to E2.2 pure compound which has basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 3 hidroxyl groups (-0H) substituted at carbon 2, 6, and 4'; a methoxy group (-OCH 3 ) substituted at carbon 4.

The fourth aspect of the present invention is directed to Fl pure compound that has basic structure of benzophenone, diphenylmethanone, diphenylketone, or benzoylbenzene with 2 hidroxyl groups (-0H) substituted at carbon 4' and 6; a methoxy group (-OCH 3 ) substituted at carbon 4, and a β-D- glycopyranoside substituted at carbon 2.

Brief Description of the Drawings

The following drawings, which are incorporated in and constitute a part of the specification of the present application, illustrate one or several embodiments of the invention. The following drawings serve to explain the principles which are taught by the present invention. Figure 1 shows a two-dimension diagram of thin layer chromatography (TLC) results of E2.2 purity test.

Figure 2 shows an MS measurement result of E2.2 compound.

Figure 3 shows an E2.2 compound structure.

Figure 4 shows a two-dimension diagram of TLC results of Fl purity test.

Figure 5 (a) shows a measurement result of ions from MS FAB Fl data.

Figure 5 (b) shows an illustration of ion fragmentation from MS FAB Fl data. Figure 6 shows an Fl compound structure.

Figure 7 shows a bar diagram indicating the effect of Fl compound against proliferation of MDA-MB-231 cancer cells.

Figure 8 shows a bar diagram indicating the effect of E2.2 against proliferation of MDA-MB-231 cancer cells. Figure 9 shows a picture of electrophoresis results indicating formation of DNA fragmentation in MDA-MB-231 cells which are caused by E2.2 compound.

Figure 10 (a) shows a picture of electrophoresis results of BCl 2 RT-PCR products. Figure 10 (b) shows a bar diagram indicating semiquantitatively BCl 2 RT-PCR products in MDA-MB-231 cells. Figure 11 (a) shows a picture of electrophoresis results of Bax RT-PCR products.

Figure 11 (b) shows a bar diagram indicating semiquantitatively Bax RT-PCR products in MDA-MB-231 cells.

Detailed Description of the Invention

The present invention will be discussed in details by giving examples without limiting the scope of the invention to the examples described.

E2.2 and Fl compounds according to the teaching of the present invention originally come from the fruits of Phaleria macrocarpa (Scheff.) Boerl . The fruits being used are ripe fruits of Phaleria macrocarpa. The plants of Phaleria macrocarpa grow in various locations inside and outside of Indonesia, preferably Phaleria macrocarpa which are planted at 10-1200 meters altitude in Java. It will be described herewith the preparation for extraction procedure of E2.2 and Fl compounds . E2.2 and Fl compounds according to the present invention function as anti proliferation, apoptosis inducer, anti cancer and anti neoplasia which can be used as treatment toward cervical intraepithelial neoplasia, anal intraepithelial neoplasia, leukemia or other diseases caused by neoplasia, including leiomyoma also other female gynecological related diseases such as cancer, adenoma, cyst related to breast, uterus, cervical, and ovary.

A. ISOLATION METHODS

1. First method of E2.2 and Fl compounds preparation (Method D Single compound of E2.2 and Fl derived from ripe fruits of mahkota dewa by some process steps: alcohol extraction, fractionation by organic solvent or liquid-liquid extraction, preparative chromatography or purification, crystallization and identification. a. Alcohol extraction:

Plant materials which are identified as ripe fruits of mahkota dewa named DLBS14, were grinded, then extracted with alcohol solvent at a ratio in the range of 1:2 to 1:20 for certain incubation time with maximum 4 days at room temperature to 7O 0 C. Undesired plant materials were separated as residue. Dry extract of this process was called DLBS1437. b. Liquid-liquid Extraction/LLE :

Certain amount of DLBS1437 were fractionated by LLE technique using mixture of organic solvent and water at a ratio in the range of 1:1 to 2:1 at room temperature, then were incubated maximum one night, thus it produced 2 layers. Upper layer, organic phase, was collected for the following process, while the remaining water fraction was discarded. c. Purification:

To the organic phase, LLE products were fractionated with preparative chromatography column (stationary phase: silica gel; mobile phase: organic solvents) where E2.2 isolate compound was obtained from an E fraction which was purified by crystallization using chloroform solvent. An Fl compound was obtained from an F fraction which was purified by re- crystallization with ethanol-water . d. Identification:

E2.2 compound physically is a yellow-orange crystal with melting point 173.1-174.7 °C. TLC assay at SiC> 2 gel media was eluted with chloroform/acetone solvent system 5/1 (v/v) migrated at Rf 0.32, analyzed with UV lamp at either long or short λ produced positive results, while derivatization using 5% sulphate acid followed by heating produced bright orange color. See Figure 1. Spectroscopy UV data showed 2 peaks, with maximum peak at 294.5 nm.

IR spectroscopy of E2.2 compound read peaks at 3288 (read as hydroxyl) , 1731 (read as conjugated ketone C=O, middle) , 1070 (read as C-O, strong), 1579, 1608 (read as C=C conjugated), 1400-1600 (read as C-C aromatic), 2858, 2913 (read as C-H aliphatic) . IR spectroscopy data of E2.2 compound above showed absorbancies as indication of hydroxyl, carbonyl, 0-ether, conjugated carbon-carbon bonds, and hydrogen-carbon aliphatic groups.

Based on NMR results, it was obtained proton NMR data which showed signals at chemical shift (δ) : 5.9 (s) ; 7.52 (d,

JHz =8.55); 6.68 (d, JHz =8.55); 3.68 (s) . Carbon NMR data showed carbon signals at chemical shift: 198.5; 165.6; 161.2;

161; 133; 132.8; 115.5; 108; 94.3; 55.5. Based on MS (mass spectroscopy) data E2.2 compound has a relative molecular mass 260.88 g/mol with formula Ci 4 Hi 2 O 5 . MS data results are shown in

Figure 2.

From all spectroscopy data showed by E2.2 isolate compound directed to benzophenone group compound (or: diphenylmethanone, diphenylketone, benzoylbenzene) . E2.2 compound structure is illustrated in Figure 3. E2.2 compound is a compound of benzophenone group with 3 hydroxyl groups (- OH) substituted at carbon 2, 6, and 4'; methoxy group (-OCH 3 ) substituted at carbon 4. Therefore, chemical name (IUPAC) of E2.2 compound is 2, 4' , 6-trihydroxy-4-methoxybenzophenone . Fl compound physically is a white crystal solid with melting point 204.5-204.7 °C. TLC assay in SiO 2 gel media eluted with chloroform/methanol/water solvent system 8/2/0.1 (v/v/v) migrated at Rf 0.35, analysis with UV lamp at either long or short λ showed positive results, while derivatization with 5% sulphate acid followed by heating produced bright orange color with stronger intensity compared to E2.2 compound. See Figure 4. UV spectroscopy data showed 2 peaks, with a maximum peak at 292.5 nm.

IR Spectroscopy data of Fl compound read peaks at 3363 (read as hydroxyl in aromatic), 3207 (read as hydroxyl in aliphatic, peak relative wide, because OH>1), 1651 (read as conjugated ketone C=O, middle) , 1087 (read as C-O, strong) , 1608 (read as conjugated C=C), 1400-1600 (read as C-C aromatic), 2970, 2920, 2872, 2821 (read as C-H aliphatic) . IR spectroscopy data of Fl compound above showed absorbancies as indication of hydroxyl group (binding to C aromatic) , hydroxyl group (binding to C aliphatic) , carbonyl, O-ether, conjugated carbon bond, hydrogen-carbon aliphatic single bond, respectively. Different with IR spectroscopy data of E2.2 compound, hydroxyl group in Fl compound was detected more than one. This was showed by absorbance peak at 3207 that is not sharp. Four absorbancies for hydrogen-carbon aliphatic single bond also give information of C-H bond in other group binding to the main structure.

Based on NMR result, proton NMR data showed signals at chemical shift (δ) : 6.2; 6.4; 6.8; 7.7; δ 6.2 (IH, d, JHz = 1.85); δ 6.4 (IH, d, JHz = 1.85); δ 6.8 (2H, d, JHz = 8.5) and δ 1.1 (2H, d, JHz = 8.5); δ 3.8 (IH, s) , δ 4.8 (2H, d, JHz = 7.9) . Carbon NMR data showed signals at chemical shift: δ 164.3; 164.10; 159.03; 158.51; 133.69; 131.84; 116.04; 111.77; 102.55; 96.80; 94.90; 78.41; 77.91; 4.83; 71.31; 62.65; 56.01 and δ 192.5 (C=O) . Based on MS data Fl compound has relative molecular mass 422.6 g/mol with formula C 2 QH 22 O 1 Q . See Figure 5a. This MS data supports the fact in Fl structure determination, with ions fragmentation illustration during FAB MS (Fast Atom Bombardment Mass Spectrometry) analysis process. Fl compound was analyzed using FAB MS machine, in the machine occurred H + atomic bombardment reaction to the Fl structure, thus it produced fragments that showed in peaks data at m/z 422.6; 260.99 and 167.15. Based on fragments value then it was reconstructed into the structure as shown in Figure 5b. Such reconstruction result was in line with MS data analysis result .

From all spectroscopy data given by Fl isolate compound directed to benzophenone group compound (or: diphenylmethanone, diphenylketone, benzoylbenzene) . Fl compound structure is illustrated at Figure 6. Fl compound is a compound of benzophenone group compound with 2 hydroxyl groups (-0H) substituted at carbon 4' and 6; a methoxy group (-OCH 3 ) substituted at carbon 4, and β-D-glycopyranoside substituted at carbon 2. Therefore, a chemical name (IUPAC) of Fl compound is 4' , 6-di-hydroxy-4-methoxybenzophenone-2-0-β-D- glycopyranoside .

2. E2.2 and Fl compounds preparation (Method 2)

As an alternative, to obtain single isolate compound of E2.2 and Fl from ripe fruits of mahkota dewa, some steps are performed including conventional extraction process and supercritical CO 2 (SCFE-CO 2 ) extraction method, fractionation by liquid-liquid extraction using combination of organic solvents that is immiscible with water, preparative/ purification column chromatography, crystallization, and identification. a. Alcohol extraction: Alcohol extraction method in this Method 2 is similar to alcohol extraction in Method 1 from DLBS14 until DLBS1437 is produced. b. Liquid-liquid Extraction (LLE) :

Liquid-liquid extraction method in this Method 2 is similar to liquid-liquid extraction in Method 1 until organic fractions are produced. c. Purification

Dried organic fractions from LLE process then will be extracted selectively using liquid SCFE-CO 2 instrument, 5% co- solvent at condition: 2 hours of operation time, 50-60 0 C of temperature. Simpler selective fractions are to be used in preparative chromatography column step. Based on chromatography orientation, an isolate was obtained from B-I fraction similar to E2.2 isolate and it was later purified by recrystallization using mixture of alcohol and water solvents. An isolate was also obtained from B-2 fraction similar to Fl isolate compound and it was also purified later by recrystallization using mixture of alcohol and water solvents. d. Identification:

Two isolate compounds obtained from SCFE-CO 2 extraction process were compared to E2.2 and Fl compounds based on the preparation according to Method 1. Identification result of SCFE-CO 2 extraction process showed that isolate compound from B-I fraction was identical with E2.2 and isolate compound from B-2 fraction was identical with Fl .

B. Anti proliferation Effect of E2.2 and Fl Compound against

MDA-MB-231 Cancer Cells

In this assay, the effect of E2.2 and Fl compounds as anti proliferation and E2.2 compound as apoptosis inducer against MDA-MB-231 cancer cells will be observed. MDA-MB-231 cells were cultured in supplemented medium cells, and then incubated at temperature 37° C, 5% CO 2 . 1 . Method a. Anti-Proliferation Assay of MDA-MB-231 Cancer Cells

This assay was performed in 96-well plates. Each well contained certain cancer cell density. After incubation for 24 hours, medium was changed into serum free medium, after that it was added with E2.2 compound sample as a single agent in various concentrations, so was with Fl compound. Cells were then re-incubated and after that the number of alive cells after treatment was determined by cell death determination assay. Absorbance value was read in microplate reader. After the absorbance value was converted with standard curve, the number of alive and death cells can be obtained.

Percentage of the number of alive cells in each treatment can be counted by counting the number of alive cells at wells that received treatment divided with the number of alive cells at healthy control and multiplied with 100%.

% The number of alive cells = B/A X 100% Notes: A is the number of alive cells at healthy control, and B is the number of alive cells that received treatment. IC50 value is a number showing a sample concentration that causes death as much as 50% in a population. Such value is obtained using BioStat statistical program.

b. DNA Fragmentation Analysis Method

Apoptosis is an active process of cell death marked with DNA chromosome cleavage, chromatin condensation, and also DNA fragmentation. In this experiment, it was observed the effect of E2.2 compound addition toward the formation of DNA fragmentation as one of apoptosis indicators. Cells were cultured in the plates. After incubation for 24 hours, it was added with E2.2 compound in various concentrations. DNA fragmentation determination was performed using DNA Apoptosis Ladder Kit. DNA fragmentation results were observed in electrophoresis gel, visualized using ethidium bromide.

c. Method of RNA isolation and RT-PCR BCl 2 and Bax genes are genes acting in apoptosis process. To identify BCl 2 and Bax mRNA expressions, RT-PCR (reverse- transcription PCR) was performed. MDA-MB-231 cancer cells were grown in plates and added with E2.2 compound samples as a single agent in various concentrations. Experiment condition was performed in serum free medium. RNA total was isolated from MDA-MB-231 cancer cells. RNA isolation products were quantified using spectrophotometer and visualized by agarose gel electrophoresis technique. The RNAs were then used in RT- PCR process using specific primers. RT-PCR process was performed in PCR machine with optimized condition. RT-PCR products were observed in electrophoresis gel which visualized using ethidium bromide.

2. Results a. Assay Results of the Effect of E2.2 and Fl Compounds Against Proliferation of MDA-MB-231 Cancer Cells

Results showed that E2.2 and Fl compounds indicated anti proliferation effect in MDA-MB-231 cells. This was shown by significant decrease of percentage of alive cancer cell along with increase of the extract concentration. This was shown in Figure 7 for Fl compound and Figure 8 for E2.2 compound. Percentage data of alive cells were used in counting IC50 in MDA-MB-231 cells. IC50 value obtained from E2.2 compound in MTT result was 36.91 μg/mL while from Fl compound in MTT result was 65.80 μg/mL. Data was obtained using BioStat statistical program.

b. DNA Fragmentation Analysis Result DNA fragments from samples obtained from MDA-MB-231 cells and treated with E2.2 compound in concentration 50, 75, and

100 μg/mL showed that E2.2 compound has an apoptosis inducer effect. One of apoptosis markers is the formation of DNA fragments as shown in Figure 9.

c. BCl 2 and Bax RT-PCR Results

BCl 2 gene is one of oncogenes of BCL-2 family member which has an antiapoptotic property. BCL-2 family member is divided into two groups activities, proapoptosis and antiapoptosis . Cell sensitivity in stimulating apoptosis depends on the balance between proapoptotic and antiapoptotic proteins. If the proapoptotic proteins are abundant, then a cell will tend to undergo apoptosis, but if the antiapoptotic proteins are abundant then a cell will tend to resist against death.

From the PCR results using BCl 2 specific primer, there was a downregulation of BCl 2 mRNA expression in MDA-MB-231 cells after treated with E2.2 compound. On the gel, it was observed that bands of BCl 2 gene amplification products were narrower by increasing the extract concentration administered. Quantitatively, the downregulation of BCl 2 in mRNA level is illustrated in Figure 10a and Ib.

Contrary to that BCl 2 , there was an upregulation of Bax in mRNA level in MDA-MB-231 cells. Analysis result showed that the higher E2.2 compound concentration, the denser the bands produced. See Figure 11a and lib.

3. Discussion and Conclusion

Conclusion of the experiment results is that E2.2 and Fl compounds could induce inhibition of MDA-MB-231 breast cancer cell proliferation. IC50 value obtained from E2.2 compound was 36.91 μg/mL while from Fl compound was 65.80 μg/mL.

A material is called active having antiproliferation effect if it has IC50 <100 μg/mL. Both compounds, E2.2 and Fl compounds, are benzophenone group which has cancer cell growth inhibition activity. The above results showed that E2.2 compound was more potent to inhibit cancer cell growth at lower dose compared to Fl compound. Therefore the apoptosis assay was more focused on E2.2 compound. The above data also showed that E2.2 and Fl compounds have potency as anti proliferation of MDA-MB-231 breast cancer cells.

Indication of apoptosis in MDA-MB-231 cells after administration of E2.2 compound is formation of DNA fragments, decrease of Bcl∑ mRNA expression and increase of Bax mRNA expression. Such results support deduction that E2.2 and Fl compounds are able to inhibit cancer cell proliferation and therefore it can be used as anti cancer. In addition, E2.2 and Fl compounds can be used to treat diseases caused by neoplasia (abnormal proliferation) also gynecological related diseases, such as cervical intraepithelial neoplasia, anal intraepithelial neoplasia, leukemia, leiomyoma, adenoma, and cyst related to breast, uterus, cervical, and ovary. Such results are also supported by the result of E2.2 compound which could stimulate apoptosis or cell death in cancer cells.

C . Pharmaceutical Dosage Forms and Nutraceutical

The present invention includes composition and pharmaceutical dosage forms that contain E2.2 and Fl compounds or one of them in an effective amount as active ingredient which is pharmaceutically acceptable and physiologically suitable . In the process of making pharmaceutical composition according to the present invention, such E2.2 compound and/or Fl (are mixed with excipient (s) , dissolved by excipient (s) , or mixed in pharmaceutical carrier which can be made in the form of capsule, sachet, paper, also other materials or packagings. If pharmaceutically approved excipient is used as a solvent, the excipient can be in the form of solid, semi-solid or liquid (oral and injection), that reacts as a carrier or medium for the active substance. Thus, pharmaceutical composition according to this invention can be made in the form of pill, capsule, tablet, powder, sachet, solution, syrup, emulsion, suspension, effervescence tablets, gel, ointment, cream, and mouthwash, massage oil, suppository, or injection. Besides , pharmaceutical composition comprising E2.2 and Fl, or one of them, according to this invention can also be made as supplement, vitamin, also food and beverage production .

Some examples of suitable excipients are microcrystalline cellulose, gelatin, lactose, dextrose, sucrose, sorbitol, mannitol, flour, calcium phosphate, calcium silicate, etc. Formulation according to this invention may also contain lubricant agent (such as talc, stearic magnesium, and mineral oil) , wetting agent, preservative agent, sweetener and flavoring.

Composition according to this invention can also be made with formulation that cause active ingredient to be released directly, sustained, or controlled after the patient receives it using methods that have been applied in pharmaceutical industry. Tablet or pill according to this invention can be coated to extend the half life of the extract thus its frequency of use can be reduced.

Method of formulating this extract in a solid form, such as tablet, E2.2 and Fl compounds or one of them can be mixed with excipient (s) to form an initial formulation containing homogeneous mixture from the composition according to this invention. The initial formulation is a mixture contained the active ingredient of E2.2 and Fl compounds or one of them dispersed homogeneously so it can be properly distributed into the required dose in a dosage form, for example capsule, tablet, or pill.

Tablet or pill according to this invention can be added with a protection layer to reduce or cover bitter taste from the composition or active substance of E2.2 and Fl compounds or one of them.

E2.2 and Fl compounds or one of them in effective amount or dose according to this invention is the concentration or dose which the E2.2 and Fl compounds or one of them able to inhibit the growth of breast cancer cells and/or other gynecologic pathologic cells. The effective concentration depends on the physical condition of the patient, including weight, age, etc. and also depends on the type, size, and amount of cancer cells and other targeted pathologies.

This present invention also anticipates the therapeutic use of E2.2 and Fl compounds or one of them as prevention, concurrent, or after intrusive surgery to carry or take out neoplasm mass. The E2.2 and Fl compounds or one of them can be given directly to or around the location of neoplasm mass carried out from such intrusive surgery.

This present invention also anticipates the therapeutic use of E2.2 and Fl compounds or one of them that is used with or after the radiotherapy. This present invention also anticipates the use of E2.2 and Fl compounds or one of them together with or additional to the composition of anti proliferation, apoptosis inducer, anti cancer, as well diseases caused by neoplasia and female related diseases or other gynecological diseases such as cancer, adenoma, and cyst related to breast, uterus, cervical, and ovary.

D. Application in Industry E2.2 and Fl compounds or one of them and its pharmaceutical dosage forms can be produced in an industrial scale in production of extract, powder extract, and/or pharmaceutical dosage forms mainly for oral dosage form such as solid, semi-solid, or liquid that is used as anti proliferation, apoptosis inducer, anti cancer, female related diseases or other gynecological diseases such as cancer, adenoma, and cyst related to breast, uterus, cervical, and ovary.