INTRODUCTION:

Cancer has become the second largest cause of death in the world after cardiovascular disease. Cancer results in 8.8 million death case in 2015. From all those kind of cancers, breast cancer positioned at the 6^th with 571, 000 death case^1,2. Breast cancer reported as the highest dead cause in woman, each year there are 1 million breast cancer case has been diagnosed and half of them end to passed away³.

Medicinal plants have become the source of the drug⁴. Plants contain a variety of chemical compounds such as steroids, alkaloids, terpenoids, flavones, phenolsetc that are responsible for many pharmacological properties⁵.

Some medicines applied now are results of derivatization of natural product⁶, and natural products play an important role in drug development programs in the pharmaceutical industry^7,8. In the last several years, research about medicinal plant as an alternatives treatment of breast cancer is being developed due to less side effects and therapy cost^3.Several compounds that successfully isolated from plants and have been applied as cancer chemotherapy treatment such as vincristine and vinblastine which isolated from Catharanthusroseus flower^{9, 10}.

Curcuma aureginosa include in Zingiberaceae family as one of plant applied as medicinal plant in Indonesia. C.aureginosa proved to have some pharmacological activities such as anti-microbe, antipyretic, anti-inflammation, and anti-androgenic^11-13. C. aureginosa applied by the inhabitant of South Konawe district, Southeast Sulawesi province empirically as breast cancer treatment and provide satisfying result.

Recently, there is not any research that explains about the activity of breast cancer to C.aureginosa rhizome. Furthermore, we conducted a research to examine phytochemical composition, anti-breast cancer activity and standardization of extract.

MATERIALS AND METHODS:

Plant Materials:

Rhizome of Curcuma aureginosa taken from South Konawe District, Southeast Sulawesi, Indonesia. Rhizomes were cleaned by running water at proper temperature. Dried rhizome that has been powdered and stored at room temperature until applied. The plants were taxonomically authenticated by Biology Department of Haluoleo University Indonesia.

Preparation of the Extracts:

Rhizome powder of Curcuma aureginosa extracted with maceration method with technical grade methanol at room temperature for 48 h. A rotary evaporator was used to evaporate the extract to dryness at 40°C to produce dried crude extract with a yield of 17.8 g (24.3%). The extract was stored in a desiccators.

GC/MS analysis:

The C.aureginosa extract was analyzed by GC-MS-QP2010 Plus(Shimadzu, Japan).The system was equipped with mass selective detector with an ion source having temperature 250°C and interface temperature 305°C. Capillary column used for MS analysis was Rt × 5MS capillary column with 30 mm × 0.25 mm (length × diameter) and 0.25 µm film thickness. The temperature of the injector was adjusted to 300°C possessing a split injection mode. The initial temperature applied was 70°C (5 minutes), which was further programmed to increase to 300°C at a ramp rate of 5°C/minutes. Helium was used as carrier gas with 25.9 cm/seconds of linear velocity. The total flow programmed was 28.0 ml/minutes, with column flow of 0.50 ml/minutes. Components were identified on the basis of retention time (RT) for GC and interpretation of mass spectrum was done by comparing spectral fragmentation obtained, to the database provided by WILEY8LIB and National Institute Standard and Technology (NIST11LIB)^14-16.

Cytotoxicity assay:

Activity assay of anti-breast cancer conducted by MTT method based on modified Graidist method (2015)¹⁷. MCF-7 breast cancer cell was incubated in 96 wellplate for 24 hours. After that, extract and control were added and incubated for 48 hours. The extract was assayed at concentration of 2000, 1000, 500, 250, 125, 62.5, 32.25, 15.625, and 7.8125 µg/ml, doxorubicin 1 µg/ml is applied as a positive control. Then cell was isolated with 1X PBS and incubated with 100 µl of 0.5 mg mg/ml MTT at 37°C. After 30 minutes, reagent stopper (10% SDS) in HCl 0, 01 N was added as much as 100 µl to each well. Formazan MTT solution was measured its absorbance at 596 nm wavelength using microplate reader. Absorbance value measured was applied for percentage of viability cell calculated by the following formula :

% cell viability = x 100%

Morphological analysis:

Analysis of cell morphology added with C.aureginosa conducted to determine cell changing caused by the extract. A changing such as membrane blebbing, shrinking of the cells, chromatin condensation, ballooning, formation of apoptotic bodies observed to predict the mechanism of cell apoptosis. Meanwhile, vacuolations of the cytoplasm and formation of double membrane vesicle containing organelles were assessed for autophagic cell death¹⁸.

Standardization of Extract:

Standardization of the extract conducted based on Indonesia Pharmacopeia consisting of specific and non-specific parameter¹⁹.

Parameter specific:

Organoleptic determination:

Extract analyzed to understand the form, color, odor and taste of the extract.

Determination the quantity of Water-dissolve compound:

5 g extract was extracted for 24 hours with 100 ml water-chloroform LP, using covered Erlenmeyer while shaking it for the first 6 hours and then left it for 18 hours, strain. Evaporate 20 ml of filtrate till dried in cup, heat the residue at 105°C until the weight is consistent. Calculated its content in compound percentage which dissolved in water to the initial weight of the extract.

The content of ethanol-dissolve compound:

5 g of extract was macerated for 24 hours with 100 ml ethanol 95% using covered erlenmeyer while shaking it for the first 6 hours and then left it for 18 hours. Strain quickly to avoid ethanol evaporation, then evaporate 20 ml of filtrate till dried in cup, heat the residue at 105°C until the weight is consistent. Calculated its content in compound percentage which dissolved in alcohol to the initial weight of the extract.

Unspecific Parameter:

Determination of water content:

Weighted carefully 1 g of extract in covered crus porcelain that has been heated at 105°C for 30 minutes and has been equalized. Even it by shaking them then it forms a layer as thick as 5 mm – 10 mm and dried at certain temperature until the mass is constant, open the cover, let crus at covered condition and the cold in desiccators until room temperature, then calculated its constant weight to calculate dried shrinkage percentage.

Determination of Ash Percentage:

Weighed carefully 2 g of extract in crus, fired slowly. Then increase the temperature gradually until 600 + 25°C, carbon free, then cooled in desiccators, weight the ash. Ash percentage was calculated in percent to initial sample weight.

Acid-insoluble ash percentage:

Ash obtained from ash percentage, boiled with 25 ml of acid chloride for 5 minutes, acid-insoluble parts are collected, filtered through free-ash filtered paper, cleaned with hot water, filtered and weighed, determined acid insoluble ash percentage in percent to the initial sample weight.

Determination of Density:

The density of extract determined to extract dilution(5% dan 10%) in particular solvent (ethanol) using picno-meter.

Determination of Total Bacteria:

Pipetted with sterile pipette 1 ml extract from 10^-4dillution, incubated in NA medium then incubated at 37°C for 24 hours. Then observed and calculated the number of colony which grow and times with dilution factor.

Determination of fungus:

Pipetted with sterile pipet 1 ml of extract from dilution 10^-4incubated in PDA medium, then incubated at 25°C for three days. Then observed and calculated the number of growing colony and multiplied with dilution factor.

Determination of lead(Pb)level:

Determination of mercury limit in extract conducted in wet destruction of extract and nitrite acid and hydrogen peroxide, mercury level determined by atomic absorption spectrophotometry

RESULT:

GC/MS analysis:

The GC/MS analysis of C. aureginosa extract was listed in Table 1. Result reveals that extract contains 19 identified constituents which represent 58.67%. The major component was Germacra-1(10), 4-diene-12-oic acid 6 alpha hydroxy gamma lactone (25.12%) and Cycloisolongifolene, 8, 9-dehydro-9-formyl- (21.61%) followed by curcuzene (1.67%), Curdione (1.66%), 2-Isopropenyl-2, 3-dihydro-7H-furo (3, 2-g) chromen-7-one (1.38%), 4-(acetyloxy)decahydro-8-hydroxy-3, 8a-dimethyl-5-methylene (1.07%), and other compounds approximately (0-1%).

Table 1: Phytochemical constituents identified in the C.aureginosa extract using GC-MS.

Compound Identified	Molecular Weight	Retention time (min)	Peak Area (%)
Champor	152	13.884	0.57
2, 4-Diisopropenyl-1-Methyl-1-Vinyl-Cyclohexane	152	21.331	0.67
Alpha-Humulene	204	22.43	0.23
Curcumene	202	23.808	0.27
Curzerene	216	24.235	1.67
Germacrene A	281	24.339	0.31
alpha-Humulene	204	24.554	0.27
Cycloisolongifolene, 8, 9-dehydro-9-formyl-	230	27.214	21.61
Isospathulenol	220	27.811	0.65
Curdione	236	29.847	1.66
Germacra-1(10), 4-diene-12-oic acid 6 alpha hydroxy gamma lactone	234	30.319	25.12
Velleral	232	30.865	0.95
Pentadecanoic acid	270	34.18	0.5
2-Isopropenyl-2, 3-dihydro-7H-furo (3, 2-g) chromen-7-one	228	35.556	1.38
Linolelaidic acid, methyl ester	294	37.576	0.35
5-Octadecenoic acid, methyl ester	264	37.703	0.91
Germacra-1	246	41.819	0.31
Trioxsalen	228	42.772	0.26
4-(acetyloxy)decahydro-8-hydroxy-3, 8a-dimethyl-5-methylene	248	43.56	1.07

Cytotoxicity assay:

Anticancer activity of C.aureginosa MCF-7 cell line extract with micro-culture tetrazolium assay (MTT) method obtained some concentrations from C.aureginosa extract to determine effective dosage. Assay result can be seen in Table 2 and figure 1. Result reveals increasing concentration of the extract directly correlated with cytotoxic activity to MCF-7 with IC50 value at 76.24 µg/ml. Anova analysis results reveals concentration 250, 500, 1000, and 2000 µg/ml has activity that are not different with Doxorubicin 1 µg/ml (p< 0.05).

Table 2: Cytotoxic activity of C.aureginosa extract on MCF-7 Breast cancer cells

Concentration (µg/ml)	Cell Viability (%)	Cell Mortality (%)
7.8125	91.43	8.57
15.625	87.62	12.38
31.25	78.23	21.77
62.5	69.66	30.34
125	13.59	86.41
250	0.16	99.84
500	0.55	99.45
1000	1.00	99.00
2000	2.69	97.31
Doxorubicin (1 µg/m)	1.91	98.09

Standardization of Extract:

Standardization of extract conducted based on Indonesia Pharmacopeia consist of specific and unspecific parameter.

Specific parameter:

Organoleptic assay:

Organoleptic assay can be seen in table 3. Organoleptic parameter of the extract aims to provide initial introduction to implicial and extract using five senses by describing form, color, smell and taste¹⁹.

Table3: Organoleptic profile of C.aeruginosa extract

Parameter	Profile
form	Thick extract
color	Blackish Brown
odor	distinctive smell
taste	Bitter

Determination of water and ethanol-dissolved extract:

Determination of water and ethanol dissolved extract aims to predict the content of polar active compounds (dissolve in water) and polar – non polar (dissolve in ethanol)¹¹. In extract, the number of compound which dissolve in water and ethanol consecutively 15 % and 13.8 % (Table 4). The number of compound in extract which dissolve in water and alcohol consecutively 49.6 % and 34.8% (table 4). The result obtained reveal that compound in C.aureginosa extract which dissolve in water is more than that in ethanol. It reveals that polar compound content in C.aureginosa extract is more plenty than non-polar compound.

Table 4: Determination the quantity of Water-dissolve and ethanol-dissolve compound

Parameter	Sample	content (%)			Average± SD %
Parameter	Sample	I	II	III	Average± SD %
Water-dissolve compound	C.aureginosa extract	15	15,6	19	49,6 ± 2,828
ethanol-dissolve compound	C.aureginosa extract	13,8	10	11	34,8 ± 0,707

Unspecific parameter:

Water content assay:

Water content is a parameter to define water residue after drying. Water content which obtained in C.aureginosa extract is fully meet the quality requirement that is ≤ 10%. Meanwhile the content of the extract is 8.33% (Table 5). Thick extract contains water content between 5 and 30%. Determination of water content also related with the purity of the extract. Too high number of water content (> 10%) causes the microbial development which decreases the stability of the ecxtract²⁰.

Table5 : Determination of water content

Para meter

Sample

Content (%)

Average± SD %

Quality requirements (%)

III

water content

C.aureginosa extract

8, 33 ±

0, 577

≤ 10%

Ash and Acid-insoluble ash content assay:

Determination of ash content aims to describe of internal and external mineral, extract was heated until the organic compounds and its derivate destructed and evaporated until mineral and organic compound left over. Ash content of the extract as many as 0, 75± 0, 0208 (table 6). Mineral is needed by human such as calcium, phosphor, and magnesium for bone growth. Different from toxic mineral (heavy metal) such as mercury, lead, cadmium and strontium. Accumulation of heavy metal in the body in long period of time may disturb blood circulation, nerve system and kidney performance²¹.

Table 6: Determination of Ash Content and Acid-insoluble ash content of C.aureginosa extract

Parameter	Sampel	Content %			Average± SD %
Parameter	Sampel	I	II	III	Average± SD %
Ash Content	C.aureginosa extract	0,74	0,78	0,75	0,75 ± 0.020817
Acid-insoluble ash content	C.aureginosa extract	0,09	0,10	0,09	0,09 ± 0.005774

Determination of Density:

Density defined as comparison of pack of a substance to water by mass value per volume. Determination of density aims to describe chemical content which dissolve in extract¹⁰. Measurement of density of extract ethanol of C.aureginosa determined using picnometer. Extract applied is extract which has been diluted 5%and 10% with aquades. Density obtained from 5% dilution is 0.762 ± 0.001 meanwhile 10% dilution is 0.775 ± 0.001(Table 7). These results describe the number of mass per volume to provide difference between liquid and thick extract, density also relates to the purity of the extract and contamination¹⁹.

Table 7: Determination extract density of C.aureginosa extract

Parameter	Dilution concentration	Density (gr/ml)			Average± SD (gr/ml)
Density	Dilution concentration	I	II	III	Average± SD (gr/ml)
	5 %	0,761	0,762	0,763	0,762±0,001
	10%	0,775	0,776	0,774	0,775 ±0,001

Determination of bacteria and fungus contamination:

Determination of bacterial contamination is one of requisite assay of the purity of the extract. The assay include determination of the number of microorganism allowed and to reveal that no bacteria in the extract. The extract was identified with 1.07 x 10⁴cfu/ml. Its result is lower than maximum limit that is 10^-6koloni/ml based on Pharmacopeia¹⁹. Similar to the assay of mold contamination in the extract as many as 4, 5 x 10³cfu/ml, that is lower than maximum limit that is 10⁴colony/ml (table 8).

Table 8: Determination bacterial and fungus contamination of C.aureginosa extract

Parameter	Total contamination (cfu/ml)	Total contamination requirements (cfu/ml)
Bacterial contamination	1, 07 x 10⁴cfu/ml	≤ 10⁶
Fungus contamination	4, 5 x 10³cfu/ml	≤ 10⁴

Determination of Lead (Pb) Content:

Determination of lead (Pb) in extract aims to guarantee that extraction doesn’t contain over limited of lead because it is toxic for body. To obtain valid data then analyzed using atomic absorption spectrophotometer (AAS). Results reveal that the contain of lead is 0 ppm (table 9). Maximum limit of lead contamination in beverages stated that maximum of lead contamination in spices is 10 mg/kg¹⁰, after conducting the assay known that extract doesn’t contain lead thus met the requirement.

Table 9 Determination of lead (Pb) level

Sample	Pb content (ppm)
Sample	I	II	III
C.aureginosa extract	0, 0000	0, 0000	0, 0000

DISCUSSION:

Discovery of new medicine from medicinal plant to treat several diseases still developed. In the middle of the development of chemical substance as discovered method and drug production. Plant has secondary metabolism that has plenty pharmacological activity. Some studies have reported several medicinal plant that act as cytotoxic. In the current decade, a study is looking for candidate of new drug substance is still developed²².

C.aureginosa extract was assayed its anti cancer activity using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide). MTT method is a common method applied for discovery of anti cancer treatment. Cytotoxic assay using MTT assay is an accurate and sensitive method to measure cell toxicity. This method can be applied for cytotoxic assay, cell viability, and proliferation in biology cell based on the reduction of Tetrazolium (MTT) through dehydrogenation of succinate mitochondria in cell ²³. The assay of cell viability play important role in toxicity assay. This is conducted to screen drug and new chemical substance and can provide initial data before conduct in vivo and clinical test²⁴.

The result reveals that extract contain cytotoxic activity in MCF-7 cell. The increasing of the dosage of the extract correlate directly to cell cancer death. IC-50 value from C.aureginosa extract to MCF-7 is 76.24 µg/ml at 48 hours of incubation time. The IC₅₀ of extract in cell line less than 100μg/ml is categorized as a potential cytotoxic substances²⁵.The American National Cancer Institute (NCI) guidelines set the limit of activity for crude extracts at 50% inhibition (IC₅₀) of proliferation of less than 30 µg/mL after the exposure time of 72 hour¹⁷. The high number of IC 50 (> 30 µg/mL) of C, aureginosa extract can be seen because of the earlier incubation time that is 48 hours. Microscopically observation result of cell morphology reveal that extract destroy cell in apoptosis pathway. There have been reported that apoptosis induction is one of ways to discontinue cell cancer proliferation. Apoptosis is a focus and main target from cancer research because cell death in this pathway does not cause inflammation²².

Analysis result of C. aureginosa extract compound using GC/MS reveal Germacra-1(10), 4-diene-12-oic acid 6 alpha hydroxy gamma lactone (25.12%) and Cycloisolongifolene, 8, 9-dehydro-9-formyl- as major compound. The study result by Chen et al report that Curcuma zedoaria Roscoe Essential Oil has cytotoxic activity to Non-Small Cell Lung Carcinoma Cells in Vitro and in Vivo, identification result of chemical substance reveals Cycloisolongifolene, 8, 9-dehydro-9-formyl- as major substance and conclude those compounds as cytotoxic agent²⁶.

Other compounds identified and proved to have cytotoxic activity include Alpha-Humulene which is toxic to colon cancer HCT-116, and murine macrophage RAW264.7 cell lines²⁷, Curcumene to ovarian cancer cell²⁸, and Curzerene to lung cell²⁹. Those compounds estimated to have cytotoxic characteristic toward MCF-7 cell cancer.

Standardization is an important step to construct consistent biological activity, consistent chemical profile and simple quality assurance as a preparation in developing extract to become traditional medicine. Research and standardization include biological and chemical parameter of the extract. In Indonesia, standardization of the extract regulated by Indonesia FDA (Food and Drug Administration). Standardization result of C.aureginosa extract has determined the profile of C.aureginosa extract and measurement reveal that extract is fully qualified as stated.

CONCLUSION:

C. aureginosa extract has cytotoxic activity toward MCF-7 cell cancer with IC₅₀ value at76.24 µg/ml for 48 hours of incubation. Result of phythochemical analysist reveals Germacra-1(10), 4-diene-12-oic acid 6 alpha hydroxy gamma lactone (25.12%) and Cycloisolongifolene, 8, 9-dehydro-9-formyl- (21.61%) as mayor compound. Cycloisolongifolene, 8, 9-dehydro-9-formyl-, Alpha-Humulene, Curcumene, and Curzerenediperkirakan which is toxic toward MCF-7 cell cancer.

ACKNOWLEDGEMENTS:

The authors wish to express gratitude to Kemenristek DIKTI as financial support for implementing this study.

CONFLICT OF INTERESTS:

There are no conflicts of interests.

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Received on 29.11.2018 Modified on 19.12.2018

Research J. Pharm. and Tech. 2019; 12(4): 1967-1973.

DOI: 10.5958/0974-360X.2019.00329.9