METHOD Collection of Plant Material

  

CHARACTERIZATION COMPOUND FROM EXTRACT CHLOROFORM

LEAVES M. umbellata (Houtt.) Stapf var. Degrabrata K. AND

TEST ANTIHYPERGLYCEMIC ACTIVITY

  1

  1

  1 Imran , Nunuk Hariani S. , and Hanapi Usman

1 Chemistry Department, Science Faculty, Hasanuddin University, Makassar, South Sulawesi,

  90245

  

Abstrak. Penelitian karakterisasi senyawa dari ekstrak kloroform daun M. umbellata (Houtt.) stapf

  var. degrabrata K. dan uji aktivitas antihiperglikemik telah dilakukan. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi senyawa dari ekstrak kloroform serta menentukan konsentrasi optimum dari ekstrak kloroform dalam menurunkan kadar glukosa darah. Isolasi senyawa dari ekstrak kloroform dilakukan dengan cara maserasi, fraksinasi, dan pemurnian. Berdasarkan hasil analisis spektroskopi IR dan NMR, senyawa yang berhasil diisolasi dari penelitian ini adalah

  β-sitosterol dan senyawa asam lemak yang terdiri dari 25 atom karbon.

  Pengujian toleransi kadar glukosa, konsentrasi 6% memperlihatkan penurunan kadar glukosa yang hampir setara dengan obat glibenklamid yang dijadikan pembanding.

  Kata kunci: β-sitosterol, ekstrak kloroform, Melochia umbellata, Uji toleransi kadar glukosa.

  

Abstract. A research about characterization of the chloroform extract of leaves of M. umbellata

  (Houtt.) stapf var. degrabrata K. and activity test as anti hyperglycemic has been done. This study aimed to isolate and characterize compounds from chloroform extract and determine the optimum concentration of chloroform extract in reducing blood glucose levels. Isolation compound from chloroform extract has been done by maceration, fractionation, and purification. Based on the data of IR and NMR spectroscopic, compounds isolated from this research were

  β-sitosterol and fatty

  acid compound consisting of 25 carbon atoms. The glucose tolerance test, concentration of 6% showed a decrease in glucose levels almost equivalent with drug glibenclamide as comparator.

  Keywords: β-sitosterol, chloroform extract, Glucose tolerance test, M. umbellata

1 Corresponding author e-mail: buhe.imran@gmail.com

  INTRODUCTION

  Exploration of medicinal plants as a source of material have been carried out. Many plants that have efficacy as traditional medicine, one of which is paliasa. Paliasa is one of the plants of the family Malvaceae used by the society, especially in South Sulawesi as a drug capable of treating liver disease, hypertension, diabetes, high cholesterol, and hepatitis by drinking boiled water leaves (Raflizar et al., 2006).

  Paliasa known in two different plant species that Kleinhovia hospita L. and M.

  umbellata (Houtt.) Stapf. M. umbellata (Houtt.)

  Stapf consists of two varieties, namely M. umbellata (Houtt.) Stapf var. degrabrata K. and M. umbellata (Houtt.) Stapf var. visenia. Among the three types of paliasa, extract of M. umbellata (Houtt.) Stapf var. degrabrata K. more toxic than the other types. It was evident from the results of research conducted by Lalo and Tayeb (2003) on the three methanol extracts of the plant species at a concentration of 10% and 15% from M. umbellata (Houtt.) Stapf var. degrabarata K. showed significant improvement of liver function. Similarly of the toxicity against Artemia salina larvae, the toxicity of methanol extract of leaves

  M. umbellata (Houtt.) Stapf var. degrabata K.

  showed highest. Bioactivity screening conducted by Nuvita (2006) from the methanol extract of free radicals and BHT as controls also showed that the leaves of M. umbellata (Houtt.) Stapf var. degrabrata K. has a higher antioxidant effect compared with the other plant species of paliasa.

  Based on the literature, chemical compounds in the plants of M. umbellata (Houtt.) Stapf var. degrabrata K. are essential oils, triterpenoids, alkaloids, and flavonoids (Heyne, 1987); and saponin compounds and antarkuinon. The lattest two compounds can be prevent liver inflammation in mice (Lalo and Tayeb, 2003).

  Research on paliasa have been done. There are many secondary metabolites compounds that have been isolated from this plant, either from the extract with a polar solvent, non-polar or semi-polar solvents such as chloroform. Two quinolone alkaloid compounds have been isolated from the chloroform extract M. umbellata (Houtt.) Stapf var. degrabrata K. namely 7,8-epoxy melochinon (3) (Erwin, 2010) and 9,10-epoxy melochinon (4) (Ridhay, 2012). Melochinon was active against leukemia cancer cells P-388 with LC

  50

  = 0.83 µg/mL. Secondary metabolites steroids also isolated from the chloroform extract Kleinhovia hospita Linn. (Soekamto, 2008) whereas for the group of terpenoid compounds 3-hydroxy-12-oleanan-28-oats (5) was also isolated from the chloroform extract of K. hospita Linn. (Purwaningsih, 2010).

  Further studies of bioactive compounds contained in extracts chloroform M. umbellata (Houtt.) Stapf var. degrabata K. has done. Although it has been many scientific reports regarding the ability of this plant in treating liver disease, but research related to the treatment of diabetes has not been done. Therefore, this report about the plant M. umbellata (Houtt.) Stapf var. degrabata K. that can lower blood glucose levels.

  METHOD Collection of Plant Material

  Leaves of plants M. umbellata (Houtt.) Stapf var. degrabata K. collected on April 2012 were taken in Hasanuddin University, Tamalanrea Campus, Jl. Perintis Kemerdekaan Km. 10, Makassar, South Sulawesi. This plant is identified by Herbarium Bogoriense, Center of Research and Development Biology, LIPI Bogor with specimen number BO-1912171.

  Materials

  To extraction, fractionation, and recrystallization used solvent with the quality of pure analys (p.a) and technis such as chloroform, methanol, n-hexane, ethyl acetate and acetone. In the process of chromatography used silica gel Merk. 7730 catalog for vacuum column chromatography (KKV), silica gel Merk. 7734 catalog for press column chromatography (KKT), silica gel Merk. 7733 catalog for gravity column chromatography (KKG), and for thin-layer chromatography (TLC) performed with silica gel-coated aluminum plate Brand Keselgel 60 F254 0.25 mm. Appear stain solution used cerium sulfate solution (Ce(SO4)

RESULT AND DISCUSSION

  Glucose Tolerance Test

  15.99 g of chloroform extract was fractionated by vacuum column chromatography with eluent n-hexane, n-hexane: ethyl acetate, ethyl acetate, acetone, and methanol with increased the polarity. The process begins with a search eluent fractionation showing stains on the value of Rf 0.3 on TLC chromatogram analysis for organic compounds separated well on the Rf value. Stain with Rf of 0.3 obtained from the comparison of the eluent [n-hexane: ethyl acetate = 9: 1]. Fractionation results obtained by 15 fractions and combined into 7 main fraction (fraction AF). Merger fractions based on TLC chromatogram analysis results that have profiles similar stains (Figure 1). Fraction B with weight 244.3 mg further fractionated by press column chromatography with eluent n-hexane, n-hexane: ethyl acetate, ethyl acetate and methanol. Fractionation resulted in 42 fractions were combined into 8 main factions. White crystals, weighing 10.9 mg were formed in fractions B4. Test purity through TLC analysis by using three kinds of systems each eluent showed one stain that appeared after heated as in Figure 2.

  Isolation and Purification

  Dried leaves powder M. umbellata (Houtt.) Stapf var. degrabata K. macerated with methanol 1 x 24 hours 4 times resulted 39.1 g of methanol extract of dark green. Then, the methanol extract was partitioned by liquid- liquid extraction with n-hexane, chloroform, and ethyl acetate that produce n-hexane extracts, chloroform extract, and ethyl acetate extract. Further isolation and purification of the chloroform extract.

  Extraction

  and the second compound suspected as fatty acid compound consisting of 25 carbon atoms.

  β-sitosterol

  In isolation study of secondary metabolites from chloroform extract of the leaves M. umbellata (Houtt.) Stapf var. degrabata K. obtained two compounds. The chemical structure of the two compounds determined by IR and NMR spectroscopic data of the first compound is suspected

  previously and had been given the originator of diabetes. Concentration varied dosage. The data obtained and recorded for further analysis.

  musculus ). The mice had been treated

  Chloroform fractions obtained tested their toxicity to mice adult male (Mus

  2

  ) 2% in 2 N sulfuric acid and for the tolerance test glucose levels (diabetic) used distilled water, 9% glucose solution, a solution of Na-CMC and glibenclamide.

  1 H and

  5 kg of dried leaves powder M. umbellata (Houtt.) Stapf var. degrabata K. macerated with methanol for 1 x 24 hours 4 times. Macerate obtained was then concentrated and the crude extract was then weighed to determine the total weight. Subsequently fractionated by liquid-liquid extraction with n-hexane and chloroform. Chloroform extracts were evaporated to dryness obtained is then weighed and then fractionated by column chromatography vacuum, press, and gravity by using the appropriate eluent. Further isolates obtained purified by recromatography, crystallization, and recrystallization. Structure determination is based on the data of IR spectra and

  Extraction and Isolation

  glucometer is used for blood glucose measurement.

  ®

  NESCO

  13 C-NMR spectrum.

  and 125.65 MHz for

  1 H-NMR

  The tools used in this study were gram scales (O'hauss), scissors, oral syringe, distillation, rotary evaporator, UV lamps, tools for thin layer chromatography, vacuum column chromatography, press column chromatography, gravity column chromatography, and tools glasses are commonly used in laboratories. For the measurement of the melting point used a Fisher John. While IR spectrometer for the analysis of variants with Shimadzu FTIR 8501, JEOL JMN A 5000 working on 500.0 MHz for

  Tools

  13 C-NMR.

  Figure 4. Chromatogram of TLC analysis

  fraction A, [n-heksan(9) : etil asetat(1)].

  Figure 1. Chromatogram of TLC analysis of

  chloroform extract, (a) the results of Fraction 4 was followed by fractionation KKV; (b) the recrystallization with warm acetone. The results combined fractionation. were then analyzed recrystallization fraction A4 TLC with eluent ethyl acetate (2): n-hexane (8)

  Furthermore, the purity test through and showed a single stain was also supported by analysis with 2-dimensional TLC with eluent the results of measurements of the melting point [n-hexane (7): ethyl acetate (3)] also showed a of 67-69 °C (Figure 5). stain that appears after heated but not fluoresce under UV light of short wave and long wave as shown in Figure 3 which indicates that the crystal is a pure isolate expressed as compound

  1 Test groups to these compounds showed that steroids purplish blue color after the addition of sulfuric acid and acetic anhydride.

  Figure 5. Chromatogram of TLC analysis

  compound 2 under UV long wave

  Compound 1

  powder with a weight of 10.9 mg showed a positive steroid test with Liebermann-Burchard reagent. This compound does not fluoresce

  Figure 2. Chromatogram of TLC analysis of

  under UV light which indicates that these compound 1, (a) [n-heksan(9) : etil compounds do not have conjugated double asetat(1)]; (b) [n-heksan(8,5) : bonds. This is consistent with the IR spectrum

  CHCl (1) : metanol (0,5)];

  3

  of the data does not show any absorption band

  3

• 1 (c) [Aseton(2) : CHCl (8)].

  in the region 3100-3000 cm (Lambert et al., 1998) as the wavelength for the aromatic group. 596 mg Fraction A further fractionated

  IR spectral data showed only aliphatic by press column chromatography with eluent CH absorption band area at 2956, 2926, and n-hexane: ethyl acetate, ethyl acetate, acetone,

• 1

  2852 cm which is supported by the presence and methanol yield 28 major fractions as in

• 1

  of absorption at 1462 and 1377 cm for CH

  2 Figure 4.

  and CH . Absorption band at wave number

  3

• 1

  3444 cm indicating the presence of OH group is supported by the absorption peak at 1058

• 1

  cm for the CO stretching vibration. In addition

• 1

  there is also absorption at 1645 cm for the C=C which indicate the presence of an olefin group.

  Figure 3. Chromatogram of TLC analysis 2

  dimention compound 1 after heated

• 1

  group that interacts with the binding carbon 2 protons (CH

  IR spectral data of compounds 2 can be seen in the Table 2.

  Table 2. Data spectrum IR compound 2

  Groups function Wavelength (cm

  ) O-H 3448 C-O 1172

  CH alifatik 2918 2848

  CH

  2

  1467 CH

  3

  1379 C=C 1636

  

1

H-NMR spectroscopic data are summarized in

  Table 3 shows the 5 peaks. The first peak with a chemical shift of 0.88 ppm is a triplet form CH

  3

  2

  ). Chemical slider with a multiplicity of 1.29 ppm multiplet with a very high intensity, demonstrating some of CH

  2

  groups in the wave number that has the same environmental conditions that led to the second peak shows a very high peak. The third peak with a chemical shift of 1.61 ppm multiplet shaped and fourth peaks with a chemical shift of 2.29 ppm triplet shaped carbon interacts with oxygen binding. Fifth peak is the peak with a chemical shift of 4.05 ppm which has a triplet multiplicity is far away from the TMS peak is caused by a group that binds oxygen.

  13 C-NMR spectral data of compound 2

  (Table 3) showed nine carbon signals consisting of a methyl group with a chemical shift of 14.31 ppm and 5 methylene on the chemical shift of 22.89; 25; 24; 26.14; 28.86, and 29.85 ppm, and 2 metin groups on the chemical shift of 32.12 and 34.63 ppm and also there is the carbon bonded to the OH group on the chemical shift of 64.59 ppm.

  Tabel 3. Data

  1 H dan

  13 C-NMR Senyawa 2

  δ

  1 H (ppm)

  δ

  13 C (ppm)

  0,88 (2H, t) 14,31 1,29 (2H, m) 22,89 1,61 (2H, m) 25,24

• 1
• 1
• 1

• 1
• 1
• 1 indicates the presence of an olefin group (C=C).

  . Absorption at region 1636 cm

  ) at a wavelength of 1467 cm

  and 1379 cm

  3

  Table 1. IR spectrum data compound 1

  Groups Function

  Wavelength (cm

  ) Compound 1

  β-sitosterol

  (Sari, 2011) O-H 3444 3452

  C-O 1058 1049 CH alifatik

  2956 2926 2852

  2954 2935 2891 2868 2850

  CH

  2

  1462 1465 CH

  1377 1381 C=C 1645 1627

  3

  Wave numbers in the IR spectrum of compound 1 did not show any significant difference with the IR spectra of compound

  β-sitosterol

  standard, so it can be concluded that compound 1 is a

  β-sitosterol.

  Figure 6. Structure compound 1 ( β-sitosterol)

  Compound 2 in the form of a white powder with a weight of 29.2 mg and a melting point of 67-69

  o

  C. IR spectroscopy showed absorption band at a wavelength of 3448 cm-1 which indicates the presence of OH groups which are supported by the bending of CO at a wavelength of 1172 cm

  . Area 2918 cm

  and 2848 cm

  indicating the presence of aliphatic CH bending backed by methylene (CH

  2

  ) and methyl (CH

  2,29 (2H, t) 26,14 4,05 (1H, t) 28,86

• 29,85
• 32,12
• 34,63
• 64,59<
• 1

  Bioactivity Test

  Effect of leaves extract of CHCl M. umbellata (Houtt.) Stapf var. degrabata K. in mice for

  3 3 hours showed a decrease in blood glucose levels (Table 4).

  

Table 4. Average blood glucose levels of mice caused by inject of the chloroform extract of leaves

of M. umbellata (Houtt.) Stapf var. K. degrabrata with control and comparison.

  Blood glucose levels every Blood glucose The rate of hour after inject of the test levels after decline in preparation treatment Treatment induction glucose levels (mg/dl) (mg/dl) (mg/dl. jam)

  1

  2

  3 Na-CMC

  202,00 132,00 141,00 101,67 29,20 1 % b/v Glibenklamid

  237,00 137,00 90,67 73,67 53,63 0,0195 mg/ml

  Chloroform extract 233,67 136,33 129,67 108,67 38,17

   M. umbellata 2% b/v

  Chloroform extract 248,67 172,67 125,67 105,00 47,80

  M. umbellata 4% b/v

  Chloroform extract 258,33 129,67 119,67 94,67 50,10

  M. umbellata 6% b/v

  The data of Table 4 obtained diagrams mice blood glucose levels decrease as the effect of chloroform extract M. umbellata (Houtt.) Stapf var. degrabrata K. leaves accompanied by a negative control and comparison as shown in Figure 7 below:

  

Figure 7. Profile decrease in blood glucose levels of mice caused by administration of chloroform

  extract of leaves of M. umbellata (Houtt.) Stapf var. degrabrata K. by comparison glibenclamide and negative controls Na-CMC 1% w/v. Hyperglycemia was defined as fasting glucose levels higher than 110 mg/dL. Hyperglycemia arises because the absorption of glucose into cells and inhibited metabolism disturbed while Diabetic Mellitus is a chronic disorder that is specifically related to glucose metabolism in the body, but the metabolism of fats and proteins are also affected.

  Chloroform extract testing is done to prove the pharmacological effects of plant leave

  stapf var. degrabrata K. (Paliasa), Unpublish Dissertasion, Chemistry Department, Postgraduate, Hasanuddin University, Makassar.

  Linn.) dan Uji Aktivitas Antibakteri

  pada Fraksi Kloroform Kulit Akar Tumbuhan Paliasa (Kleinhovia hospita

  6. Purwaningsih, E., 2010, Isolasi Senyawa

  Unpublish Thesis, Biomedical /Pharmacology Department, Hasanuddin University, Makassar.

  Ekstrak Daun Paliasa terhadap Radikal Bebas Penyebab Penyakit Degenaratif ,

  5. Nuvita, T., 2006, Uji Aktivitas Antioksidan

  Structural Spectroscopy Second Edition, Pearson, United States.

  4. Lambert J.B., Gronert, S., Shurvell, H.F., and Lightner, D.A., 1998, Organic

  Mencit yang Diinduksi dengan Karbon Tetraklorida, Majalah Farmasi dan Farmakologi , 8 (2), 25-32.

  umbellata var. visenia terhadap Fungsi Hati

  3. Lalo dan Tayeb, 2003, Efek Ekstrak Metanol Paliasa Jenis Kleinhovia hospita, M. umbellata var. degrabrata dan M.

  Kehutanan, Yayasan Sarana Warna Jaya, Jakarta.

  Indonesia , Jilid III, Translate by Balit Bang

  2. Heyne, K., 1987, Tumbuhan Berguna

  Isolat Kayu Batang M. umbellata (Houtt.)

  M. umbellata (Houtt.) Stapf var. degrabrata K.

  CONCLUSION

  as a drug that can treat diabetic situation and determine the optimum dose. This study used a chloroform extract of the plant with a concentration of 2%, 4%, and 6% w/v. Na-CMC suspension 1% w/v was used as a negative control and a positive control used glibenclamide 0.0195 mg/ml.

  Antihyperglycemic effect was determined using the glucose tolerance and the treatment condition was kept uniform with the aim to reduce the factors that may affect the outcome of the experiment.

  Animals used in this study were mice (Mus musculus) male sex in a healthy condition. Female mice were not used because of their hormonal system is unstable compared with male mice. Nevertheless, the biological variation factors of the test animals can not be removed so it can affect research results.

  Before treatment, the mice were fasted beforehand to avoid the influence of food at the time of measurement of blood glucose levels and to increase the speed of drug absorption.

  Measurement of blood glucose levels in mice were performed for 3 hours with 1 hour intervals. It is based on literature indicating that the absorption of glucose in the body takes about 30-60 minutes and will decline after 2-3 hours, then to see a decrease in glucose levels more obvious use for a period of 3 hours after administration of the test preparation (Syahrul 2010 ).

  After the measurement of blood glucose levels for 3 hours, from the diagram it appears that the higher the concentration of chloroform extract M. umbellata (Houtt.) Stapf var. degrabrata K. the rate of decrease in blood glucose levels also increase. The biggest influence is shown by a comparison giving chloroform extract M. umbellata (Houtt.) Stapf var. degrabrata K. at a concentration of 6% w/v.

  Two compounds from the chloroform extract of the leaves M. umbellata (Houtt.) Stapf var. degrabrata K. been isolated. Compound 1 is suspected as

  1. Erwin, 2010, Penentuan Struktur Molekul

  β-sitosterol and

  Compound 2 is suspected as a fatty acid compound having 25 carbon atoms.

  Chloroform extracts of the leaves of plants Melochia umbellata (Houtt.) Stapf var. K. degrabrata can lower blood glucose levels, which is consistent with the use of this plant in ethnobotany.

  ACKNOWLEDGEMENTS

  Thank you so much to DIKTI that has supported fund this research through PKM-P and also to Prof. Dr. Nunuk Hariani S., MS. as the main supervisor and Prof. Dr. Hanapi H. Usman, MS. as the first supervisor.

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