Folia Medica Indonesiana Vol. 45 No.

1 January - March 2009 : 28-31

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

ABSTRACT

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--L-arabinopyranosyl-4,5,7-trihydroxyflavone or 6,8-di-C--L-arabinosyl-apignine. Minor compounds is 6-C-
-L-arabinopyranosyl-4,5,7-trihydroxy-8-C--D-ylopiranosyl-flavone or 6-C--L-arabinosyl-8-C--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,
Surabaya, Indonesia

INTRODUCTION used HPLC method connected with the diode array

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), -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 mens spermatozoa
penetration in vitro (Prajogo et al. 1998). LD50 of
methanol extract was given orally didnt 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
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

28
 Isolation of Male Antifertility Compound in N-Butanol Fraction of Justicia Gendarussa Burm. F. Leaves (Bambang Prajogo EW et al.)

Extraction used by percolation method from dry leaves

powder (4 kg) with n-Hexane, Methanol, Chloroform
and n-Buthanol successively (Figure 1).

Figure 2: LC-UV Chromatogram major and minor

compound
Figure 1: Scheme of flavanoid extraction from Justicia
gendarussa The major and minor compounds were analyzed with
1
H-13C-NMR. For the structure elucidation from the
For isolation, the n-Buthanol fraction was separated data that were obtained such as Chemical Carbon
with HPLC preparative Shimadzu SCL-8A used by movements, Proton, HBMC, HSQC-TOCSY and the
Universal LC open column with isocratic system others compared with Flavonoid 1H and 13C-NMR data,
Methanol: Water = 30:70. It also could be obtained from 6,8-di-C--arabinosyl-apigenin (Prajogo et al. 1998;
Methanol fraction which has been separated with Diaion Prajogo et al. 2002), glycoside 13C_NMR data in C and
open column before, continued with gradient system flavonoid O-glycoside binding (Reny et al. 1997),
Methanol: Water MPLC and RP18 as immobile phase. estimate moving chemical structure data used
ChemNMR 1H-13C in 5,7,4 trihidroxy flavone. The
results were shown in table 1 and 2.
RESULTS AND DISCUSSION
Table 1: Major compound spectrum from 1H and 13
C-
Fist detection from n-Butanol fraction used LC-MS NMR correlated with 1H-1H and 1H-13C
showed 12 flavanoid compounds with retention time
from 38 until 50 minute which have same molecule C atom Corelation
weight (534-535). Purification was held by using HPLC Number 1
H-NMR 13
C-NMR between 1H-1H
Preparative from n-Butanol obtained one major or 1H-13C
compound, and then with MPLC from Methanol
2 - 164.8
fraction obtained major and minor compounds.
3 6.94s 102.9 Hmbc : 183.0,
164.8, 121.8,
104.2
4 - 183.0
4a-10 - 104.2
5 5- 159.2
OH13.90
s

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Folia Medica Indonesiana Vol. 45 No. 1 January - March 2009 : 28-31

6 - 108.8 4 161.3
7 7-OH9.60 162.2 1 4.82 74.4* Hsqctocsy :
bs 79.0 74.4
8 - 105.5 2 4.07 70.9
8a - 155.6 3 3.37 79.0 Hsqctocsy :
1 - 121.8 70.6
2 8.23 brs 130.1 4 3.67 70.2 Hsqctocsy :
3 7.03 d 8.2 116.7 Hmbc : 161.9, 79.0
Hz 130.1, 121.8, 5 3.28-3.30 70.6 Hsqctocsy :
116.8 3.98-4.00 79.0
4 - 161.9
1 4.90 brs 75.3
2 3.91- 69.3 CONCLUSIONS
4.01*m
3 3.54- 75.8 The major compound of flavonoids in Justicia
3.60*m gendarussa Burm. f. was: 6,8-di-C--L-arabinocyl-
4 3.91- 69.7 4,5,7 trihidroxy-flavon or 6,8-di-C--L-
4.01*m arabinocylapigenin and names purposed for this
5 ~3.71- 71.7 compound was gendarusin A
3.73*m
~3.91- OH

4.01*m OH
1 4.79 brs 74.8 O
OH
2 3.91- 69.3
4.01*m HO
HO O
3 3.54- 75.8
3.60*m
4 3.91- 69.7
4.01*m HO O OH O
5 3.71- 70.9 OH

3.73*m HO

3.91- Gendarusin A

4.01*m Figure 3: Molecular structure of gendarusin A

Table 2: Minor compound spectrum from 1H and 13

C- One of the minor compounds in Justicia endarussa
NMR correlated with 1H-1H and 1H-13C Burm. f. was -C--L-arabinopyranocyl-4,5,7-
trihidroxy-8-C--D-cylopyranocylflavone or 6-C--L-
C atom 1
H-NMR 13
C- Correlation arabinocyl-8-C--D-ylocilapigenin and names purposed
Number NMR between for this compound was gendarusin B.
1
H-1H or HO
1
H-13C OH

2 163.9
3 6.93 102.7 103.3 or .4
4 182.3 HO OH
O
4a 103.4
5 5- 158.4* HO O
OH13.78
s
6 108.3 HO O OH O
7 7-OH9.4 161.4 OH
bs HO
8 104.9 Gendarusin B

8a 155.0 Figure 4: Molecular structure of gendarusin B

1 121.5
2 8.04 d 128.6
3 7.04 d 116.0

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 Isolation of Male Antifertility Compound in N-Butanol Fraction of Justicia Gendarussa Burm. F. Leaves (Bambang Prajogo EW et al.)
ACKNOWLEDGMENT
The author was grateful to IRR UNIL Director,
Switzerland for the chance to done this research,
generous assistance and discussion.
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