ISOLATION AND CHARACTERIZATION OF STEROID COMPOUND FROM LEAF OF LIMAN SITE (Elephantopus scaber L.)

Steroids are a class of secondary metabolic compounds are quite important in the medical field. Until now more than 150 species have been listed as steroid medication (Suryelita, 2000). Several types of steroid compounds used in the world of drugs such as estrogen is a type of steroid sex hormones are used for contraception as inhibiting ovulation, progestin is a synthetic steroid used to prevent miscarriage and pregnancy testing, as an anti-inflammatory glucocorticoid, allergies, hay fever, leukemia, and hypertension as well as cardiac glycosides kardenolida a steroid used as a diuretic and cardiac amplifier (Doerge, 1982). Due to the increasing need for steroid medication, it should be pursued quest of more raw materials to synthesize steroid drugs in the future.

Targeted in this study is the plant footprint liman (Elephantopus scaber L). Liman footprint is not alien plants in human life since time immemorial. These herbs are abundant grass field, dikes, and can be found in large quantities. At this time liman footprint has grown because it has a high economic value in traditional medicine (www.iptek. Net.id). tread liman also have pharmacological effects such as fever, antibiotic, anti-inflammatory, laxative urine, eliminate swelling, and neutralize the toxins (Wijayakusuma, 1992) Adfa (2002) reported that the steroid content of the preliminary test reagents burchard Lieberman declared that leaves footprint liman positive for steroid (+ +), but the isolation of steroids from the leaves tread liman not been reported.

EQUIPMENT AND MATERIALS

The tools that are used

A set of distillation equipment, a set of tools soklet, kromatograpi column, camber, oven, ultraviolet spectrophotometer (agilent 8453, German), infrared spectrophotometer (BioRad, Digilab FTS), Melting point-john fisher, analytical balance (mettle), and glass tools chemicals commonly used in the laboratory.

The materials used

The materials used were n-hexane, chloroform, ethyl acetate methanol, concentrated sulfuric acid, anhydrous asetata acid, filter paper, silica gel (60 mesh) for column chromatography and silica plates for thin-layer kromotografi (GF254 20 X 20 cm).

Sample Preparation

Samples tread liman (Elephantopus scaber L) taken the leaves, the leaves are then finely chopped and left to dry in the air open for 2 weeks.

Introduction Test Compounds Steroids

A total of 4 grams of crushed sample with chloroform, then fitratnya taken and put in a drip plate and the solvent allowed to evaporate, then add 2-3 drops of acetic acid anhydride and stirred until all the residue is dissolved menjadin then added 1-2 drops of concentrated sulfuric acid. The emergence of green to blue indicates the existence of steroids. In comparison to use powder that spilled 0.1 mg kolosterol reagents burchard Lieberman, marked (+ + +) (Manjang, 1988).

Extraction of steroids

Dried samples of 500 grams disokletasi using n-hexane solvent. Sokletasi with n-hexane was stopped when the solvent n-hexane extract obtained clear again and n-hexane. N-hexane extracts obtained were then tested using a reagent Lieberman steroid Buchard. Positive test results followed by a layer chromatography and column chromatography using a suitable eluent.

Thin Layer Chromotografy

Chromotografy thin layer made to extract concentrated n-hexane, and fractions - fractions on column chromatography using silica plates 60 GF254. Eluent used was n-hexane, chloroform, and various comparisons of n-hexane with ethyl acetate.

Concentrated extracts amounted amid lower limit of the plate using a capillary tube and allowed to dry in the air. Eluents were prepared and incorporated into the camber using filter paper saturated. Then the plate is inserted into the camber and camber closed. After the eluent reaches the upper limit plate, the plate is removed and allowed to dry. Sightings stain using an ultraviolet lamp, Lieberman burchhard reagents and I2 vapor. The way it is done repeatedly using different eluents, so in particular eluent obtained a separate stain well and then used as the eluent for column chromatography.

Column Chromogtografy

Column chromatography is used washed first with water until it is completely clean, then dried in the oven, then rinsed column chromatography with n-hexane.

As the absorber (stationary phase) used silica gel, which first made into a slurry with solvent n-hexane. Column chromotografy clamped by clamps on the vertical position of the column ran dank chromotografy closed. The solvent n-hexane added a third column chromotografy. Furthermore, cotton or glass wool that has been soaked incorporated into chromotografy, then compacted until no air bubbles can enter again into chromotografy. The slurry of silica gel that was created entered into chromotografy column carefully so that there are no more air bubbles. At the silica slurry valves inserted column chromotografy be left open, as it solidified silica slurry in the column by passing a solvent chromotografy repeatedly.

Samples to be separated first performed pre absorption, by dissolving it with n-hexane and added to the silica gel, after which the solvent allowed to evaporate. Samples were introduced into the column and eluted chromotografy using mobile phase n-hexane and ethyl acetate as elution (step gardien polarity)

Results chromotografy column housed in a small bottle containing 10 ml and given a serial number. To monitor the results of this column chromogtografy done with a thin layer and appearance chromogtografy stains do with ultraviolet light, Lieberman Burchard reagent and I2 vapor. Of trials that provide a thin layer chromotografy Rf prices (factor essence) the same, then combined into one fraction and the solvent evaporated.

Recrystallization

Recrystallization performed with methanol. At Crystal added methanol and heated, so the crystal and soluble impurities. Then settling at room temperature. Once formed crystals back the crystal and the filtrate separated. Further crystals dissolved in ethyl acetate and allowed to evaporate the solvent. So it will form needle crystals.

Insulation Compound Purity Test Results

1.      Thin Layer Chromatography

Thin-layer chromatography carried out on the isolated crystals were dissolved with ethyl acetate. If the stain with a single eluent gave the compound can be said that the isolated pure. Eluent used was n-hexane, chloroform, ethyl acetate, n-hexane with ethyl acetate (2:8), n-hexane with ethyl acetate (8:2), n-hexane with ethyl acetate (1:1). Sightings stain used is a physicist with ultraviolet light and chemical reagents as well as the Lieberman Burchard I2 vapor.

2.      Melting Point Test

Tests carried out by means of melting point melting Point Fisher John. Few solid crystals inserted into a capillary tube and placed into the device. Then the temperature was raised and observed when crystals start to melt entirely.

Repeated the above manner by slowing the increase 2-5 C per minute around the melting point earlier. Temperatures observed in crystals begin to melt at all. If the melting point range during melting until it melts completely no more than 2? C the compounds are said to be pure (Manjang, 1988).

Characteristics of Insulation Compounds Steroids Results

The existence of the Association of Multiple determined by Ultraviolet Spectrophotometer.

The isolated compound in the form of crystals dissolved in methanol. The samples were in the form of this solution is put into the ultraviolet spectrophotometer cuvette for tools. Maximum absorption measurements performed at a wavelength of 200-400 nm to determine the presence or absence of the double bond berkonyugasi contained in the isolated compound.

The existence Force Characteristics (Functional Groups) Determined by Infrared Spectrophotometer.

Samples in the form of crystals mixed with KBr powder. The mixture was crushed, and then made a thin and transparent pellets. Pellet was placed in the cell samples in an infrared spectrophotometer device. Measurements were taken at wavenumber 4000-600 cm ¹ to determine functional group owned the isolated compounds.

RESULTS

Isolation of Steroids From Leaves Tread Compound Liman

In this study the extraction of steroids sochletase than 500 grams of leaves tread liman using the solvent n-hexane to obtain ekstak n-hexane and dregs. N-hexane extracts obtained and concentrated by evaporating the solvent, so that the concentrated extract obtained in the form of pasta as much as 5 grams. Having obtained the concentrated extract followed by specific steroid test using Lieberman Burchard reagent and gave a positive test is characterized by the formation of a blue color. Against this concentrated extract made chromotografy thin layer separation of steroids to get the condition of other chemical components.

Test chromotografy thin layer with different eluent showed that the separation of the chemical components contained in the extract of n-hexane is relatively good with eluent n-hexane-ethyl acecat (8:2). This is consistent with the recommended system for the separation of steroids (Harborne, 1987). Thin-layer chromatography of the concentrated extract was obtained four separate stain well with Rf: 0.7; 0.55; 0.35 and 0.15. Furthermore, for the separation of the chemical components of each column chromotografy done as much as 4 grams of concentrated extract using eluent n-hexane and ethyl acetate as elution (Step Gradient Polarity). The results of column chromatography gave 14 fractions.

The results obtained chromotografy column was then monitored by thin-layer chromotografy using eluent n-hexane: ethyl acetate (8:2) and the appearance of stains by using ultraviolet light, I2 vapor, and reagents burchard Lieberman. From the results of only a fraction of that column chromotografy 2.4 and 7, which gave a positive test for steroids. Evaporation of the solvent to the fraction 2 (vial No. 3-5) gives the results in the form of oil, the fraction 4 (vial 13) as an orange solid whitish stains and give 3 as monitored by thin-layer chromotografy, so

Further work is focused on the fraction 7. Washing crystals at fraction 7 using n-hexane and then recrystallized from methanol, to obtain a pure compound.

Determining Shape Crystals, Color Crystals and crystal melting point compounds Steroid Isolation results.

Compounds found in the isolated fraction 7 in the form of white needle crystals as 0.48 grams with 0.096% crystal bath. To ensure that the isolated compound is completely pure then test them with the melting point and thin layer chromotografy.

christal the isolated needle gives the melting point from 170.1 to 170.5? C so that the isolated compound can be said to be purely due to the distance numbers obtained from testing the melting point of not more than 2? C subsequent isolation of crystalline needles in the test results of thin-layer chromatography using different eluent and various stains apparition gave single spots with Rf as shown in Table 2, the apparition Lieberman Burchard reagent stains provide a single stain colors blue spots on thin-layer chromatography plate, so it can be said that the isolated compound is a steroid.

Characteristics of Insulation Compounds Steroids In The spectrophotometer UV and IR

From the measurement results UV spectrophotometer steroid compounds provide the isolated absorption band at 204 nm as shown 16, this absorption is a transition from the double bond? to? * is not conjugasion because according woodward maximum absorption for the double bond conjugasion greater than 217 cm. (Cresswell et al., 1982).

Further structural characteristics is done by providing an infrared spectrophotometer absorption peaks at v max 3433 (wide); 2937; 2866; 1651 (weak), 1382; 1061; 1023; 970; 800 cm? ¹. Wide peak at 3433 cm region? ¹ are characteristic for hydrogen bonded OH stretching vibrations from alcohol and not from charbosilat acid as hydrogen bonded carboxylic OH absorbing region 3300-2500 cm ¹. This is reinforced by the presence of peaks in the region 1023 cm ¹ and 1061 cm ¹ are characteristic for the CO stretching vibration. The existence of the peak 1651 cm ¹ supposed to be stretching vibrations of the C = C groups are not conjugate, and supported by the bending vibrations of CH bond ring 970 cm ¹. and stretching vibrations of the CH (sp ³) at 2937 cm ¹ and 2866 cm ¹ and a weak peak of 800 cm? ¹ CH bending vibrations which are outside the field of non-aromatic ring, because it will give the aromatic ring CH stretch peak at 3030 cm ? ¹ and CH bending out the field with sharp bands 900-675 cm ¹. While the absorption band 1459 cm ¹ and 1382 cm ¹ referred as gem-dimethyl system. CH bending vibration absorption band at 970 cm ¹ suggests a double bond is not conjugasion the steroid side chain unit. While the absorption band 1651 cm ¹ an absorption band of the double bond in the steroid framework (Silverstain et al., 1991).

Based on the data from the characteristics of the ultraviolet and infrared spectrophotometers and infrared spectrum compared to the general sterol compounds that give absorption peaks at wave numbers: 3300-3450 cm ¹ (OH) 0.1460 to 1465 cm ¹ and 1350-1387 cm ¹ (gem-dimethyl), 1640-1670 cm ¹ (C = C) is supported by the absorption peak 800-860 cm and 970-980 cm ¹? ¹ (Tarin, 1980; Fish 1968), who showed that the isolated compound is a compound steroids containing hydroxyl groups, methyl groups, and double bonds are not berkonyugasi, it turns out the data is similar to the data steroid sterol group.

CONCLUSIONS AND RECOMMENDATIONS

From the results of research on the isolation of steroids from the leaves footprint liman (Elephantopus scaber L) can be concluded:

Pure steroid compounds have been isolated from the leaves can tread liman (Elephantopus scaber L.).

Steroid compounds the isolated form of white needle crystals as much as 0.48 grams with a melting point of 170.1 -170.5? C.

Based on the characterization of the ultraviolet and infrared spectroscopy
alleged that the isolated steroid compounds including sterols class.

From the research that has been done to complete spectroscopy suggested the isolated compound this further by MS, H-NMR and C-NMR to ensure that the structure is owned by the isolated steroid compounds. Besides, it is necessary to test the bioactivity of the steroid compound isolated yield.