ISOLATION, CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF TRITERPENOID COMPOUNDS FRACTION
CHLOROFORM BARK PHYLLANTHUS NIRURI LINN
I. W. G. Gunawan1*, I. G. A. G. Bawa2 and A. A. Bawa Putra3
1,2Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, University of Udayana, Bali.
3Laboratory of Anorganic Chemistry, Department of Chemistry, Faculty of Science, University of Udayana, Bali.
ABSTRACT
The use of traditional medicine as Phyllanthus niruri compounds have been reported to contain triterpenoids, alkaloids and flavonoids are efficacious as a cure dysentery, rheumatic, anti-inflammatory and intestinal worms in children. This study aims to isolate, characterization and determine the antibacterial activity of the triterpenoids compounds of chloroform fraction bark of Phyllanthus niruri. A total of 700 g of dry powder of bark Phyllanthus niruri macerated with n-hexane for 24 hours. Subsequently extract thick n- hexane in the partition with chloroform produce a yield of 12.446 (1.778%). The results showed that isolates compounds Fi chloroform fraction bark contains compounds Phyllanthus positive niruri
triterpenoids with Rf 0.411 and is able to inhibit the growth of bacteria Staphyloccocus aureus at a concentration of 1000 ppm with inhibition of 12 mm. Identification using a spectrophotometer mass and HNMR showed that the compound isolates F1 possibilities include compound group triterpene steroids with the characteristics of the molecular ion m/z 424 [M +], m/z 381 [M+-C3H7], m/z 341 [M+- C3H4], m/z 313 [M+- C2H4], m/z 287 [M+- C2H2], 229 [M+- C3H6O], m/z 151 [M+-C6H6], m/z 109 [M+ –C3H6], m/z 95 [M+- CH2], m/z 81 [M+- CH2], m/z 69 [M+-C], m/z 55 [M+- CH2], m/z 43 [M+-C] and a singlet peak δ 7.337 [aromatic], multiplet δ 0.808 to 1.682 [CH2 and CH3], doublet 5.8 δ [C = CH], doublet 4.6 δ [OH].
SJIF Impact Factor 6.041
Volume 5, Issue 6, 357-364 Research Article ISSN 2278 – 4357
*Corresponding Author I. W. G. Gunawan Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, University of Udayana, Bali.
Article Received on 13 April 2016,
Revised on 03 May 2016, Accepted on 23 May 2016 DOI: 10.20959/wjpps20166-7018
KEYWORDS: Isolation, Characterization, Antibacterial, Triterpenoids, Phyllanthus niruri.
INTRODUCTION
The development of the use of traditional medicines in particular are derived from plants to help improve public health is quite widespread. The types of plants that come from the genus Phyllanthus widely used as traditional medicine, such as Phyllanthus urinary in the Solomon Islands is used to treat pain in the chest, Phyllanthus acidus in Indo China used to treat smallpox and cough, Phyllanthus wilfordii in China is used to treat cough and mucus from the throat. Niruri Phyllanthus plants in Indonesia has been widely used as a remedy dysentery, inflammation of the liver, colitis, rheumatism, injuries hit, inflammation of the kidneys and intestinal worms in children (Tanaka, 1993; Matsunaga et al, 1993).
Plant species of the genus Phyllanthus reported to contain triterpenoid compounds such as Phyllanthus sellowianus known to contain triterpenoid alcohol, Phyllanthus flexuosus contains triterpenoids diol and the type of Phyllanthus watsonii also known to contain a lot of triterpenoids, but so far Phyllanthus niruri unknown chemical content and activities as an antibacterial against bacteria Staphyloccocus aureus, Based on the literature, Phyllanthus niruri secondary metabolites known to contain flavonoids, terpenoids, alkaloids and steroids (Kardinan and Kusuma, 2004). Some research indicates a terpenoid compound has antibacterial activity that is monoterpenoid linalool, hardwicklic diterpenoid acid, phytol, triterpenoids and triterpenoid saponin glycosides (Grayson, 2000; Bigham et al; 2003;
Anonymous, 2007).
Triterpenoids in plant serves as a protection to resist the attack of insects and microbes.
Triterpenoids used in medicine as an anti-allergy, anti-inflammatory, and anti-neoplastic.
Triterpenoids is one of the compounds found in plants that are derived from the biosynthesis of six isoprene units and are derived from C30 acyclic hydrocarbons is squalene. This compound is colorless, crystalline, high melting point and is optically active (Parekh, 2006).
According to Lim, et al (2006) triterpenoid compounds can be divided into four categories, namely: triterpen fact, saponins, steroids and cardiac glycosides. Chemically, triterpenoids are generally soluble in fat and contained in the cytoplasm of plant cells. These compounds are known in plants and to date only a few are known to spread widely. The compound is a pentacyclic triterpene α-Amyrin and β-Amyrin well as their derivatives serves as a protection to resist insects and microbial attack. Some triterpene steroid structures of Phyllanthus plant can be seen as follows (Brown, 1998):
Phytostrol Campesterol
Stigmasterol Sitosterol
Lanosterol
MATERIALS AND METHODS Preparation Sample
The materials used in this study is the bark of Phyllanthus niruri obtained from Tabanan-Bali region in a fresh state, the next determination made in plant conservation UPT Eka Karya Botanical Garden Bedugul Bali. The bark of Phyllanthus niruri drawn at random by not taking into account the age of the plant. Then be cut small pieces and left to dry under direct sunlight. The bark is dried subsequently pulverized and blended to form a fine powder.
Extraction of Secondary Metabolites
A total of 700 grams of powder of dried bark of Phyllanthus niruri extracted by 3 x 1000 ml macerated using n-hexane solvent respectively for 24 hours. N-hexane filtrate obtained combined, then evaporated with a rotary vacuum evaporator at 40°C until a thick n-hexane extracts are dried or reaches a constant weight. Furthermore partitioned with chloroform.
Chloroform extract obtained is evaporated until thick and tested to determine the content of phytochemical triterpenoidnya the Liebermann-Burchard reagent and are determined
antibacterial activity against bacteria Staphylococus aureus. The extracts showed antibacterial activity best resumed in the process of separation and purification of active compounds by thin layer chromatography analysis method and column chromatography using solvent benzene-chloroform (10:1).
Antibacterial Activity Test
Taken as a colony of bacteria Stapphylococus aureus using a needle ose conducted aseptically. Inserted into tubes containing 2 ml Mueller-Hinton broth, then incubated for 24 hours at 37°C. Homogenous bacterial suspension that was incubated ready smeared on Mueller-Hinton media surface evenly using a sterile swab. Then affixed to the disk that contains the sample, the standard tetracycline and chloroform were used as controls. Then measured inhibitory substances against bacteria.
Extracts positive triterpenoids and most active antibacterial resumed in the separation and purification process analysis using thin layer chromatography and column chromatography with silica gel G60 stationary phase and a mobile phase of benzene: chloroform (10:1).
Fractions obtained from column chromatography in phytochemical screening and antibacterial activity test. Isolates relatively pure characterization by mass spectrophotometer analysis and HNMR to determine the structure of compounds triterpenoidnya.
Analysis of Nuclear Magnetic Resonance Spectroscopy
F1 isolates compounds were made in liquid form with a concentration of 2-15%, dissolved in CDCl3 and plus a number of TMS as standard. Then characterized using Nuclear Magnetic Resonance spectrophotometer.
Analysis Mass Spectroscopy
Some samples isolates F1 (chloroform fraction) was added to purified through direct inlet ion source in a mass spectrophotometer. The positive ions formed will be separated based on the price of m/z, where each will be recorded as a mass spectrum.
RESULTS AND DISCUSSION
Extraction by maceration bark Phyllanthus niruri using n-hexane was obtained yield of 12.446 g (1.778%). Subsequently extract thick n-hexane in a partition using chloroform yield of 10.94 g (1.563%) to form a green color. Phytochemical screening results using the Liebermann-Burchard reagent showed that the chloroform extract thick bark contains
Phyllanthus niruri positive triterpenoids compound and provides a color change from red to pink-purple-green-blue while the reagent H2SO4 showed positive purple and brown triterpenoids. Further test results antibacterial activity against bacteria aureus Stapphylococus viscous chloroform extract at a concentration of 1000 ppm gives a strong inhibition of 12 mm. It can be concluded that the isolated triterpenoids chloroform extract of the bark of Phyllanthus niruri potential as an antibacterial. Therefore the chloroform extracts followed by purification and separation processes using thin layer chromatography and column chromatography resulted in four fractions as shown in Table 1.
Table 1. Results of chromatographic columns on each fraction chloroform Fraction The Number
of Stains Rf Large Discoloration Information
F1 (1-25) 1 0,411 Brown-red purple +++ (strong color intensity) F2 (26-31) 2 0,516, 0,580 yellowish-brown + (weak color intensity) F3 (32-48) 2 0,659, 0,775 Brown-purple ++ (moderate color intensity) F4 (49-137 1 0,856 light yellow + (weak color intensity)
The test results showed that the antibacterial activity Isolates F1 with a concentration of 1000 ppm could inhibit the growth of bacteria with strong inhibition of 12 mm to Stapphylococus aureus bacteria. The result of antibacterial activity against isolates F1 presented in Table 2.
Table 2. Results of antibacterial activity test isolates of F1 chloroform extract the stem bark of Phyllanthus niruri
No The concentration of the test material
Diameter barriers (mm)
Category Stapphylococus aureus
1 2 3 4 5
F1 (100 ppm) F1 (500 ppm) F1 (1000 ppm) Control (chloroform) Control (tetracyclin 30 µg)
0 6 12
0 25
Weak Moderate Strong weak Very strong
HNMR Spectroscopy Analysis
The results of nuclear magnetic resonance spectroscopy measurements shown that the compounds F1 isolates chloroform extract of the bark of Phyllanthus niruri provide two separate signals. This is due to the molecule containing two different types of proton electronic environment with relative intensity proton ratio 0.119: 0.965 (1: 8). This comparison is interpreted as the ratio of the overall number of protons in a molecule. The presence of singlet peak at δ = 7.337 are characteristic aromatic proton and proton indicates that no other on its neighbors. While peak multiplet at δ = 0.808 to 1.682 were difficult to
interpret whether this peak is derived from one type of proton or power pisahnya not good enough, judging from the number of peaks existing proton neighbors means there are six C-H aliphatic proton/cyclic. Of the price range of chemical shifts indicates that there are protons CH2 and CH3. Doublet peak at δ = 5.8 on the spectrum probably derived from the type of proton olefin C = CH, while the doublet peak δ = 4.6 is likely to come from OH proton.
Figure 2. HNMR spectrum Isolate F1 Compound
Mass Spectroscopy Analysis
The results of the mass spectroscopy measurements provide clues to the fragments occur in the molecular ion can be determined so possible structure and relative molecular mass compounds F1 isolates chloroform extract of the bark of Phyllanthus niruri. Results fragmentation mass spectrum with a retention time of 3.38 minutes and 2.29 minutes isolate compound F1 as a potential antibacterial provides molecular ion M at m/z 424. The other fragment ions can be interpreted in Figure 1 and details are presented in Table 3.
Figure 3. The mass spectrum peak Isolate F1(A)
A
B
The figures show that isolates F1 has a molecular ion carriers with mass number m/z 424 [M+], then experience the release of molecules C3H7 giving m/z 381, followed by ion fragmentation of m/z 341 [M+-C3H4], ion fragment m/z 313 [M+-C2H4]+, fragment ions m/z 287 [M+-C2H2], fragment ions m/z 229 [M+-C3H6O], fragment ions m/z 206 [M-C16H26]+, ion fragment m/z 151 [M+-C6H6], fragment ions m/z 109 [M+-C3H6], fragment ions m/z 95 [M+- CH2] +, fragment ions m/z 81 [M+- CH2], fragment ion m/z 69 [M+-C], fragment ion m/z 55 [M+-CH2] and fragment ion m/z 43 [M+-C].
The results of the analysis of both mass spectroscopy and nuclear magnetic resonance spectroscopy, it can be presumed that the compound isolates F1 (chloroform fraction) bark of Phyllanthus niruri positive for triterpenoids steroid derivate of stigmasterol compounds with the following structure:
OH HO
14-stigmast-5,7,24(28)-trien-3,15-diol
CONCLUSIONS AND SUGGESTIONS
1. From the analysis of mass spectroscopy and HNMR and phytochemical screening reagents Liebermann-Burchad, then isolate the compounds F1 chloroform fraction bark Phyllanthus niruri a class of compounds derived triterpenoids steroid stigmasterol with m/z = 424, molecular formula C29H44O2.
2. Compound F1 isolates chloroform extract can inhibit the growth of bacteria in a concentration of 1000 ppm with a strong inhibition of 12 mm for Staphylococcus aureus ATCC®25293.
3. In connection with the discovery of one of the compounds derived triterpenoids steroid of chloroform fraction of the bark of Phyllanthus niruri which have antibacterial activity, it needs further study to identify the active isolates using 13CNMR analysis, so as to set the structure of the proposal of the F1 isolates.
AKNOWLEDGMENTS
The author would like to thank all those who have helped and supported the implementation of this study. Mainly To the Head of Unit Plant Conservation Center Eka Karya Botanical Garden Bedugul Bali who have helped in the determination of plant Phyllanthus niruri
REFERENCES
1. Anonymous., 2007. Eclipta prostata, dumenat. smbh. Univ_paris 13.fr/med/tradmed 200 1.htm, 4 januari 2007.
2. Bigham, A.K., Munro, A.T., Rizzacasa, M.A., Roy, M and Browne, R., 2003.
Divinatorins A-c, New Neoclero Diterpenoid from the controlled sage silva divinorum, Melbourn University, Victoria, Australia.
3. Brown, G.D., 1998. The Biosynthesis of Steroids and Triterpenoids, Nat. Prod. Rep., 15:
653-95.
4. Grayson, D. H., 2000. Monoterpenoid, University Chemical Laboratory, Trinity College, Dublin 2, Ireland.
5. Hnatyszyn, O and Ferro, G., 1985. Phylanthol from Phyllanthus sellowianos in Planta- Medica, p. 467.
6. Kardinan, A. dan Kusuma, F. R., 2004. Meniran Penambah Daya Tahan Tubuh Alami, Agromedia Pustaka, Jakarta.
7. Lim, S. Y., Bauermeister, A., Kjonaas, R. A. and Gosh, S. K., 2006. Phytol-Based Novel Adjuvants in Vaccine Formulation: 2. Assessment of Efficacy in the Induction of Protective Immune Responses to Lethal Bacterial Infections in Mice, Departement of Life Science, Indiana State University, Terre Haute, IN 47809, USA.
8. Matsunaga, S., et al., 1993. Triterpenes from Phyllanthus watsonii in Phytochemistry, 27(11): 165-70.
9. Nigom, S.K., et al., 1992. Triterpenoidal Saponin from Madhica butyracea in Phytochemistry, 31(9): 3169-72.
10. Parekh J, Karathia N, Chanda S. Evaluation of antibacterial activity and phytochemical analysis of Bauhinia variegata L. bark. African Journal of Biomedical Research, 2006; 9:
53-56.
11. Pavia D.L., Lampman, G.M., Kriz, G.S., dan Vyvian, J.R., 2007. Introduction to Spectroscopy, Sauders College, Philadelphia.
12. Tanaka, R and Matsunaga, S., 1988. Triterpene Diols and Other Constituen from Phyllanthus flexuosus in Phytochemistry, 18(7): 2273-77.
