Effects of stress on alkaloid metabolism in Crinum asiaticum

Phytochemistry, Vol. 29, No. 3, pp. 805-811, 1990.

0031-9422/90 %3.00+0.00 PergamonPressplc

Printedin Great Britain.

EFFECTS

OF STRESS ON ALKALOID METABOLISM ASIATZCUM* SHIBNATH GHOSAL,

Pharmaceutical

Chemistry

Research

Laboratory,

SUSHIL K. SINGH

Department

and

IN CRINUM

SANKARA G. UNNIKRISHNAN

of Pharmaceutics,

Banaras

Hindu University,

Varanasi-221005,

India

(Received in revised form 18 August 1989) Key Word Index-Crinum

asiaticurn; Amaryllidaceae;

phosphatidyllycorines;phosphatidylpseudolycorines; C. asiaticurn; chemical

fruit; glucosyloxy alkaloid; lycorine-1,2-0-fi-o-diglucoside; stress metabolism of alkaloids; flux of alkaloids and CAMP in

elicitors

Abstract-The biochemical role of free and conjugated alkaloids in the fruits of Crinum asiaticurn during strees (incisional injury and attack by an insect) is appraised. Wounding of C. asiaticurn fruits caused almost complete hydrolysis of the alkaloidal conjugates and also produced oxidized metabolites of lycorine and its analogues. Prior treatment of fruits with anaesthetic agents, e.g. ether and lidocaine, not only protected the alkaloidal conjugates from hydrolysis but also prevented their oxidation. The qualitative and quantitative changes observed in the alkaloids, in response to the stress, suggest their role in protective and repair mechanisms of the producer plant, notably, augmentation of a ‘second messenger’, CAMP, in the fruits in response to injury and systemic administration of theophylline was observed. Prior administration of anaesthetic agents (ether or lidocaine), kept the concentration of CAMP significantly low despite inflicting subsequent injury.

RESULTS AND DISCUSSION

INTRODUCTION

This study was undertaken with a view to determining just how natural are the Amaryllidaceae alkaloids that are isolated by means of the conventional techniques. It was considered likely that during the isolation of alkaloids from fresh plant materials through grinding, macerating and solvent extraction procedures, deleterious changes might take place due to mixing of the contents of normally separate cellular components (e.g. the vacuolar contents with those of cytosols), and by chemical interactions between them. In order to minimize these effects, the recommended [l] gentlest procedure of hand-grinding in a mortar and pestle, was adopted. Also, an ideal homogenization medium (see Experimental) was used to neutralize the acidic vacuolar contents, to maintain isotonicity with the cytosol and to prevent oxidation of the readily oxidizable molecules. The products so isolated were compared by TLC and HPLC with those isolated from the same plant parts following the conventional procedures [2]. Considerable variations were observed between the two sets of products. We then examined, by application of some anaesthetic agents, the impact of the stress stimuli under normal and anaesthetic conditions on the fruit alkaloids. Once again, distinct qualitative and quantitative variations were observed in the alkaloids (free and conjugated) isolated from the anaesthetized and un-anaesthetized fruits. It was, therefore, thought worthwhile to study the mechanism of alkaloid metabolism, under different experimental conditions, and their biochemical significance.

*Part 32 in the Series ‘Chemical Constituents of Amaryllidaceae’. For Part 31 see ref. [15].

Four developing green fruits, of approximately equal weights, from the fruit-stalks of 10 C. asiaticurn plants, were used. Fruit 1, on each stalk, was covered with a small plastic bag whose opening was plugged with a cotton ball moistened with ether. Fruit 2, was wrapped in a fine cloth moistened with a solution of lidocaine-HCl (in Pi buffer, pH 7.2). Fruit 3, was injected with the lidocaine-HCl solution by a hypodermic syringe. Fruit 4, was washed with distilled water and the excess water wiped off. This fruit was kept as the control. Ten replicates were prepared. After 15 min, an equal number of incisions (l&12) were made in each fruit with a knife. After a further 30 mitt, the fruits were harvested and separately homogenized in a mortar and pestle with the homogenization medium. The extracts were centrifuged and the supernatants lyophilized. Each residue was subjected to TLC and analytical HPLC using authentic markers. A portion of the lyophilized residue was fractionated into basic, neutral and acidic constituents in the usual way [2]. The basic fraction, containing alkaloids, was again subjected to analytical HPLC. Considerable variations in nature and abundance were observed in the free and conjugated (glucosyloxy, phosphatidyl) alkaloids and in their metabolites, isolated from the four differently treated fruit samples (Table 1). The injury to the control fruit 4 (by incision) caused almost complete hydrolysis of the alkaloidal conjugates and also produced several oxidized metabolites of lycorine and its analogues. Prior treatment of the fruits with the two anaesthetic agents, i.e. ether (fruit 1) and lidocaine (topically applied, fruit 2), not only protected the alkaloidal conjugates from hydrolysis but also prevented oxidation of lycorine and its analogues (Table 1). Additionally, three new alkaloidal conjugates 805

S. GHOSAL

806

et

al.

0

1

R1,R’

=

P-D-+

28

R’, R3 =

pahitoyl,

13

R’.R*

=

H

2b

R’,R’

palmitoyl,R2

3a

R’,R’=

palmitoyl,R3

=

stearoyl,

3b

R’, R3 =

palmitoyl,R’

=

oleoyl,

=

R’ = =

stearoyi. oleoyl,

R4 +

R’ =

CH,

R4 + R5 = CH, R* =

Me,

Rs =

R4 = Me, R5 =

H H

OR3

0

4 12

R’ =

Me,

RI =

0,

9

R’,R” Ra,R3 =

=

H

6 7 8

H

10

R’ +

11

R’.R2

(compounds 1-3) were isolated from the extracts of fruits 1 and 2 in high yields (Table 1). Another interesting difference was observed when lidocaine was administered systemically (fruit 3). The treatment protected the alkaloidal conjugates from hydrolysis, albeit partially, and also caused noticeable oxidation of lycorine and analogues (Table 1). The dual response seems to be due to a combination of stress (prick of the hypodermic needle before anaesthetization) and increased local concentration of lidocaine at one point in the fruit. A similar dual response was observed when chloroforni was injected into C. asiaticurn fruits. The characterization of the three new compounds (l-3) only is described here. Compound 1, C28H37N014 (FABMS and elemental analysis), obtained as an amorphous powder, was optically active. It responded to Dragendorffs reagent for alkaloids and benzidine-metaperiodate reagent for poly01s. The physical and spectral (UV, IR, ‘H NMR) properties of the compound showed close similarities with those of lycorine-1-0-/3-D-glucoside [3]. It was freely soluble in water from which it could be re-extracted with n-butanol. The compound gave no molecular ion peak in its EI mass spectrum, the identifiable fragment ion peaks were due to

R2 = CH, = Me

R’t

Rz =

(‘If,

R’= Me. R2= II R’ = ti. R’ = Me

14

the aglucone moiety (m/z 287) whose base peaks appeared at m/z 227 and 226. These are typical of those of lycorine [2]. It formed an octa-acetyl derivative which exhibited a small, but identifiable, [MI’ peak at ml-_ 947 and fragment ion peaks of appreciable abundance at m/z 33 1,289, 271, 211, 169, characteristic of a tetraacetylglucosyl moiety. The ‘H NMR spectrum of the compound showed the presence of two 0-glucosyl functions whose anomeric protons are in /Y-configuration, as was suggested from their coupling constants (J = 7.5 Hz). Methylation of the compound with sodium hydride and methyl iodide, in tetrahydrofuran, according to ref. [4] gave the permethylether (m/z 723, [M] ‘). Cautious hydrolysis of this product followed by acetylation of the aglucone afforded 1,2-di-O-acetyllycorine [S]. The glycone moiety was identified and estimated, according to ref. [6], as lhydroxy-2,3,4,6-tetra-0-methylglucose (aglucone- glucose 1 :2). Hence, compound 1 was assigned the 1,20-fl-D-diglucosidelycorine structure (1). Compound 2, consisting of a mixture of two closely related entities, C,,H, Ix NO,,P, and C,,H, ,hNO,ZP, exhibited UV, IR and ‘H NMR spectra characteristic of a fully acylated 2-0-glycerophosphoryllycorine [7]. It re-

Alkaloid Table

1. Changes

in the alkaloidal

metabolism

Lycorine G(-and /?methocations (4) Anhydrolycorinium (5) Hippadine (6) Pratorinine (7) Pratorimine (8) Trisphaerdine (9) Ungeremine (10) Criasbetaine (11) Lycorine N-oxide (12)

Lycorine (13) 2-Epilycorine (14) Compound 1

807

constituents of fruits of C. asiaticurn on wounding and anaesthetic conditions Amount

Alkaloid

in Crinum asiaticurn

Controlt 14.22 f 1.03

004~0.03 0.07 + 0.02 0.09 f 0.03 0.04 + 0.01 0.02 + 0.005 6.27 + 0.64 4.08+1.11 5.18+0.55 20.74_+2.82 10.54+ 1.92 1.04+0.12

Compound 2

2.82 + 0.47

Compound 3

0.89+0.10

of alkaloids

Ether $

1.02*0.01 P
t

(mg/lOO g fresh fruit)* Lidocaines (topical)

2.74 f 0.02 P
t

Lidocaine 11 (systemic)

0.88&0.05 0.05 +0.001 t 0.84+0.03 0.02 + 0.005 0.88 f 0.07 3.55 kO.72

t

1.20*0.11 22.44 f 2.92 P
under normal

t t 18.30+ 1.88 PcO.001 16.22+ 2.68 PcO.001 8.54kO.24 PcO.001

0.71+0.08 8.22 _+2.30 t 12.40f2.22 10.88k2.13 7.045 1.82

*Values (by analytical HPLC) are means k s.d. for 10 determinations. t Unanaesthetized fruits subjected to incisional injury were macerated in homogenization medium (see Experimental). f Fruits were kept under a plastic cover plugged with cotton, moistened in ether, prior to incisional injury. §Fruits wrapped in fine cloth moistened with a soln. of lidocaine hydrochloride (in Pi buffer, pH 7.2). /IPi buffer soln. (2%, 1 ml) of lidocaine hydrochloride injected to fruits by a hypodermic syringe. -, absent; t, traces; total amount of alkaloid in control, 66.04 mg/lOO g fruit. Level of significance (P) compared to control group.

mained unaffected on treatment with acetic anhydride-pyridine at 95”. Deacylation of the compound with sodium methoxide-methanol gave 2-O-glycerophosphoryllycorine [7] and a mixture of methyl esters of palmitic, stearic and oleic acids (10: 5 : 1, GC). Hydrolysis of the compound with phospholipase D gave palmilycorine [8] and phosphatidic acid. The latter, on graded hydrolysis, according to ref. [7], afforded glycerol, three fatty acids (palmitic, stearic and oleic acids in ca 5 : 5 : 1) and phosphoric acid. Selective deacylation at the C-2 position of the glycerol moiety, with phospholipase A,, gave a mixture of stearic and oleic acids. On the basis of these observations, compound 2 was assigned the l-Opalmitoyl-2-O-(l’-O-palmitoyl-2’-O-stearoyl/oleoyl)-glycero-phosphoryllycorine structures (2a/2b). Compound 3, like compound 2, contained a mixture of two closely related entities, C&H, 20N0,2P and C6,H,,,N0,2P. It showed, in its ‘HNMR spectrum, one Ar-OMe (63.98) in lieu of the methylenedioxy signal (65.9) of compound 2. Also, on acetylation it gave a mono-0-acetyl derivative (Ar-OAc; vmax 1762 cm- ‘). Methylation of the compound by treatment with ether-diazomethane, in methanol, followed by cautious acid hydrolysis of the product afforded methylpseudolycorine [2] from the aglycone fraction. Deacylation of compound 3, with sodium methoxide-methanol, in tetra-

hydrofuran, afforded pseudolycorine and methyl esters of palmitic, stearic and oleic acids (in ca 10: 5: 1). Hydrolysis of compound 3 with phospholipase D gave 1-O-palmitoyl-pseudolycorine and a phosphatidic acid. The latter product was identical with the one obtained from similar enzymatic hydrolysis of compound 2. The linkage of the phosphatidyl moiety at C-2 of pseudolycorine was then established. Thus, treatment of the acetyl derivative of compound 3 with Pd(PPh,), in tetrahydrofuran, and sodium methoxide in succession, according to ref. [7], produced 1-O-palmitoyl-lo-0-acetylpseudolycorine, mp 227-229”. The displacement ofthe phosphatidyl group by the Pd(O)-catalysed reaction suggested its attachment to an allylic oxygen function (C-2 in the pseudolycorine moiety). On the basis of these observations, compound 3 was assigned the 1-O-palmitoyl-2-O-( l’-O-palmitoyl2’-0-stearoyI/oIeoyl)-glycerophosphorylpseudolycorine structures (3a/3b). The above experiments regarding the fluctuations of alkaloids, were repeated several times during the growing of C. asiaticurn fruits in different seasons (rainy and winter) when strikingly similar results were obtained. These findings suggested that something more than chance was in operation. The investigations of the Indian Savant, Acharya Jagadish Chandra Bose, with living plants over many years led him to conclude that there was

S. GHOSAL et al

808

a fundamental unity of physiological responses in plants and animals. Acharya Bose discovered that ether and chloroform vapour would exert a narcotic effect upon Mimosa, weakening its rapid closure response to mechanical stimulation [9]. Our findings are germane to the discovery of Acharya Bose in respect of similar biochemical behaviour of plants and animals, at least in the observed area of influence of anaesthetic agents. We further tested as to what extent the observed interface between them was fortuitous or genuine. The secondary metabolites of plants, e.g. the alkaloids, are the products of special biosynthetic activities of specialized cells and are ecologically important for the producer plants. Alkaloids defend their plant sources from predators (e.g. animals, microorganisms, and pathogenic/parasitic plants) and also serve as intra- and interspecific signals. Any form of stress (e.g. injury to plant or contact between pathogen and plant surface) would disturb the balance of the production of natural alkaloids and their metabolism in the producer plant. An interesting plant-insect interaction observed with C. asiaticum, where alkaloids play a very significant biochemical role to both, is described here to lend credence to the above contention. The host cells of Amaryllidoideae, in response to invasion by insects, form structures that ordinarily restrict further invasion. The phenanthridine alkaloids, lycorine and its analogues, normally present in the form of conjugates (glycosides and phosphatidyl derivatives in Amaryllidaceae plants), are released when the producer plants are invaded by insects. The free alkaloids provide protection to the producer plants by virtue of their cytotoxic action [lo]. However, caterpillars of a smoky-grey moth, Polytela gloriosu Fab., rear almost exclusively on C. lutifolium Linn. and also on C. asiaticum. The alkaloids of these plants are converted into new conjugates in the larvae and pupae of P. gloriosa [l I]. Thus, inspite of the potent cytotoxic effects of the alkaloids upon other insects and herbivores, P. gloriosa absorbed these alkaloids and also adopted them for the purpose of its defence and growth. Interestingly, infestation of the leaves of C. asiaticum with this insect, in Table

September-October, significantly augmented the concentration of lycorine and its metabohtes in the fruits. Intentional infestation of the leaves of C. asiaticum with the caterpillars of P. gloriosa. transmitted the warning signals to the fruits. This was revealed from the surge of free phenanthridine alkaloids and their oxidized metabelites, in the fruits, within 3 hr of the insect infestation (Table 2). However, when the fruits were anaesthetized with ether, 1 hr before infestation with P. gloriosu, no change was observed in the concentrations of the free alkaloids and their oxidized metabolites compared to those observed in the control. Thus, the warning signals transmitted from the leaves to fruits under unanaesthetized state was no longer available under the influence of the anaesthetic agent. These findings are reminiscent of the different responses to stimuli under conscious and unconscious states in animals and in man. In addition to playing a protective role to producer plants. the phenanthridine alkaloids of the Amaryllidaceae seem to assist in the biosynthesis of some other compounds that are required for the cell-to-cell communication and repair mechanism in the host cells. Many living cells receive instructions and warning signals through chemical agents, e.g. hormones, which in turn activate the synthesis of a ‘second messenger’, CAMP. In this study, we appraised if the injury to fruits of C. asiaticum, under anaesthetized and unanaesthetized conditions, caused any change in their CAMP contents. It is now well documented that CAMP is ubiquitously present in plants although its exact biochemical role in plants is not clear. The estimation of CAMP in the fruits (supernatant of the centrifuged extract) of C. asiaticurn was carried out by both HPLC and radioimmunoassay [ 121. A statistically significant increase in the concentration of CAMP was observed in the control (unanaesthetized) compared to the ether- and hdocaine-anaesthetized groups. Systemic administration of theophylline also produced a statistically significant increase in CAMP (Table 3). It seems likely that the elevation ofcAMP in the control fruits is associated with the cell repair mechanism. It is also conceivable that the oxidation of the alkaloids

2. Flux of alkaloids in fruits of C. asiaticurn in response infestation of leaves with P. gloriosa

to intentional

Amount of alkaloid (mg/lOO g at times shown)* Alkaloid Lycorine X- and /I-methocations (4) Lycorine (13) Ungeremine

(IO)

Criasbetaine

(11)

Lycorine

N-oxide

(12)

Ohr

3 hr

6 hr

12hr

24 hr

1.28 (1.52)t 1.16 (1.02) 0.12 (0.08) 0.02 (0.02) 0.04 (0.06)

2.87 (3.05) 3.84 (nd.) 4.42 (0.11) 0.55 (0.07) 0.88 (nd.)

2.02 (3.18) 5.45 (n.d.) 8.33 (n.d.) 2.22 (n.d.) 1.74 (0.05)

2.18 (2.88) 8.57 (1.82) 15.x9 (0.28) 3.04 (0.09) 2.83 (0.07)

2.44 (2.84) 15.55 (1.44) 18.24 (0.07) 4.94 (0.05) 2.88 (0.03)

*Caterpillars ofP. glorisa were allowed to settle on the leaves of fruit-bearing C. asiaticum plants (0 hr); alkaloidal constituents in the fruit homogenates were determined by HPLC; Values are means of 10 determinations. tin parentheses, amounts of alkaloids of fruits of insect-free plants; n.d.. not determined.

Alkaloid Table

metabolism

in Crinum asiaticurn

809

of CAMP in fruits of C. asiaticum

3. Concentration

CAMP Group 1. 2. 3. 4.

treatment

Controlt Ether-anaesthetized Theophylline-treated3 Lidocaine-anaesthetizedg

?l

(nmol/g fruit)*

p II

1014 1014 1014 814

20.0&2.80/18.89 f 1.87 7.5 *0.82/ 8.58 +0.70 36.35k3.7 /33&l&3.12 14.5 *2.02/12.44* 1.02


*Values [by analytical HPCL/radioimmunoassay by commercially available kit (Amersham Corpn)]. tunanaesthetized fruits subjected to incisional injury and then macerated in homogenization medium; fTheophylline crystalline (Sigma) (1% aq. soln., 2 ml) injected to fruit-stalk about 2.5 cm below the fruit-bunch; §Lidocaine hydrochoride (in Pi buffer saline, pH 7.2; 1% soln.) injected as in c; I/Significance in relation to Group 1.

(already present in the free form and those liberated from the conjugates) is associated with the synthesis of CAMP by the reaction sequence: ADP+ ATP+cAMP, and is indirectly assisting the cell repair mechanism of the stressed producer plant. The increase in CAMP in response to systemic administration of theophylline (Table 3) is consistent with this contention. Theophylline is known to stimulate CAMP-dependent processes via increase in oxygen consumption in animal cells [ 131. Faster metabolism of lycorine and analogues into corresponding oxidized products could thus be viewed as an attempt to annul the toxic effects of the alkaloids generated during the injury. Furthermore, it has been observed that two biosynthetic processes that are specifically impaired by an uncontrolled surge of alkaloids are those of carotenoids and ascorbic acid [13]. Carotenoids are one of the important agents that elevate the concentration of intracellular CAMP. During the process, the carotenoids are metabolized into xanthophylls. As expected, the concentration of xanthophylls (oxidized metabolites of carotenoids, e.g. zeaxanthine dipalmitate), was found to increase appreciably in the injured unanaesthetized fruits of C. asiaticurn. It is possible that the transformation of lycorine into its oxidized metabolites, that of carotenoids into xanthophylls and the enhanced synthesis of CAMP in response to injury in C. asiaticurn fruits are inter-connected. Further study in this direction is currently in progress. When lidocaine-anaesthetized (systemic) fruits, were not removed from the flower stalk and the control injured fruits were also left on the flower stalk, for three to four days, no qualitative or quantitative difference in the alkaloidal patterns was observed between the two sets of samples. The concentrations of the free alkaloids were reduced in both and the alkaloidal conjugates reappeared, albeit marginally. The vigour of biochemical response in both tissues was considerably reduced at this stage since a fresh incisional injury did not elicit any noticeable change in terms of alkaloids. However, systemic administration of theophylline or noradrenaline, even at this stage, significantly augmented the formation of lycorine and its analogues and their oxidized metabelite! (Table 4). Both theophylline and noradrenaline are known to stimulate CAMP-dependent processes and to increase oxygen consumption in animal cells. The pat-

terns that stress and the elicitor chemicals evoked in C. asiaticurn in respect of alkaloids seem to convey the message that: ‘cell-cell communication in plants may be inherently similar to those in animals and in man’. It is possible that all living plants, like animals and man, react similarly towards aversive stimuli to annul their effects and fail to do so when anaesthetized. EXPERIMENTAL The genera1 methods are the same as reported recently [7]. The statistical analyses were carried out by standard procedures

c141. Plantmaterial. Ten fruit-bearing

plants of C. asiaticurn, grown in the gardens of the Banaras Hindu University Campus, were selected for the experiments. The experiments were carried out between August and September (rainy season) and January-February (winter) for three consecutive years. In fully grown plants, there were usually 12-15 fruits (average wt 14 g) in each stalk. In a typical experiment, 4 fruits (15-16 g, each), on each stalk, were marked fruit 1 to 4 and were treated as described in Results and Discussion. Sample preparation. The harvested fresh fruits were sliced and immersed in homogenization medium [mannitol (0.25 M), Tris buffer (pH h 7.5,50 mM), mercaptoethanol(10 mM), Mg (OAc), (10 mM) and Ca (OAc), (1 mM)] for 10 min and then homogenized with a mortar and pestle. The mixture was centrifuged (1.1 x lo4 9 for 1 hr) and the supernatant collected. This was divided into three portions for monitoring by TLC for all common secondary metabolites; estimation of CAMP, and for detection and estimation of alkaloids by HPLC. One portion was lyophilized and the residue was fractionated into neutral, basic and acidic components in the usual way [Z]. The residue from the basic fraction was dissolved in MeOH-H,O (4: 1) and subjected to analytical HPLC. HPLC analyses. PBondapak C,, analytical column (30 cm x 4 mm i.d.) and ?jiO, C,,-RP column (25 cm x 4 mm i.d.); MeOH-H,O (4: 1); flow rate, 0.5 ml/min. Each 10 ~1 of the soln (MeOH-H,O), injected onto the column, contained ca 20-25 ng residue. For assay of CAMP (PBondapak C,, column), the mobile phase was 0.01 M-KH,PO,-MeOH (43.7); flow rate, 1 ml/min; and the detector sensitivity was 0.02 a.u.f.s. Isolation ofalkaloidal conjugates. The lyophilized residue from the supernatant was partitioned between EtOAc-H,O (1: 1). The aq. layer was then extracted with n-BuOH.

810

S. GHOSAL

Table

4. Effects of theophylline

and noradrenaline on alkaloidal fruits of C. asiaticurn Amount Control t

Alkaloid

3.40+0.7

Lycorine a- and pmethocations (4) Anhydrolycorinium (5) Ungeremine (IO)

0.01 +0.001 1.12kO.25

Criasbetaine (11) Lycorine (13)

0.21 kO.08 5.27 & 1.22

2-Epilycorine

7.33 + 2.45

(14)

et al.

of alkaloids

flux in senescent

(mgj100 g fruit)*

Theophylline: 8.22i 1.5 P
Noradren+line$ 7.88 i. 1.82 P
*Values (by analytical HPLC) are means &s.d. for 10 determinations; t Pi buffer (2 ml) administered to previously injured fruits after recovery $Systemic administration (I mM, 2 ml) by a hypodermic syringe to previously injured fruits SSystemic administratidn (1 PM, 2 ml in’Pi buffer) to previously injured fruits Level of significance (P) in relation to control. Total amounts of alkaloids: in control, 22.7 mg/lOO g fruit; theophylline-treated, 55.3 mg/lOO g fruit; noradrenaline-treated, 57.9 mg/lOO g fruit.

Treatment of the n-BuOH $~~ction. This fraction, on evapn, gave a brown gummy material which showed one major and several minor Dragendorff and benzidine metaperiodate positive spots on TLC. It was repeatedly extracted with hot Me,CO to remove the alkaloidal aglycones (e.g. lycorine and analogues). From the Me&O-insoluble fraction compound 1 was obtained. Compound 1. The Me,CO-insoluble residue was dissolved in MeOH and passed through a column of silica gel-H. Elution was carried out with CHCI,, CHCI,-MeOH, MeOH, and CHCI,-MeOH-H,O (8: 3: 1). The CHCI,-MeOH-H,O eluates were combined and evapd under red. pres. to give a straw coloured solid (22 mg from 100 g of Et,O-anaesthetized fruits); R, 0.15 (CHCl,-MeOH-HOAc, 190:9: I); [r];’ -27.5” (H,O; ~0.58); UV: A::;” nm (log E) 235 sh (3.02), 288 (3.47); IR: “;:?I cm- 1: 34OO(br), 1650_16OO(br), 1590,1510,1040,945,938; FABMS (using glycerol matrix): m/z 612 [MH]+; ‘H NMR (D,O):66,68(lH,s, H-l 1),6.60(1H,s, H-8), 5.90(2H,s,OCH,O), 5.55 (IH, m, H-3), 4.98 (IH, d, .J=7.5 Hz, H-l’), 4.90 (lH, d, J = 7.5 Hz, H-l”), 3.15-3.80 (m, sugar H + HDO), centred at 2.80 (2H, m, H, H-5), centred 2.60 (2H, tn. H, H-4). (Found: C, 54.6; H, 6.1; N, 1.9. C,,H,,NO,,.H,O requires C, 54.9; H, 6.3; N, 2.2%). The octaacetyl derivative, prepared by treatment of 1 with Ac,O-pyridine (under reflux; the excess reagents were removed by chasing with N,), crystallized from EtOH-hexane as microcrystals, mp 105-108” (dec.); EIMS: m/z 947 ([Ml’, rel. int. 1.8%); fragment-ion peaks at m/z 331 (35), 289 (8). 271 (ll), 227 (lOO), 226 (95), 211 (7), 169 (18). The permethyl ether was obtained as a glassy solid. It did not exhibit any OH band in its IR spectrum (in Nujol); EIMS: m/z 723 (M+, 2.5%). Treatment ofthe EtOAcfiaction. The residue from the EtOAc fraction was triturated with hot hexane, CHCI, and MeOH, in succession, when compound 2 (2a/Zb) and compound 3 were obtained from the CHCI, and MeOH-soluble fractions, respectively. Compound 2 (2a/2b). This compound was further purified by semi-prep. HPLC (FBondapak C,, column; mobile phase, MeOH-H,O-MeCN, 90:7:3; flow rate 4 mlimin). It was ob-

tained as a straw coloured amorphous powder (26 mg from 100 g Et,O-anaesthetized fruit); R, (min) in analytical HPLC 14.5, 15.8; FABMS: m/z 1184 [MH]+ (28); 1182 [MH] + (2b); deacylation (NaOMe-MeOH) and GC-MS analyses established the fatty acid composition as C,,: O/C, s: 0 (palmitic/stearic acid, 2: 1) in 2a and C,,:,/C,,:, (palmitic/oleic acid, IO: 1) in 2b; UV I,,, 22G-222 (3.47), 232 sh. 280 nm (3.08): IR: Y,,, 3400 (br), 1750, 173.5, 1600 (br), 1205, 1035, 938, 828, 732cm-‘; ‘HNMR (CDCI,): 66.84 (IH, s, H-l I), 6.72 (lH, s, H-8), 5.95 (2H, s, OCH,O), 5.70 (lH, hr, H-3), 5.28 (m. CH=CH), 5.W.2 (m, H-1, glycerol methine and methylene protons), 1.23 (fatty acid methylenes), 0.88-0.9 (methyl protons). (Found: C, 69.3; H, 10.4; N, 1.0; P. 2.4. Ch9HllsNOtzP requires C, 69.9; H. 9.9; N, 1.2; P, 2.6%). Compound 3 (3a/3b). This compound was obtained from the MeOH-soluble fraction and was further purified by semi-prep. HPLC, as before, as a light-brown amorphous solid (11 mg from 100 g Et,O-anaesthetired fruits); R, 28.5, 29.8; deacylation followed by GC-MS of the methyl esters of mixture of fatty acids showed their composition as C I6 :()/C,, : 0 (palmiticjstearic acids, 2: 1) in 3a; C,,:,/C,,: 1(palmitic/oleic acids; ratio) in 3b; UV AnI,, 22(f222 (3.01), 230 sh, 283 nm (3.63); IR: Ymai 3350, 3300, 25OG-2350, 1755, 1612, 1600, 1205, 905, 730cm-‘; ‘HNMR (CD,OD): 66.88 (lH, s, H-11), 6.62 (lH, s, H-8), 5.74 (IH, br, H-3), 5.3 (m, CH=CH), 3.90 (3H, s, Ar-OMe), 1.22 (poly-CH,-), 0.90,0.88 (CH,); FAB-MS: m/z 1186 [MH]+ (3a); 1184 [MH]’ (3b). (Found: C, 69.5; H, 10.2; N, 0.98; P, 2.3. C,,H,Z,NO,,P requires C, 69.9; H, 10.2; N, 1.17; P, 2.6%). The corresponding aglucone, isolated from the product of complete hydrolysis of compound 3, was converted into the acetyl derivative in the usual way. The acetyl derivative, crystallized from EtOH, was found to be identical with l,lO-di-O-acetylpseudolycorine, mp and mmp 203-205” (co-HPLC, MS) [7]. Acknowledgements-We are indebted to the Regional Sophisticated Instrumentation Centre, Central Drug Research Institute, for some of the analytical facilities. S.K.S. and S.U. thank

Alkaloid

the University fellowships.

Grants

Commission,

metabolism

New Delhi, for research

REFERENCES

1. Goodwin, T. W. and Mercer, q. I. (1988) Introduction to PIant Biochemistry, 2nd Edn., Chap. 3. Pergamon Press, New York. 2. Ghosal, S., Saini, K. S. and Razdan, S. (1985) Phytochemistry 24,214l. 3. Ghosal, S., Kumar, Y. and Singh, S. P. (1984) Phytochemistry 23, 1167. 4. Ghosal, S., Srivastava, A. K., Srivastava, R. S., Chattopadhyay, S. and Maitra, M. (1981) Planta Med. 42, 279. 5. Ghosal, S., Saini, K. S. and Frahm, A. W. (1983) Phytochemistry 22, 2305. 6. Ghosal, S. (1985) Phytochemistry 24, 1807.

in Crinum asiaticurn

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S. (1987) Phyto7. Ghosal, S., Singh, S. K. and Unnikrishnan, chemistry Xi, 823. 8. Ghosal, S., Shanthy, A., Kumar, A. and Kumar, Y. (1985) Phytochemistry 24, 2703. 9. Bose, J. C. (1926) The Nervous Mechanism of Plants. Longman Green, London. IO. Singh, R. P. and Pant, N. C. (1980) Experientia 36, 552. Il. Ghosal, S., Kumar, Y., Srivastava, R. S., Singh, S. K. and Lal, J. (1989) Proc. 32nd. IUPAC Cong., Stockholm, Sweden III, 3533. 12. Asakawa, T. A., Russell, T. R. and Ho, R. J. (1976) Biochem. Biophys. Res. Commun. 68, 682. 13. Robinson, T. (1981) 7’he Biochemistry ofAlkaloids. 2nd Edn. Springer, New York. 14. Lembeck, F. and Sewing, K.-Fr. (1966) Pharmacological Facts & Figures, pp. 104109. The English University Press, London. 15. Ghosal, S., Mittal, P., Kumar, Y. and Singh, S. K. (1989) Phytochemistry 28, 3193.