GC-MS of Crinum latifolium L. alkaloids

Abstract

A GC-MS analysis of underivatized alkaloids from leaves of Crinum latifolium was performed. From the identified 15 alkaloids, 9 were found for the first time in this plant. Almost all alkaloids belonged to the crinane type. Substantial changes in the methylation and oxidation pattern of the alkaloids at and after flowering were observed.

Full text

GC-MS of Crinum latifolium L. Alkaloids
Nguyen Thi Ngoc Tram
a
, Maya Mitova
b
, Vassya Bankova
b
, Nedyalka Handjieva
b
and Simeon S. Popov
b,
*
a
Vietnam Pharmaceutical Corporation, Laboratory for Chemistry and Technology of
Natural Substances, 24 Nguyen Thi Nghia Str., Dist.1, Ho Chi Min City, Vietnam
b
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of
Sciences, 1113 Sofia, Bulgaria. Fax: ++3 59-700-225. E-mail: simpopov@orgchm.bas.bg
* Author for correspondence and reprint request
Crinum latifolium L., Amaryllidaceae, Alkaloids
Z. Naturforsch. 57 c, 239Ð242 (2002); received October 2/November 29, 2001
A GC-MS analysis of underivatized alkaloids from leaves of Crinum latifolium was per-
formed. From the identified 15 alkaloids, 9 were found for the first time in this plant. Almost
all alkaloids belonged to the crinane type. Substantial changes in the methylation and oxida-
tion pattern of the alkaloids at and after flowering were observed.
Introduction
The plants of the genus Crinum (Amaryllida-
ceae) are used in Asian folk and traditional medi-
cine as rubefacient, tonic and for treatment of al-
lergic disorders and tumor diseases (Ghosal et al.,
1985). These activities are attributed to the pres-
ence of Amaryllidaceae alkaloids known to pos-
sess moderate antitumor and immunostimulating
activities (Ghosal, Saini, & Razdan 1985). Aque-
ous extracts of Crinum latifolium L. leaves are
used in Vietnamese folk medicine as an anticancer
remedy (Tram et al., 1999). Recently, aqueous ex-
tracts from C. latifolium leaves from Vietnam
showed in vitro and in vivo T-lymphocyte activa-
tion (Tram, et al., 1999) and retarded growth of
chemically induced tumors (sarcomas) in rats
(Tram et al., 2000).
Till now, the chemical investigations on C. lati-
folium alkaloids were concentrated mainly on
bulbs (Ghosal and Singh, 1986; Ghosal et al., 1984;
Ghosal et al., 1983). The present paper deals with
the GC-MS analysis of the alkaloid fraction from
C. latifolium leaves (water extract). The leaves
were investigated at and after flowering, because
significant changes in the alkaloid content of C.
latifolium during different stages of plant growth
have been observed (Ghosal et al., 1985).
0939Ð5075/2002/0300Ð0239 $ 06.00 ”2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com ·
D
Experimental
Plant material
Leaves from Crinum latifolium L. at and after
flowering were collected in April and July respec-
tively, at Dist. Go Vap, Ho Chi Min City, Vietnam.
A voucher specimen is kept in the Nguyen Thi
Ngoc Tkam Herbarium. The identity of the plant
was confirmed by Prof. Dr. Sc. Tkan Cong Khanh,
Department of Botany ÐHanoi College of Phar-
macy, Center for Research and Development of
Ethnomedicinal Plants (CREDEP).
Isolation of the alkaloid fractions
250 ml boiling water was added to ground C. lat-
ifolium leaves (30 g), after 30 min the extract was
filtered and acidified to pH 4 with acetic acid. The
acidic solution was extracted successively with
light petroleum and chloroform. The acidic aque-
ous phase was made alkaline (pH 9) with 25%
aqueous ammonia. It was extracted with chloro-
form (3¥). The chloroform extract (0.02 g, the
same at and after flowering) was subjected to GC-
MS investigation.
GC-MS analysis
Total alkaloids were investigated by GC/MS on
a Hewlett Packard gas chromatograph 5890 Series
II Plus linked to Hewlett Packard 5972 mass spec-

240 N. T. N. Tram et al. · GC-MS of Crinum latifolium L. Alkaloids
trometer system equipped with a 30 m long,
0.25 mm id, 0.25 µm film thickness HP1-MS capil-
lary column. The temperature was programmed
from 150 ∞C to 270 ∞C at a rate of 5 ∞C.min
Ð1
with
a 10 min hold. Helium was used as a carrier gas
with a constant flow at 0.9 ml.min
Ð1
. The ioniza-
tion voltage was 70 eV.
Identification of compounds
The alkaloid identification was performed by
comparisons of RT and mass spectra with authen-
tic samples. When such samples were not available
tentative structures were proposed on the basis of
the mass spectral fragmentation.
Results and Discussion
We subjected to GC-MS analysis the underivat-
ized alkaloid mixture, encouraged by the excellent
results of Kreh et al. (1995). These authors applied
for the first time GC-MS to underivatized Amaryl-
lidaceae alkaloids (from Narcissus pseudonarcis-
sus) and demonstrated its advantages over the
analysis of silylated samples, especially in identi-
fying minor components. Using this method, we
Table I. GC-MS data of the alkaloid mixture from C. latifolium leaves.
Alkaloid M
+
and characteristic ions (%) RT At After
[min.s] flowering flowering
(%)
a
(%)
a
9Ð0ctadecenamide
b
(1) 281(10), 126(21), 112(18), 98(90), 72(95), 59(100) 15.39 0.5 0.2
Dihydro-oxo-demethoxy 271(22), 243(100), 214(75), 186(60), 115(25) 16.03 0.7 3.5
haemanthamine
b
(2)
Augustamine
b
(3) 301(66), 300(100), 244(72), 215(30), 201(27) 16.43 1.8 0.2
Oxoassoanine
b
(4) 281(100), 266(12), 250(8), 238(20) 16.66 0.3 0.7
Crinane-3α-ol
b
(5) 273(100), 256(27), 229(42), 201(50), 185(32), 115(25) 16.98 Ð1.1
Buphanidrine
b
(6) 315(100), 300(31), 284(34), 260(45), 245(63), 231(37), 18.78 2.2 Ð
130(46)
Powelline (7) 301(100), 258(20), 246(20), 220(61), 217(40) 19.31 <0.1 0.3
Undulatine (8) 331(100), 258(41), 205(62), 189(43), 173(39) 20.68 19.9 <0.1
Ambelline (9) 331(98), 299(38), 287(100), 260(97), 255(70), 211(75) 20.92 6.2 3.0
6-Hydroxybuphanidrine
b
(10) 331(55), 276(100), 261(28), 229(78), 91(35) 21.08 8.5 Ð
6-Hydroxypowelline (11) 317(100), 299(34), 262(28), 244(90), 233(60) 21.65 Ð1.5
Crinamidine
b
(12) 317(60), 288(100), 244(29), 217(42), 205(38), 203(37) 21.75 14.1 30.0
6-Hydroxyundulatine
b
(13) 347(30), 276(32), 256(29), 229(31), 219(100), 204(20) 22.76 4.8 0.7
1β,2β-Epoxyambelline(14) 347(35), 318(100), 274(32), 231(30), 205(52) 23.02 1.6 1.8
Epoxy-3,7-dimethoxycrinane- 345(100), 316(46), 286(63), 270(71), 231(90) 23.59 Ð0.8
11-one
c
(15)
6-Hydroxycrinamidine (16) 333(80), 304 (45), 286(100), 274(98), 256(58), 231(87) 23.84 Ð2.8
a
% of the total ion current. The area of the GC/MS peaks depends not only on the concentration of the correspond-
ing compounds, but also on the intensity of their mass spectral fragmentation, so the data given in the table is not
a true quantitation but can be used for comparisons between the two samples, which is the objective of this work.
b
New for C. latifolium.
c
Tentative structure.
identified 16 alkaloids (one of them tentatively)
(Table I). Some components remained uniden-
tified due to the lack of reference substances and
library spectra.
Until now, 7 alkaloids have been isolated and
identified in C. latifolium leaves (Kobayashi et al.,
1984; Kobayashi et al., 1984; Jeffs. et al., 1985; Vo,
1997). In our samples, we found only 3 of them: 6-
hydroxycrinamidine (16), 6-hydroxypoweline (11),
undulatine (8). From the remaining 12 alkaloids, 9
are found for the first time in C. latifolium (Ta-
ble I, Fig. 1). Ambelline (9), 1,2-β-epoxyambelline
(14) and powelline (7) have been found in other
plant parts of C. latifolium.
Contrary to other reports on C. latifolium leaves
(Kobayashi et al., 1984; Kobayashi et al., 1984;
Jeffs. et al., 1985; Vo, 1997), in leaves of Viet-
namese C. latifolium we identified almost ex-
clusively alkaloids of the crinane type. The main
alkaloids appeared to be undulatine (8) and crin-
amidine (12), which contain 1β,2β-epoxy ring.
Other important components of the alkaloid mix-
tures were 6-hydroxybuphanidrine (10), ambelline
(9) and 6-hydroxyundulatine (13). According to
the structures of the identified alkaloids most of

N. T. N. Tram et al. · GC-MS of Crinum latifolium L. Alkaloids 241
Fig. 1. Alkaloids in C. latifolium leaves: dihydro-oxo-demethoxyhaemanthamine (2), augustamine (3), oxoassoanine
(4), crinane-3α-ol (5), buphanidrine (6), powelline (7), undulatine (8), ambelline (9), 6-hydroxy-buphanidrine (10),
6-hydroxypowelline (11), crinamidine (12), 6-hydroxyundulatine (13), 1β,2β-epoxyambelline (14), epoxy-3,7-dimeth-
oxycrinane-11-one (tentative) (15), 6-hydroxycrinamidine (16).
them are biogenetically related and can be pro-
duced by an oxidation or O-methylation of crinine.
Two of these alkaloids are know to possess biolog-
ical activities. Epoxyambelline (14) moderately ac-
tivated mouse spleen lymphocytes; a mixture of
epoxyambelline (14) and ambelline (9) (1:1) pro-
duced pronounced activation of the spleen lym-
phocytes (Ghosa et al., 1984).
Significant differences in the alkaloid composi-
tion at flowering and after flowering were ob-
served (Table I). Some components considerably
prevail at flowering (undulatine (8), 6-hydroxybu-
phanidrine (10), 6-hydroxyundulatine (13), ambel-
line (9), buphanidrine (6), and others after flower-
ing (6-hydroxycrinamidine (16), crinamidine (12),
6-hydroxypowelline(11).
According to Table I it is evident that there are
substantial changes in the methylation and oxida-
tion of alkaloids at different ontogenetic stages.
The 3-O-methylation of powelline (7) to buphani-
drine (6), of 6-hydroxypowelline (11) to 6-hydro-
xybuphanidrine (10), of crinamidine (12) to undu-
latine (8) and of 6-hydroxycrinamidine (16)to6-
hydroxundulatine (13) prevails at flowering. Oxi-
dized products of buphanidrine (6): undulatine (8)
(1,2-epoxidation), ambelline (9) (11-hydroxyla-
tion), 6-hydroxybuphanidrine (10) (6-hydroxyla-
tion) and 6-hydroxyundulatine (13) (1,2-epoxida-
tion and 6-hydroxylation) are present in
significantly higher concentrations at flowering.
On the other hand, the oxidized products of pow-
elline (7) (6-hydroxypowelline (11), crinamidine
(12), 6-hydroxycrinamidine (16) dominate after
flowering.
The results obtained here differ from previous
results on C. latifolium alkaloids (Ghosal et al.,
1985). This could be caused by presence of plant
varieties or hybridization, or to specificities of the

242 N. T. N. Tram et al. · GC-MS of Crinum latifolium L. Alkaloids
collection site. The ontogenetic stage at the mo-
ment of collection of plant material is also of im-
portance. Taking into account the low concentra-
tion of alkaloids in leaves (lower than 0.1%) and
the complexity of the alkaloid mixture, GC-MS of
Ghosal Sh., Saini K. S. and Frahm A. W. (1983), Alka- Kobayashi Sh., Tokumoto T. and Taira Z. (1984), Lati-
loids of Crinum latifolium. Phytochemistry 22, 2305Ðfine, a biogenetic isomer of cherylline, from Crinum
2309. latifolium L. J. Chem. Soc., Chem Commun.,1043Ð
Ghosal Sh., Saini K. S and Arora V. K. (1984), 1,2-β- 1044.
Epoxyambelline, an immuno-stimulant alkaloid from Kreh M., Matusch R. and Witte L. (1995), Capillary gas
Crinum latifolium. J. Chem. Research (S), 232Ð233. chromatography-mass spectrometry of Amaryllida-
Ghosal Sh., Saini K. S. and Razdan S. (1985), Crinum ceae alkaloids. Phytochemistry 38, 773Ð776.
alkaloids: their chemistry and biology. Phytochemistry Tram Ng. Th. Ng., Zvetkova E., Nikolova E., Katzarova
24, 2141Ð2156. E., Kostov G., Yanchev I. and Baicheva, O. (1999), A
Ghosal Sh. and Singh S. K. (1986), Chemical constitu- novel in vitro and in vivo T-lymphocyte activating
ents of Amaryllidaceae. Part 24. Crinafoline and cri- factor in Crinum latifolium (L.) aqueous extracts. Exp.
nafolidine, two anti-tumor alkaloids from Crinum lati- Pathol. Parasitol. 3,21Ð26.
folium. J. Chem. Research (S), 312Ð313. Tram Ng. Th. Ng., Yanchev I., Zvetkova E., Katzarova
Jeffs. P. W., Abou-Donia A., Campau D and Staiger D. E., Kostov G., Svilenov D. and Shalamanov P. (2000),
(1985), Structures of 9-O-demethylhomolycorine and Retarded growth of chemically induced with 20
5α-hydroxyhomolycorine. Alkaloids of Crinum de- methylcholanthrene in rats under the action of cold-
fixum, C. scabrum and C. latifolium. Assingment of hot aqueous extracts (decoctions) from Vietnamese
aromatic substitution patterns from
1
H-coupled
13
C plant Crinum latifolium (L.). Exp. Pathol. Parasitol.,
spectra. J. Org. Chem. 50, 1732Ð1737. in press.
Kobayashi Sh., Tokumoto T., Kihara M., Imakura Y., Vo, T. B. H., Nguyen, K.,Q.,C. and Ngo, V. Th (1997),
Shingu T. and Taira. Z. (1984), Alkaloidal constituents Hydroxycrinamidine, a new alkaloid from leaves of
of Crinum latifolium and Crinum bulbispermum Crinum latifolium(L.). Tap Chi Duoc Hoc 11,9Ð10
(Amaryllidaceae). Chem. Pharm. Bul. 32, 3015Ð3022. (Vietnamese), from CA128: 292716u.
underivatized samples is the method of choice for
rapid analysis of Crinum alkaloids. It requires min-
imum amount of plant material and allows the
identification of numerous compounds.