NPC
2011
Vol. 6
No. 3
349 - 352
Natural Product Communications
The Quaternary Indole Alkaloids from Two Sumatran
Lerchea species
Dayar Arbaina, Nova Syafnia, Friardia, Deddi Prima Putraa, Ismiarni Komalab,
Kentaro Yamaguchic and Yoshinori Asakawab
a
Faculty of Pharmacy, Andalas University, Kampus Limau Manis, Padang 25163, West Sumatra,
Indonesia
b
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
c
Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Sanuki-city,
Kagawa 769-2193, Japan
d.arbain48@yahoo.com
Received: January 16th, 2011; Accepted: February 7th, 2011
The Sumatran forest plants Lerchea cf. bracteata and L. parviflora were found to contain alkaloids and their extract showed siginificant
activity toward some testing pathogenic microbes. Isolation work on L.cf. bracteata yielded known quaternary alkaloid N(b)-methylantirhine
(2) while L. parviflora gave 5,6-dihydroflavopereirine (3).
Keywords: Lerchea cf. bracteata, Lerchea parviflora, N(b)-methylantirhine, 5,6-dihydroflavopereirine.
Lerchea spp. (Rubiaceae) is an endemic and endangered
plant of Sumatra, Indonesia. There are 19 species of this
genus so far reported exist in the world and only 8 of them
found in Indonesia, 2 species in Java and 6 species in
Sumatra [1]. One of these, Lerchea bracteata Val. a small
tree attaining 2 m was found during a field trip in the late
1980 in the Anai Reserved forest, West Sumatra. Later on,
the only alkaloid detected on this plant was isolated and
identified as the new quaternary alkaloid named lercheine
(1) [2].
As a continuation of our study on the Sumatran
Rubiaceous plants [3], during an inventory of Sumatran
forest plants in Kerinci Seblat National Park (TNKS) in
Sumatra in 2006, special attention was given toward the
presence of this family. In this way, two alkaloid-bearing
plants Lerchea parviflora North. and another species
which looked similar to that of Lerchea bracteata [2]
previously investigated but could not identified completely
were found. This second species, looked similar except
shorter as well as smaller and its methanolic extract also
showed totally different TLC patterns compared to that of
Lerchea bracteata [2]. This second Lerchea species was
temporarily identified as Lerchea cf. bracteata.
No traditional value has been recorded so far on these
two species; however, people in the region occasionally
used it as poultice for pain relieving or treatment of skin
infection. During the preliminary work, the ethanolic
extract of these two plants showed strong inhibition toward
5
6
H
21
9
N
2
N
20
3
15
N
H
12
N
H
H
16
HO
2
1
OH
H
14
H
19
17
18
the growth of some common pathogenic microbes
Staphylococcus aureus (Sa, ATTC: 6538), Bacillus subtilis
(Bs, clinically isolated), Pseudomonas aeruginosa (Pa,
ATTC: 1542), Klebsiella (Kl, clinically isolated),
Micrococcus luteus (Ml, ATCC: 9342), Escherichia coli
(Ec, ATTC:8739), Pseudomonas aeruginosa (Pa, ATTC:
1542), Staphylococcus epidermidis (Se, ATTC) but did not
showed activity towards the testing fungi Trichophyton
mentagrophytes (Tm, ATTC: 5431) and Candida albicans
(Ca, ATTC: 10231) [4]. Inhibition Diameter (ID) and
Minimum Inhibition Concentration (MIC) of extracts and
fractions are as shown in Table 1 and Table 2.
Lerchea cf. bracteata Val. is a small woody shrub with the
height of up to 1m. Extraction and isolation work on this
species using air dried material was unsuccesful, only a
trace of crude polar alkaloids were found and no sign of
the presence of alkaloids in the semi-polar fraction.
Repeated extraction by using the fresh aerial parts then
column chromtographed in the usual way led to isolation
of the only detected major quaternary indole alkaloid
350 Natural Product Communications Vol. 6 (3) 2011
Arbain et al.
Tabel 1: Growth Inhibition Assay of MeOH extracts of Lerchea cf. bracteata and L. parviflora and their fractions.
__________________________________________________________________________________________________________________________________________________________________________
Species
Extract/fraction
Bs
Diameter of inhibition (DI) zone (mm, 5 mg/disk)
Ec
Pa
Kl
Sa
Ml
Tm
Ca
_____________________________________________________________________________________________________________________________________________________________________________
L. parviflora MeOH`
Hexane
EtOAc
n-BuOH
L. bracteata MeOH
Hexane
EtOAc
n-BuOH
6.0
5.0
7.5
8.5
9.5
6.0
8.5
6.0
9.5
-
5.0
5.0
10.5
8.5
8.5
6.5
10.5
7.5
7.5
14.5
7.5
14.0
6.0
9.5
-
6.5
6.0
9.0
15.5
8.5
7.5
11.5
6.5
12.0
15.5
7.5
11.5
6.5
8.5
-
-
-
_
_______________________________________________________________________________________________________________________________________________________________________________
Table 2: Minimum Inhibitory Concentration (MIC) of MeOH extract of
Lerchea spp and its fractions.
Species
Extract/
MIC (μg/mL)
Pa
Ec
Se
Sa
MeOH
Hexane
EtOAc
n-BuOH
MeOH
5
2.5
5
10
20
20
20
5
20
20
20
2.5
2.5
20
20
20
2.5
10
20
20
Hexane
EtOAc
n-BuOH
10
20
20
20
5
20
5
5
20
20
10
20
fraction
L. parvifloraa
L.cf. bracteata
Bs (Bacillus subtilis), Ec (Escherichia coli), Pa (Pseudomonas
aeruginosa), Kl (Klebsiella sp), Sa (Staphylococcus aureus), Ml
(Micrococcus luteus), and fungi; Tm (Thrichophyton mentagrophytes)
and Ca (Candida albicans). Se
Table 3: NMR data of N(b)-methylantirhine (2) (CD3OD, 500 MHz).
_____________________________________________________________________________________________________________________
No.C δC(ppm)
δH (ppm), multipilcity, J (Hz)
___________________________________________________________ __________________________________________________________
2.
3.
5.
128.32
64.59
64.77
6.
18.84
7.
8.
9.
10.
11.
12.
13
14.
105.77
127.37
118.53
123.71
120..98
112.70
138. 48
24.48
15.
16.
17.
31.36
46. 82
60.34
18.
118.53
19.
20
140.26
46. 82
21.
22.
64.77
37.85
5.17, d(br), J3a-14b 12.9
3.80, dd, J 5α− 5β12.3, J 5α−6α 6.5
3.94, ddd, J 5β−5α 12.3, J 5β−6β 12.3, J 5β−6α 6.0
3.28, m, J 6α−6β 17.0, J 6α−5β 12.3, J 6α−5α 12.0
3.18, dd, J 6β−6α 17.0, J 6β−5β 6.0
7.40, ddd, J 9-12 0.9, J 9-111.9, J 9-10 8.2
7.18, ddd, J 10-9 0.9, J 10-11 7.0, J 10-9 8.2
7.09, dd, J 11-9 0.9, J 11-10 7.0, J 11-12 7.9
7.50, ddd, J 12-9 0.9, J 12-10 1.9, J 12-11 7.9
2.79, d (br), J 14a-14b 13.6
2.21, ddd, J 14β-14α10.4, J H14 β -H3β 13.6, J H14 β -H15 β 8.2
2.19, m
2.67, m 1H
3.61, ddd, J gem 13.3, J 3.4, 13.3
3.51, ddd, J gem 13.3, J 2.0, 5.7
5.28, ddd, J trans 16.2 J gem 1.7, J 18-20 0.7
5.24, ddd, J cis 10,0, J gem 1.7, J 18-20 0.7
5.78, ddd, J trans 16.2, J cis 10.0, J 19-20 9.6
2.01, d (br), J 16α -16β 16.0
2.37, m, J16β -16α 16.0, J 16β -17α 10.0, J 16β -17β 4.9
3.77, d, 2H, H17, J 4.9
3.30, s, Me
_____________________________________________________________________________________________________________________
from n-butanolic fraction in a low yield which was
identified as N(b)-methylantirhine (2). Spectroscopic data
as well as optical rotation of this compound also looked
very similar to that of lercheine [2] except that instead of
detecting an ethyl functional group like in lercheine, in
this compound a vinyl group was clearly observed.
Table 4: NMR data of 5,6-dihydroflavopereirine (3) (CD3OD,500 MHz).
_____________________________________________________________________________________________________________________
No.C δC
δH, multipilcity, coupling
(200 MHz) [12] J (Hz)
______________________________________________________________________________________________________________________
2.
3.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
18.
19.
20.
21.
124.98
142.03
56.41
19.27
117.66
125.16
112.37
126.53
121.09
120.23
140.25
120.51
145.21
13.64
26.36
140.43
143.25
4.88, 2H, m
3.42, m
7.46, 1H, dd, J 8.0,, 0.8
7.71, 1H, ddd, J 8.3, 8.0, 0.8
7.36, 1H, ddd, J 8.3, 8.0, 0.8
7.67, 1H, dd, J 8.3,0.8
8.07, 1H, d, J 8.0 Hz
8.36, 1H, dd, J 8.0, 2.0
1.38, 3H, t, J 7.6
2.85, 2H, q, J 7.6
2.85, 2H, q, J 7.6
4.98, 2H, m
7.82, d, J 8
7.26, t, J 8
7.45, t, J 8
7.64, d, J 8
8.33, J 8
1.38, t, J 7.5
1.38, t, J 7.5
2.88, q, J 7.5
9.01, 1 H, s
___________________________________________________________________________________________________________________
Unfortunately the COSY, HMBC, HSQC correlation did
not clearly showed the exact correlation of position of
protons and carbon atoms in the molecule. Surprisingly,
single crystal X-ray diffraction analysis revealed that the
skeleton of this compound is different from lercheine (1)
[2] where H3 was in the α position while the vinyl group
was not attached to the C20 but to C16. This compound was
previously found in Hunteria eburnea Pichon [5],
Strychnos camptoneura (Loganiaceae) [6], Amsonia
elliptica (Apocynaceae) [7], Strychnos usambarensis [8]
and from Alstonia angustifolia [9]. Compound (2) showed
moderate activity against Plasmodium falciparum (strain
K1) in vitro [10] while antirhine itself was first found in
Antirhea putaminosa (F. Muell.) Bail. [11].
Another species L. parviflora Val. [12] looked totally
different from L. cf. bracteta. The height attained 60 -70
cm with thinner and greener leaves. Extraction and
fractionation work on this plant in the usual way gave a
brownish gum. As above, the alkaloids containing
n-butanolic fraction was chromatographed on silica gel but
no pure compunds obtained. Column chromatographic
separation on silica gel using an increasing amount of
conc. aquaeus ammonia in n-butanol gave major fraction
of which showed one spot on TLC. Based on its
spectroscopic data particularly 1H NMR COSY, HSQC,
HMBC and NOESY this compound was identified as
5,6-dihydroflavopereirine (3) (see the NMR assignment,
Table 3). Preparative TLC of more polar fraction gave
more of 3 and another more polar one with a slight
inadmixture with 3.
Alkaloids from Lerchea species
Natural Product Communications Vol. 6 (3) 2011 351
Plant material: L. cf. bracteta (DA-RT 2074) was
collected in Kerinci Seblat National Park (TNKS) near
Kambang, Pesisir Selatan District, West Sumatra, while L.
parviflora (DA-RT 6159) was collected in Teluk Kabung
Forest near the City of Padang, West Sumatra, Indonesia.
Both herbarium specimens were identified by Mr. Rusjdi
Tamin and lodged at Herbarium Andalas University
(ANDA).
Figure 1: ORTEP diagram of N(b)methylantirhine (2).
Surprisingly the mass and NMR spectra of these two
compounds in deuterated methanol were identical. It was
concluded that these two compounds were a quaternary
indole alkaloids in the form of (3) and its zwitter-ionic
form (3a) which was identical to that of the one
synthesized by Lounasmaa et al. which they named
5,6-dihydroflavopereirine (3) following the preferred
biogenetic numbering system of Corynanthe alkaloids as in
geissoschizine (4) [13]. Compound (3) was previously
isolated from Strychnos usambarensis Gilg [14] originally
named 6,7-dihydroflavopereirine as it was considered as a
quinolizine derivative alkaloid (3b) [14].
6
9
10
7
5
7
8
1
12
13
N
H
OH
Cl
N
11
3
21
4
20
2
14
N
15
19
N
H
6
N
N
H
18
3
3a
6
9
5
10
8
7
N
11
12
13
3b
1
4
21
20
3
2
N
H H
14
18
19
15
Extraction and isolation: Fresh aerial parts of L. cf.
bracteta (2.5 Kg) was macerated with MeOH for 2 days.
After separation of solvent the process was repeated twice
more. The combined methanolic extracts were evapolated
in vacuo to give 55.4 g thick brownish gum. This thick
extract (55 g) was disssolved in MeOH (500 mL) and
water (500 mL) was added then fractionated in turn with
hexane (3 x 1 L), EtOAc ( 3 x 1 l) and n-butanol (4 x 1 L)
respectively and evaporated in vacuo to give fractions of
hexane (11.0 g), EtOAc (6.4 g) and n-butanol (18.7 g) and
water (19 g) respectively. The n-butanolic fraction
(12.5 g) was preadsorbed on SiO2 (12.5 g) then eluted with
n-butanol (1 l) then with the organic layer of admixture of
n-butanol-AcOH-water (4:1:5) to give only one series of
fractions which gave (+)-ve Dragendorff test. The
combined fractions were evaporated and recrystallized
from MeOH to give colorless plates (115 mg) of N(b)methylantirhine (2).
MP: 164-166ºC.
[α]D: +172 (c 0.013, MeOH).
IR (KBr): 2963, 1643, 1575, 1476, 1458, 1437,1323, 1262,
1199, 1171, 1118, 972, 851, 809, 776, 733, 619 cm-1.
UV λmax (ΜeΟΗ) nm (log ε): 231 (2.19), 229 (1.91), 312
(1.60).
1
H NMR (500 MHz) and 13C (125 MHz): Table 3 (based
on COSY, HSQC, HMBC and NOE experiments).
MS (CI, 70 eV): m/z (%) = 311 (CI,M+1, C20H26N2O with
loss of HCl by thermal Hoffman elimination from
C20H27ClN2O).
16
17
4
H 3COOC
OH
Experimental
General: Melting points were measured on a Sybron
Thermolyne Melting Point Apparatus MP-12615 and are
uncorrected, optical rotations were aquired on JASCO P
1030 polarimeter, FT-IR spectra on Perkin Elmer FT-IR
Spectrum One spectrophotometer. UV spectra on
Shimadzu Spectrophotometer UV-VIS Pharmaspec 1700,
the 1H and 13C NMR spectra were measured using Bruker500 (500 MHz for 1H and 125 MHz for 13C) instruments
with chemical shift values expressed in δ (ppm) downfiled
from TMS as internal standard. Mass spectra were
obtained on a JEOL JMS 700 instrument. TLC was carried
out using silica gel 60F254 (Merck) and visualized under
UV light (254 nm) or sprayed with Dragendorff reagent.
Column chromatography was performed on silica gel 60
(0.063-0.200 mm) (Merck). Preparative TLC were done
by using silica gel PF254 (Merck).
Fresh aerial parts of L. parviflora (1.5 kg) was treated as
above to give fractions of hexane (4.5 g), EtOAc (12.5 g),
and BuOH (16.1 g). The n-butanolic alkaloid containing
fraction (11 g) was chromatographed on silica gel as above
but no pure compounds were isolated. The work was
repeated by using an increasing amount of conc. aquaeus
ammonia solution in n-butanol as a mobile phase to give
two major fractions of alkaloids. The less polar factions
were combined and recrystallized from methanol to give
210 mg colorless feathery needles which was identified as
6,7-dihydroflavopereirine (3).
MP: 246ºC (dec.).
IR (KBr): 3022, 1638, 1589, 1557, 1432, 1313, 1286,
1242 cm-1.
UV λmax (ΜeΟΗ) nm (log ε): 247 (2.52), 314 (1.82), 392
(1.79).
1
H NMR (500 MHz) and 13C (125 MHz): Table 4 (based
on COSY, HSQC, HMBC and NOE experiments).
352 Natural Product Communications Vol. 6 (3) 2011
MS (CI, 70 eV): m/z (%) = 248 (M+1), (C17H15N2 with loss
of HCl by thermal Hoffman elimination from C17H16ClN2).
The more polar fractions was chromatographed on
preparative TLC eluted with 10% aqueous NH4OH in
n-butanol which after recrystallization yielded 90 mg of
(3) and 110 mg of more polar quaternary indole alkaloid
(3b) with a slight inadmixture with 3.
Antimicrobial test: Bacterial cultures, namely Echerichia
coli (Ec, ATTC:8739), Pseudomonas aeruginosa (Pa,
ATTC: 1542), Staphylococcus aureus (Sa, ATTC: 6538),
Micrococcus luteus (Ml, ATCC: 9342), Bacillus subtilis
(Bs, clinically isolated), Klebsiella (Kl, clinical isolate),
and fungi; Candida albicans (Ca, ATTC: 10231) and
Thrichophyton mentagrophytes (Tm, ATTC: 5431) were
obtained from Biofarma-Bandung Indonesia and grown in
Arbain et al.
nutrient agar media at 37ºC for bacteria and Sabaroud
dextrose agar at room temperature for fungi. Each
bacterium and fungus strain was transferred from stored
media to fresh media 24 hour before being used as well as
for a preculture on nutrient broth media. Assay for
antimicrobial activity was performed using agar diffusion
methods. A paper disk 5 mm in diameter (Whatman No.3)
was used as reservoir [16]. MIC was determined using
dilution method in 96-well microtiter plate and nutrient
broth used as media [17].
Acknowledgment - Competency Grant from the
Directorate General of Higher Education, the Ministry of
Education of the Republic of Indonesia (to DA) and
Research grant from International Foundation for Science
(IFS) and from Organization for Prevention of Chemical
Weapons (OPCW) (to DPP) are gratefully acknowledged.
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