Isolation and Identification of Polysaccharide Compound by Qualitative
Test in Aqueous Extract of Safflower Petals (Carthamus tinctorius L.)
from Bone Region. Rahmawati Syukur 1 , Gemini Alam 2 , Arfiana 3
1 Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia 2 Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia 3 Laboratory of Phytochemistry, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia
Abstract Isolation and identification of polysaccharide compound by qualitative test from aqueous extract of safflower petals (Carthamus tinctorius L.) has been conducted. This research aims to get polysaccharide compound from aqueous extract of safflower petals, which acquired from Bone Regency. Safflower petals were extracted by infusion method with aqua destillata. The resultant extracts were centrifuged, and the supernatants was added with 70% ethanol and placed in the refrigerator overnight. The precipitate were collected, dissolve in water and adjusted to pH 3 by HCl. Insoluble materials were removed by centrifugation, and the supernatants were again adjusted to pH 7 by NaOH. After that, the precipitates were collected by centrifugation with addition 70% ethanol, then dried. The dried precipitate were hydrolyzed with HCl at 100oC, and identified with TLC multiple development, and identifications by test for polysaccharides compounds. The isolate obtained about 0.0705 gram, characterized by FTIR. The isolate has characteristic functional groups such as -CH (aliphatic), -OH (hydroxy), -C=O (carbonil), and -C-O- (ester). The isolate is a polysaccharide compound.
Key word : Safflower, Carthamus tinctorius L., polysaccharide.
INTRODUCTION
One of the plants used in traditional medicine by the people especially in the district of South Sulawesi is Safflower (Carthamus tinctorius L.) from the Asteraceae tribe. Safflower an annual plant, spiked on branches and leaves, the flowers are yellow or reddish. generally planted in hot, dry climates [1]. Empirically, Kasumba steeping the dried flowers, used by the community for the treatment of measles. In the Chinese medical system, in addition to the treatment of measles, Turate Kasumba flowers are also used for the treatment of menopausal disorders, fever, and improve blood circulation system [2]. Safflower contains a number of compounds such as carthamin, carthamidin, glycosides, coumarins, fatty acids, steroids, and polysaccharides. Based on the study of modern pharmacology, it is known that the extract Safflower have several biological activities, such as anticoagulants, antihypertensives, antioxidants, immunosuppressants, and antitumor. And most of these effects, there is the water extract [3.4]. It has been reported from several studies, that the bioactive polysaccharide compounds that have been isolated from fungi, bacteria, algae, and from the plant, has antitumor and immunomodulatory effects [5]. Polysaccharides are compounds that consist of a combination of a large number of monosaccharide molecules (containing ten or more monosaccharide) that these compounds can be hydrolyzed into monosaccharides many molecules [6]. Safflower polysaccharide composed of xylose, fructose, galactose, glucose, arabinose, and ramnosa uronik acid residues, was reported to stimulate the immune system in mice [7]. Based on the information that has been obtained, it can be seen that the water extract of polysaccharide compounds Safflower can boost the immune system. From the literature search results, in Indonesia has not been a lot of reports of research on the polysaccharide content of the active compounds of interest Safflower, and its potential as munomodulator. Therefore, need to be isolated from the aqueous extract polysaccharide compound interest Safflower, and identify isolated compounds. In this study, Safflower used comes from Wempubbu village, in Bone region. The study was conducted in accordance polysaccharide compound isolation procedure, where the sample is extracted with infundation method. Matter content was determined by the method of polysaccharide hydrolysis. Identification of isolated compounds performed thin layer chromatography, using a reagent identification. Then do the characterization of isolated compounds using FT-IR. MATERIALS AND METHODS
Safflower petals (Carthamus tinctorius L.), taken from the District Amali, Bone regency. Safflower petals (Carthamus tinctorius L.) is obtained in the form of dry, ready to be extracted. The chemical used in the present study were obtained from the following source. Cellulose TLC plates, Molisch Reagents, and Seliwanoff Reagent. Chamber (Camag ), FT-IR (Bruker ), UV light (254 nm and 365 nm), pH meter (Sartorius ), centrifuged (Sorvall RC 5B Plus), and a set of tools infundation. Extraction and Isolation Dried petals of safflower (Carthamus tinctorius L.) were extracted with boiling water for 15 min, then filtered while still hot. The resultant extracts were centrifuged at 4000 rpm for 20 min, and the supernatants were precipitated with 70% ethanol (4 times the volume of aqueous extract), and placed in the refrigerator overnight . The precipitates were collected by centrifugation (4000 rpm, 20 min), The precipitate obtained were dissolved in water, and the aqueous solution was adjusted to pH 3 by HCl. The insoluble materials were removed by centrifugation , and the recovered supernatants were again adjusted to pH 7 by NaOH. Active compounds were collected by 70% ethanol precipitation. Ethanol precipitate was dried, and then proceed to stages of hydrolysis [5]. Hydrolysis of Polysaccharides Compound Dried precipitate obtained, weighed and then put into a covered Erlenmeyer meeting, added 0.25 N HCl, placed in a preheated oven at 100C for 3 to 5 hours. Tubes are removed from the oven, cooled to room temperature. The filtrate was taken, then added absolute ethanol, to precipitate saccharide. Depositionthe precipitate was taken, and then evaporated [8,9]. Identification and Characterization of Isolates Identification of polysaccharide compound by Thin Layer Chromatography on cellulose plates with different mobile phase. After that, used a spray reagent, for polysaccharide compound identification. For qualitative tests, used Seliwanoff, Molisch, and iodine reagents. Isolates obtained were then characterized by FT-IR. RESULTS AND DISCUSSION In the early stages, extraction with infundation, is intended to separate the target compounds, polar polysaccharide, from other compounds present in the sample. The liquid extract from the results of infundation, conducted by precipitated polysaccharide compounds using 70% ethanol. It is based on the nature of the polysaccharide compounds have very low solubility rate of the ethanol, so the only other compounds with low molecular weight dissolved, and the polysaccharides were precipitate [10]. The precipitate polysaccharides, separated from the filtrate using a centrifuge. The precipitate obtained is dissolved then adjusted pH to pH 3, to release proteins that bind to polysaccharides, which is then neutralized again by NaOH and precipitated with ethanol [11]. Qualitative and quantitative analysis of carbohydrates by using TLC, is a method that is faster, easier and cheaper than previous methods, such as HPLC and GC. However, polysaccharides can not be identified by TLC. However, polysaccharide constituent monosaccharides can be determined by TLC, after hydrolysis [12]. Qualitative analysis of the polysaccharide compound by TLC and spray reagent, however, do not support the results obtained. This was confirmed by the results of a literature search of that has been described previously, that the polysaccharide compound identification can not be done by TLC. Therefore, carried out using acid hydrolysis of polysaccharide compounds, polysaccharide compounds to break down into simpler molecules, such as monosaccharides so that they can test their identification. On acid hydrolysis, dilute acid solution usually used, where the speed of the reaction is proportional to the concentration of acid. In general, soluble components contained in the results of acid hydrolysis of polysaccharides is xylose, glucose, cellobiose, furfuraldehida, hidroksimetilfurfural, and organic acids such as formic acid, levulinat acid, and acetic acid, [13,14]. From the results of the test compound identification results of hydrolysis, obtained that states that the test results seliwanoff hydrolysis compounds do not contain fructose compounds. Test molisch stated that isolates a carbohydrate compound, and for iodine test, the test results showed a brown color, which leads to the compound dextrin, which is a breakdown product of the polysaccharide. However, positive results for iodine test still requires a more specific test. Isolates obtained from the hydrolysis were identified by TLC. Carbohydrates are a very hydrophilic compound, so it can bind strongly on TLC plates, such as silica gel, alumina, and cellulose. Therefore, it takes a mobile phase that more polar on TLC development [15]. Effective with cellulose TLC plates to separate a mixture of monosaccharides, including amino acids and sugars. Nevertheless, TLC multi eluent or two-dimensional TLC needed to confirm the results of isolation, which is then detected by reagent class. Based on the literature study, the multi TLC eluent used some kind of comparison eluent for compounds such as polysaccharides and their derivatives: ethyl acetate: n-propanol: acetic acid: water (4:2:2:1), acetonitrile: water (3:1), acetone: water (3:1), ethyl acetate: ethanol: water (2:2:1) and methanol: water (2:1). There showed IR spectral data interpretation (Table 1). The IR spectrum (Figure 1) shows a broad absorption band at wave number 3344 cm -1 , believed to be the-OH (hydroxyl), which usually appears in the wavenumber region 3600-3200 cm -1 . Absorption at 2946 cm -1 and 2836 cm -1 are sharp and feeble alleged aliphatic CH groups are common in the region 3200-2700 cm -1 uptake, and the uptake in the area of the curve of the wave number 1453 cm -1 and 1425 cm -1 which represents a group-CH, which usually appears in the catchment area from 1470 to 1430 cm -1 . Absorption appears at wave numbers 1741, 1647, and 1546 cm -1 , thought to be the carbonyl group (-C = O). whereas the indentation area, there is a sharp absorption at wave numbers 1158, 1105, and 1024 cm -1 is thought to be group-CO-which usually appear at wave numbers 1300-1000 cm -1 . Carbonyl group appears at wave number 1741 cm -1 , probably derived from esters, which usually appears at wave number 1750-1725 cm -1 . This is indicated by the presence of the-CO-in the curve of the wave number 10241 cm -1 are sharp and strong, where the absorption in the region 1500-600 cm -1 is the finger print area, which can provide information about the presence in the mixture of polysaccharide compounds [16,17]. Table 1. FTIR spectra data of polysaccharide compound isolated from safflower Wavenumber (cm -1 ) Band appears Intensity Possible functional group 3344 broad Medium -OH 2946 sharp weak -C-H 2836 sharp weak -C-H 1741 sharp weak -C=O 1647 sharp weak -C=O 1546 sharp weak -C=O 1453 broad weak -C-H 1425 broad weak -C-H 1158 sharp weak -C-O- 1105 sharp weak -C-O- 1024 sharp strong -C-O-
Figure 1.FTIR spectrum of polysaccharide compound from Safflower aqueous extract.
CONCLUSION From the research that has been done, as many isolates obtained 0.0705 gram. Based on the identification test polysaccharide compound, and compound characterization by FT-IR showed that isolates containing group -CH (aliphatic),-OH (hydroxy), -C=O (carbonyl), and -CO- (ester), isolated compounds believed to be polysaccharides. REFERENCES [1] Longe, JL. The Gale Encyclopedia of Alternative Medicine Vol.4. 2 nd ed. Thomson Gale. New York. 2005. p. 19. Available as PDF file. [2] Syukur R, Usmar, dan Fatmawati A. Uji Aktivitas Imunomudulator Kasumba Turate (Carthamus tinctorius.Linn) sebagai Upaya Pembuatan Sediaan Terstandar Menuju Prototipe Skala Industri Kecil. Majalah Farmasi dan Farmakologi. Vol.14. No.[1] 2010. hal. 17-20. [3] Chengaiah B, Mallikarjuna R, Mahesh KK, Alagusundaram M, Madusudhana C. Medicinal Importance Of Natural Dyes-A Review. International Journal of PharmTech Research Vol.2 No.1. 2010. pp. 144-154. [4] Zhou F, Zhao M, and Tu PF. Simultaneous determination of four nucleosides in Carthamus tinctorius L. and Safflower injection using highperformance liquid chromatography. Journal of Chinese Pharmaceutical Sciences. 2009. pp. 326-330. [5] Wakabayashi T, Hirokawa S, Yamauchi N, Kataoka T, Woo JT, and Nagai K. Immunomodulating activities of polysaccharide fractions from dried safflower petals. Cytotechnology 25. 1997. pp. 205-211. [6] Robinson T. Kandungan Organik Tumbuhan Tinggi. Edisi VI. Terjemahan oleh Padmawinata K. Penerbit ITB.1995. hal. 27. [7] Khare, CP. Indian Medical Plants. Springer. NewDelhi. 2007. p.124. Available as PDF file. [8] Worsfold P, Townshend A, Poole C. Encyclopedia of Analytical Science. Germany. pp. 400-401,441. Available as PDF file. [9] Manullang M dan Yohani V. Ekstraksi dan Analisis Polisakarida Buah Sukun (Artocarpus altilis). Teknik dan Analisis Pangan. Vol VI no.3. 1995. [10] Cooke M. Encyclopedia of Separation Science. Academic Press. USA. pp. 2235, 3921. Available as PDF file. [11] Chihara G, Hamuro J, Yukiko Y, Maeda. Fractination and Purification of Polysaccharides with Marked Antitumor Activity, Especially Lentinan, from Lentinus edodes (Berk.) Sing. (An Edible Mushroom). Cancer Research.Vol.30. National Cancer Center Research Institute. Japan. 2012. pp. 2776-2781. [12] Wozniak KS, Widelski L, Glowniak K. Plant Materials in Modern Pharmacy and Methods of Their Investigations. In: Hajnos MW, Sherma J, and Kowalska T, editors. Thin layer chromatography in phytochemistry. CRC Press. USA. 2008. p.28. [13] Striegel ME, Hill J. Thin-Layer Chromatography for Binding Media Analysis. The Getty Conservation Institute. USA. 1996. p. 49. [14] Mastuti E, Setyawardhani. Pengaruh Variasi Temperatur dan Konsentrasi Katalis pada Kinetika Reaksi Hidrolisis Tepung Kulit Ketela Pohon. Ekuilibrium. Vol. 9. Universitas Sebelas Maret. 2010. hal. 23-27. [15] Bassett, J, Denney RC, Jeffery GF, & Mendham J. Buku Ajar Vogel Kimia Analisis Kuantitatif Anorganik. Edisi 4. Terjemahan oleh Pudjaatmaka AH & Setiono L. Penerbit Buku Kedokteran EGC. Jakarta. 1994. hal. 228. [16] Silverstein RM, Webster FX, Kiemle DJ. Spectrometric Identification of Organic Compounds. 7 th ed. New York: John Wiley & Sons Inc. 2005. pp:80-100. [17] Coates J. Interpretation of Infrared Spectra, A Practical Approach. Encyclop[edia of Analytical Chemistry. USA. 2000.