Standardization of Eleutherine bulbosa Urb. Bulbs and Total Flavonoid Content from Three Locations in Kalimantan, Indonesia

The use of traditional medicines which has not been tested in the efficacy and safety of herbal medicines, cannot be used like modern medicine1. Considered herbal medicines have an important role in the health sector, it should be to determine the quality and safety standards of medicinal plants extracts2. Standardization of medicinal plant extracts is one of the important stages in the development of natural medicines3.


INTRODUCTION
The use of traditional medicines which has not been tested in the efficacy and safety of herbal medicines, cannot be used like modern medicine 1 . Considered herbal medicines have an important role in the health sector, it should be to determine the quality and safety standards of medicinal plants extracts 2 . Standardization of medicinal plant extracts is one of the important stages in the development of natural medicines 3 .
To develop this potential, standardization of extracts were carried out. It consisted of nonspecific and spesicfic parameters 14 . Beside it, bulbs of Eleutherine bulbosa Urb. were examined for the organoleptic, macroscopic and microscopic parameters 15 . Standardization of Eleutherine bulbosa Urb. bulbs had been carried out but from three different locations, that were Malang, Bogor, and Purbalingga (Java Island) 16 and also the standardization of this plant had been done used different solvent, thas was ethanol 96 % which the plant only from east borneo 17 . Therefore this research needed to complete the standardization data for 70% ethanol extract of Eleutherine bulbosa Urb. bulbs and also to determined the total flavonoid content.

Macroscopic and Organoleptic Extract
Observations were carried out with the five senses to describe the shape, color, taste and odor of the extract 14 . The statements "odorless", "practically odorless", "a faint characteristic odor", or variations there of, were determined by observation after the material has been exposed to the air for 15 minutes. Freshly opened package of apportion of about 25 g of the article to an open evaporating dish of about 100 ml capacity 15,18 .

Microscopic Test
This test used aquabidest reagent. Powder microscopy was also carried out and the specific characteristic were recorded 23 . Plant parts that can be observed include starch, transport bundles, endodermis, epidermis and parenchyma tissue 21 .

Water/Ethanol Soluble Content
Determination was done by permeating 1.0 g extract with 25 mL waterchloroform (39: 1) for 24 hours, while shaking it repeatedly during the first 6 hours. Then allowed to stand for 18 hours and filtered. The filtrate is evaporated, the residue was heated at 105°C until the weight remained. Replicated 3 times. For Ethanol soluble content, the solvent used 96% ethanol 2,18,20 .

Chromatography Profile
The method used Thin Layer Chromatography used n-hexane: ethyl acetate (7: 3 v/v) as a mobile phase and silica gel 60 GF 254 as a stationary phase. Bottle extract with a concentration of 0.5% TLC plate GF254 with a size of 8 x 1.5 cm with a distance of 1 cm from the bottom edge and 0.5 cm from the top edge. Spotted on UV light of 254 nm and 366 nm. Sprayed with 10% sulfuric acid (H 2 SO 4 ) solution in methanol 18 .
Determination Non Spesific Parameter of 70% ethanol extract of Eleutherine bulbosa Urb. Bulbs

Specific gravity
The 1 g extract was diluted by 5% with 70% ethanol. Empty pycnometer is weighed then added with water at 25oC weighed by water weight. Liquid extracts at 20 o C are introduced, adjusted at 25 o C and weighed 14 .

Water content
Determination is done by distillation. A total of 5 g of extract was put into a round bottom flask and 200 ml of xylol which had been saturated with water and then heated at a temperature of 110 o C for 1 hour. After the layers separate completely, the volume of water is read and calculated 14,19. Water content is calculated in % v/w 20 .

Total ash content
Accurately 2 g of the extract was put into the silicate crucible then heated with a hot plate followed by a furnace at 650 o C until the charcoal was used up. After that, the silicate crucible weighed after cooled to room temperature in a desiccator then calculated the results, expressed %w/w 14,15 .

Acid insoluble ash content
The ash obtained as directed under Total Ash Content was boiled with 25 ml of dilute sulfuric acid P for 5 minutes, the acid insoluble part was collected, the filtered ash was filtered with ash-free filter paper, washed with hot water, put into a silicate crucible, glowed with a furnace at a temperature of 650 o C to charcoal was gone. Acid insoluble ash content was calculated to the material weight in %w/w 14,15 .

Residual solvent
Concentrated extract was diluted to a concentration of 0.1% with methanol as a solvent. Samples were injected into the GC-MS at temperatures of 70 o C to 200 o C. Analysis of the presence of ethanol gropus through the similar index and the re resulting cromatogram pattern 14,21 .

Heavy metal contamination
The instrument used to perform this test was Atomic Absorption Spectrophotometry (AAS) with the calibration curve method. Create a standard curve for lead (Pb) and Cadmium (Cd) with a concentration of 1000 ppm. Dilution was carried out gradually until a contentration of 1 ppm was obtained. Series levels of 1, 5, 10 and 15 ppm for lead (Pb) and 0,2; 0,4; 0,6 and 1 ppm for Cadmium (Cd) were made. Concentration of the sample solution was measured after absorption 21 . Weighed 2.5 g of extract and added 20 ml of concentrated HNO 3 and allowed to stand for 24 hours, heated to 100 o C for 10 minutes then cooled then added 2 ml of 30% H 2 O 2 , heated until a clear yellow solution and filtered to a 50 volumetric flask and added aquadest until border mark. Samples were measured by means of AAS then heavy metal content was calculated 2 , 22 .

Microbial contamination
Pipette 1 ml from each dilution into a sterile (duplo) petri dish. Plate Count Agar (PCA) media was poured as much as 5 ml into each petri dish which had been melted at 45 o C. Leave it until the mixture is frozen and put in an incubator cabinet at 37 o C for 48 hours in an upside down position. Colony growth was recorded after 24 hours 2, 21 . Observed and counted the number of colonies that growth on petri dish.

Mold and yeast contamination
In a sterile (duplo) petri dish, 5 ml of diluted Potato Dextrose Agar (PDA) media was poured at 45 o C, then 1 ml was pipetted from each dilution. Leave to freeze in a saucer and incubated at room temperature or 25 o C for 7 days. Results recorded 2,21 .

Total Flavonoid Content
Total flavonoid content was determined by aluminium chloride spectrophotometric method. Determination of Operating Time 0.5 mL of a quercetin solution with concentration 60 µg/mL added to the vial. Then added 0.1 mL AlCl 3 , 0.1 mL of sodium acetate 1 M and 2.8 mL aquadest, shaken and read the absorbance continuously at intervals 3 minutes for 60 minutes 25 .

Quercetin Standard Curve
Quercetin was used to make a standard calibration curve. 100 mg quercetin was dissolved in 100 mL of ethanol (1000 µg/mL) and then diluted to get the concentration 20, 30, 40, 50, 60 µg / mL. 0.5 mL of each solution diluted standard solutions were pipette out and added with 0.1 mL AlCl 3 , 0.1 mL sodium acetate 1 M and 2.8 mL aquadest then shake it to stand for operating time and read the absorbance at the maximum wavelength 25 .
Determination of Total Flavonoid Content 0.5 mL extract solution with concentration 1000 µg/mL was added to the vial, added with 0.1 mL AlCl 3 , 0.1 mL sodium acetate 1 M and 2.8 mL aquadest, then shaken and allowed to stand during operating time and read the absorbance at the maximum wavelength obtained 24 .

RESULTS AND DISCUSSION
In this study, bulbs of Eleutherine bulbosa Urb. extracted with maseration method used 70% ethanol. The yield extraction of sample from three locations presented at Table 1. Standardization of medicinal plants is an important step in conducting research and development of natural medicines to ensure the quality and safety of drug preparations 15 . Spesific parameter of 70% ethanol extract of bulbs of Eleutherine bulbosa Urb. tested consist of extract identity, organoleptic extract, microscopic test, water/ethanol soluble content and chromatography profile.
Previous research results, the yield extract from Melak, West Kutai district, East Kalimantan used 96% ethanol as solvent produced yield 1,49% w/w 16 . Based on these research, the yield used 70% ethanol was greater than 96% ethanol. This result because the polarity level of 70% ethanol higher than 96% ethanol so that was able to atrracted more compounds.
Specific parameter desribe the identity an extract. The identification process is an important part of quality control of traditional medicine product because ingredients usually come from different cultivated areas, and have many physical similarities with other plants that are still of the same genus. The first parameter determined was extract identity. With the extract identity, it can be a specific clue to differentiate between plant extracts from one another. Then the organoleptic determination of the extract was the second step to check the quality of the extract by obsercing color, staste and odor. Water soluble content or ethanol soluble content were the next test. Each plant contains different compound, which of these chemical sbstances can be dissolved or attracted based on their respective polarity. In the Table 2, showed extract from three location were more soluble in ethanol compared water so it can be concluded the attracted compound were semipolar. The results of spesific parameter of extract identity, organoleptic and water/ethanol soluble content presented of Table 2.
Macroscopic and microscopic characters are one of the important criteria for identification 25 . Bulbs of Eleutherine bulbosa Urb between three location Kalimantan have the save from. The sample have whole bulbs in groups, each group consists of several bulb, part of bulb base is hard, the bulb surface is smooth, pointed ends and have oval form. At microscopic characters between three location have similarity, their have parenchyma with oil drops and isolated schlerencyma. The results of spesific parameter of macroscopic and microscopic presented of Figures 1 and 2.
The next parameter in extract standardization is chromatography profile.The determination of the chromatogram pattern was carried out by the TLC method which aimed to ceparated the compounds in the extract based on spot pattern and color after being observed on UV light and H 2 SO 4 as spray reagents.The TLC profile is a qualitative analysis to show the presence of chemical compounds present in the sample 19 . The results showed there are four spot in TLC plate. The results of spesific parameter of TLC profile presented of Figure 3.
Non spesific parameter of 70% ethanol extract of Eleutherine bulbosa Urb Bulbs tested consist of specific gravity, water content, total ash content, acid insoluble ash content, residual solvent, heavy metal     contamination (Pb dan Cd), microbial contamintation and mold yeast contamination. The result showed at Table 3.
Specific gravity relates to purity and contamination. These results spesific gravity from three location almost the same with the result of previous resarch from fridayanti et. al., that was 0,9347 ± 0,00 16 . In this research, determination of water content used destilation method. The results appropriate requirment but its value almost value standard. One of the reason it can be happen because the solvent used was 70% ethanol which contains a high water content.
Next determination were total ash cotent and acid insoluble ash content. This determination aims to provide an overview of the internal and external mineral content originated from the initial process until the extract formed 13 . At this stage the extract was heated until the organic compounds and their derivatives are destructed and evaporated until only the mineral and inorganic elements remain. Another nonspecific parameter was determined the residual solvent. If the residual solvent still high in the extract, it is possible to enter the body and give the side effect 2 . This method used GC-MS for analyze. Based on chromatogram pattern, the sample from three location proven negative.
Heavy metal contamination determination aims to ensure that the extract does not contain certain heavy metal exceeding the specified values which are harmful to health. Two heavy metals tested were lead (Pb) and cadmium (Cd). Based on the result, the extracts accordance with the requirment. And the last non specific parameter were microbial contamination, mold and yeast contamination. This parameter aims to provide assurance that the extract does not contain microbes, mold and yeast exceed the requirment because it affects the stability of extract and harmful to healthy 13 . In this determination, the extract also accondace with the requirment.
Based on metabolit secondary and activity from Eleutherine bulbosa Urb, total flavonoid content was determined. In this method used quercetin as standard. The results for maximum wavelength was 435 nm, with operating time 30 minutes 26 . The maximum wavelength accordance with literature that stated the wavelength maximum for quercetin with this method was 415-440 nm 26 .
Quercetin standard curve have regression y = 0,0132x + 0,0152, R 2 = 0,9998. Quercetin standard curve showed at Figure 4. Total flavonoid content used aluminium chloride as reagent. AlCl 3 will reacted with C-4 at ketone group and C-3 or C-5 at hydroxyl group from flavonoid structure 28 . The reaction between AlCl 3 and quercetion showed at Figure  5. Furthermore determination of total flavonoid content for Eleutherine bulbosa Urb from three location. The result for total flavonoid content presented at Table 4 showed the highest total flavonoid content from palangkaraya city as 7,585 ± 0,0437 mg QE/g extract.