Isolation Of Male Antifertility Compound In N-Butanol Fraction Of Justicia Gendarussa Burm. F. Leaves
Bambang Prajogo EW1, David Guliet2, Ferreira Queiroz E2, John-Luc Wolfender2,
Noor Cholies Z1, Aucky H3, K Hostettmann2
1Department of Nature Product, Faculty of Pharmacy Airlangga University, Surabaya, Indonesia 2Institute of Pharmacognosy and Phytochemistry, University of Lausanne, Switzerland 3Deparment of Biomedic, Faculty of Medicine, Airlangga University, Surabaya, Indonesia
The isolation has been done on major compound of Justicia gendarussa Burm.f. (Acanthaceae) leaves, it used as male contraceptive in Papua as ethno medicine. Preclinical study showed activity as a male contraceptive. First information, J. gendarussa was known containing flavonoid beside alkaloid, steroid and mineral. Extraction used by percolation method with n-hexane, methanol, chloroform and n-butanol successively. Isolation and separation were done by column chromatography (silica, diaion and sephadex), MPLC and preparative HPLC. For the analyzing purpose, TLC, LC-UV and LC-MS were used. For the structure elucidation, 1H-13C NMR Inova 500 Mhz was used. Major compounds is 6,8- di-C-α-L-arabinopyranosyl-4’,5,7-trihydroxyflavone or 6,8-di-C-α-L-arabinosyl-apigénine. Minor compounds is 6-C- α-L-arabinopyranosyl-4’,5,7-trihydroxy-8-C-β-D-ylopiranosyl-flavone or 6-C-α-L-arabinosyl-8-C-β-D-xylosyl- apigenine. Nevertheless that compounds were not the novel compound, but this is for the first time known in J. gendarussa, so names proposed for both compound were gendarusin A and gendarusin B.
Keywords: Justicia gendarussa, flavone, apegenine, male contraceptive
Correspondence: Bambang Prajogo EW, Department of Nature Product, Faculty of Pharmacy Airlangga University,
From the ethnomedicine study, was known that Justicia gendarussa Burm.f. (acanthaceae) have been used by society of Papua as male contraception (Moeso and Agus 1985). The other names of this plant is Gendarussa vulgaris Nees and Gandarusa as local name,could growth at elevated place 0-500m above the sea. Justicia gendarussa containing Flavonoid, sterol (Prajogo et al. 1997), β-cytosterol, lupeol, friedeline (Chakravarty and Dastidar 1982), iridoid, triterpene, coumarine, alkaloid (Hegnauer 1964), and simple amine aromatic. Antifertility bioactivity study has been done, in the decreased motility and viability of rabbits, mice and men spermatozoa in vitro (Hartati et al. 1997), decreased disperse ability of cumulus oophorus in human in vitro (Sri et al. 1997; Reny et al. 1997) could decreased testosterone concentration in mice serum (Prajogo et al. 1994), Pursue men’s spermatozoa penetration in vitro (Prajogo et al. 1998). LD50 of methanol extract was given orally didn’t influence renal, lever, intestines and histopathology of mice (Prajogo et al. 1998). Beside that, it could decrease Hyaluronidase activity of mice spermatozoa.
One of Flavanoid isolation containing Flavonol 3- glycoside (Prajogo et al. 1988) and methanol extract used HPLC method connected with the diode array containing 8 Flavonoid compounds (Prajogo et al. 1997). Continuation research from extraction and isolation result obtain sediment from n-Buthanol (1,2%) and flavanoid total concentration from leaves simplicia 0,18% (Prajogo et al. 2002). The aim of this research was to show which Flavanoid compound in Justicia gendarussa which have antifertility activity in men.
MATERIALS AND METHODS
Leaves sample was taken from plants which grow at Pacet, Mojokerto. The herbarium was stored in The Departement of Natural Product, Faculty of Pharmacy Airlangga University. Chemical materials used were n- Hexane, n-Buthanol, Methanol, Chloroform, Silica gel GF 254 (Merck), Silice 60 A.C.C (SDS. France), Methanol pro PLC (Merck), and Daion (Mitsubhisi). Instruments used in this study were Rotavapor BUCHI R-114, Rotavapor BUCHI R-153, HPLC-MS TSQ 700 Finigan MAT, HPLC HP 1090, HPLC Analitic Shimadzu LC –10AD, HPLC Preparative Shimadzu SCL-8A, MPLC BUCHI Gradient former B-667, 1H and 13 C_NMR Varian Unity Inova 500 Mhz Extraction used by percolation method from dry leaves powder (4 kg) with n-Hexane, Methanol, Chloroform and n-Buthanol successively (Figure 1).
For isolation, the n-Buthanol fraction was separated with HPLC preparative Shimadzu SCL-8A used by Universal LC open column with isocratic system Methanol: Water = 30:70. It also could be obtained from Methanol fraction which has been separated with Diaion open column before, continued with gradient system Methanol: Water MPLC and RP18 as immobile phase.
RESULTS AND DISCUSSION
Fist detection from n-Butanol fraction used LC-MS showed 12 flavanoid compounds with retention time from 38 until 50 minute which have same molecule weight (534-535). Purification was held by using HPLC Preparative from n-Butanol obtained one major compound, and then with MPLC from Methanol fraction obtained major and minor compounds.
The major and minor compounds were analyzed with 1H-13C-NMR. For the structure elucidation from the data that were obtained such as Chemical Carbon movements, Proton, HBMC, HSQC-TOCSY and the others compared with Flavonoid 1H and 13C-NMR data, 6,8-di-C-α-arabinosyl-apigenin (Prajogo et al. 1998; Prajogo et al. 2002), glycoside 13C_NMR data in C and flavonoid O-glycoside binding (Reny et al. 1997), estimate moving chemical structure data used ChemNMR 1H-13C in 5,7,4’ trihidroxy flavone. The results were shown in table 1 and 2.
Table 1: Major compound spectrum from 1H and 13C- NMR correlated with 1H-1H and 1H-13C
Table 2: Minor compound spectrum from 1H and 13C- NMR correlated with 1H-1H and 1H-13C
The major compound of flavonoids in Justicia gendarussa Burm. f. was: 6,8-di-C-α-L-arabinocyl- 4’,5,7 trihidroxy-flavon or 6,8-di-C-α-L- arabinocylapigenin and names purposed for this compound was gendarusin A
One of the minor compounds in Justicia endarussa Burm. f. was -C-α-L-arabinopyranocyl-4’,5,7- trihidroxy-8-C-β-D-cylopyranocylflavone or 6-C-α-L- arabinocyl-8-C-β-D-ylocilapigenin and names purposed for this compound was gendarusin B.
The author was grateful to IRR UNIL Director, Switzerland for the chance to done this research, generous assistance and discussion.
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