Formation of alkaloids in suspension-cultured Colchicum autumnale

0031-9422/88 $3 oO+OOO PergamonPress plc

Phytochemrstry, Vol 27, No 5, pp 1371-1374, 1988 Prmtedm Great Brltam

FORMATION

OF ALKALOIDS COLCHICUM

TAKAHISA HAYASHI,*

IN SUSPENSION-CULTURED AUTUMNALE

KEIICHIRO YOSHIDA

Basic Research Laboratory, Central Research Laboratories, Ajnomoto

and

KONOSUKE

SANO

Co. Ltd, l-l Suzuki-cho, Kawasakr 210, Japan

(Receloed24 August 1987) Key Word Index-Colchrcum colchline

autumnale,Liliaceae, plant tissue culture, growth substances; nutritional factors;

alkaloids.

Abstract-Undifferentiated callus tissues were induced from flowering shoots of Colchicum autumnale by treatment with 2,4-D The callus tissues producing colchicine have been grown in modified Murashige & Skoog medium contammg IBA and kinetin as growth factors. The type of nitrogen source in the medium influenced alkaloid formation. Although nitrate or ammonium as the sole nitrogen source inhibited the formation of colchicine as well as growth, the formation and growth were better with 20mM ammonium plus 40mM nitrate. Addition of SOi-(20mM) markedly increased the formation of colchdne. At high concentrations, PO:-, CaZ+, and Fez+ were inhibitory for formation. The identity of colchicine formed by callus was confirmed by UV and mass spectrometry analyses.

INTRODUCTION Colchicum

species produce alkaloids which have been included in many pharmacopoeias Colchtcine is used m the treatment of gout and demecolcme is an antineoplastic agent. Colchicine was originally used m biological experiments to induce polyploidy or multiplication of the chromosomes in a cell nucleus [l]. Currently, the utilization of this alkaloid is mcreasmg m the area of plant breeding; for instance, to produce either female or male sterility to make first filial generations by hybridization. Recently, many useful secondary metabohties have been obtamed in large amounts from tissue cultures of higher plants, e g shikonin from Lithospermum erythrorhizon [2], saponm from Panax ginseng [3], berberme from Berberls wtlsonrae [4], Rosmarmic acid from Coleus blumel [S] The levels of the metabolites in the above cases are higher than those of Intact plants. Using the same rationale, we may expect Colchicum cultures to be able to produce colchicme alkaloids in a fermentor tank. The callus of Colchrcum autumnale has already been induced and cultured in Lmsmaier & Skoog [6] medium containmg 2 ppm 2,4-D and 0.3 ppm kinetin as reported by Hunault [7]. However, no data were presented on the formation of alkaloids. The present communication describes the production of alkaloids m suspension-cultured Colchlcum cells and examines the effect of growth substances and nutritional factors on the formation of colchicine. This is the first report on the production of colchicme alkaloids by plant tissue culture RESULTS

Plant hormones Callus tissues induced by 10m5 M 2,4-D and grown for two months was cultured m Murashtge & Skoog [lo]

*Author to whom correspondence should be addressed.

medium containing 0.5 PM IBA plus 0.5 PM kinetin because cell growth was inhibited by 2,4-D and levels of colchicine alkaloids markedly decreased in the medium containing 2,4-D. The calluses were transferred to solid medium containing 0.5 PM IBA plus 0.5 PM kinetin at two month intervals. The formation of colchicine and callus growth was stable m the medium containing IBA and kmetm during growth. Although IAA could be used instead of IBA as an auxin, the callus generated mostly rootlets during growth. The morphological phenomenon is similar to that of callus cultured m auxin-free medium. The results suggest that 2,4-D is required for the formation of callus from Colchicum tissues but represses the formation of alkaloids, and IAA is an unstable auxm, probably due to the degradation by IAA oxidase [ 111. Effects of IBA and kmetin on colchicine formation and growth of suspension-cultured Colchlcum cells were examined. Colchicine formation was stimulated by 0.5 FM kinetin, but htgh concentrations (5 PM) of kinetin were inhibitory (Fig. 1) IBA stimulated cell growth in the presence of kmetin. We therefore conclude that 0.5 PM kmetm plus 0 5 PM IBA is optimal for colchicine formation and cell growth. The time course of colchicme formation in callus cultures on medium with 0.5 /*M IBA plus 0.5 PM kinetin is shown m Fig 2. The formation was parallel to growth until the cessation of cell growth after seven week.

Carbohydrates Several carbohydrates, e g. xylose, arabinose, glycerol, galactose, maltose, and cellobiose, were less effective than sucrose for growth and alkaloid formation. Glucose and fructose supported growth to a limited extent (80 and 60% of sucrose control, respectively) and lower levels of colchicine than sucrose as carbon source. The results showed that sucrose is the only effective carbon source for colchicine formation m suspension-cultured Colchlcum cells.

1371

1372

T

0 pM

HAYASHI

et al

Kbnetin

6

ij

6-

Nf+ 4_

”

Yi t

2-

0 I /

I 3

I 5

Sucrose

(%I

Fig 3 Effects of sucrose concentrdtlon

t -

6

I

0 5 pM

formatmn

\ . 7-J:

4

0

on colchunc

and cell growth

Klnetin

;

2

I IO

r 8

z

,

Z6-

F

~ 5 pM

N 0 -4-

K,net,n

.

;

L iL 0

IBA

(pM1

\

2-

\.

I 20

I i”,,

I 40

I 60

I

I 40

I 30

I 20

I 0

60

1 Effects of IBA and kmetln on colchlcme cell growth

formatlon

-

I 0

NO,

Fig

l

l

(mM)

ana Fig 4 Effects of NH:

and NO, on colchlcme cell growth

formalIon

and

Nttroyen sources

1

0

2

I 4 Weeks

Fig 2 Time cource of cell growth

I 6

I 8

and colchlcme

formanon

High concentrations of sucrose (more than 5%) were mhlbltory to growth (Fig 3) Both colchlcme formatlon and growth were highest with a 3% sucrose concentratIon

The blosynthesls of shlkomn derlvatlves IS markedly inhibited by NH:, while NO; has no slgmficant effect [S] Recently, Yazakl et ~11.[9] reported that the represslon of shlkonm production m Lrthospermzcm cells cultured m a medmm contammg NH: IS closely related to the accumulation of glutamme m htgh concentrations. Therefore, the effect of each nitrogen ion on the formation of colchxme alkaloids was tested The ratio of NH,’ and NO; m the basal medium (60 mM as N) was changed without altering the concentration of nitrogen sources When NH: or NO; was used as the sole nitrogen source, formatlon of coichlcme and cell growth were markedly inhibited (Fig 4) Cell5 grew hctter III the presence of NH,’ and NO, m the proportion of 1 1. but the formation of colchlcme was inhibited at a higher ratio of NH,f to that of NO; The concentration (O--l00 mM) of nitrogen sources m the medium was changed without altering the ratio of NH; and NO; (1 2) The optimal nitrogen concentration was 60 mM for both growth and colchlcme formation The result\ suggert that both NH; and NO, are reqmred for the formation of colchlcme and growth We have concluded that 20 mM ammomum

Alkaloids

plus 40 mM nitrate and cell growth.

1s optimal

for colchicme

of cultured

Colch~cum

1373

formation

Inorganzc ions Sulphate ton as sodium sulphate markedly increased colchicine formation (Fig. 5). The mcrease was observed at a certain concentration between 20 mM and 50 mM. The effect of phosphate as potassium hydrogen phosphate (KH,PO,) and calcmm as calcium chloride on colchitine alkaloid formation were tested. Both PO:and Ca’+ inhibited the formation at high concentrations, 2.5 mM and 10 mM, respectively (Ftgs 6 and 7). Mtzukami et al. [ 121 reported that Fe2 + stimulated growth but inhibited shikonin derivative formatton at htgh concentrations (more than 100 PM). The effect of Fe’+ as ion sulphate on colchtcme alkaloid formation was to increase formation at 100 /.LM and inhibit it at higher concentrations (1 mM) (Fig. 8)

0 PO:-( mM Fig 6 Effects of PO:-

on colchlcme

)

formatton

and cell growth

Identzjcatzon of Colchzczne In order to identify the colchicine formed m suspension-cultured Colchzcium cells, large quantities of cells were cultivated in modified Murashtge & Skoog’s medium modified as above and named ‘Co1 medium (Table 1). Ten grams of cells were extracted with 30 ml of ethanol and the extracts were analysed by HPLC on acetowith elution RP-18 and Ltchrosorb nitrtle-methanol-water, (7.1: 12) The fraction corresponding to colchtcine was colleted and further purified by HPLC usmg gradient elution with 5% to 90% acetonitrile. The isolated sample had an A maxima at 245 and 350 nm and mmtma at 220 and 292 nm. The sample was further subjected to mass spectrometry. The spectrum of the sample exhibited the most mtense fragment peak at m/z 312 [13]. This is probably formed by loss of acetamide from the [Ml’. The ion further decomposes either by loss of a methyl group or by loss of an aromatic methoxyl group to sire peaks at m/z 297 and 281, respectively. The peak at m/z 371 is formed by the loss of carbon monoxide from the [M] +. The UV and mass spectra of the isolated sample were identical to those of colchtcme [13]

4-H+7@ Ca"(mM) Fig 7. Effects of Ca*+ on colchlcme

0

001

formatlon

01

and cell growth

I

Fe’+ (mM)

Fig. 8. Effects of Fe’+ on colchlcme

formation

and cell growth

DISCUSSION

0

Fig 5. Effects of SOi-

PHYTO27:5-J

20

40

60

SOf

( mM1

on colchlcme

80

100

formatlon

and cell growth

We have developed a culture medium for C autumnale tissues allowing productton of colchicme for the first time. The medium developed is given m Table 1 as COL medium, notably, it requires high concentrattons (2&50 mM) of sodium sulphate for colchtcine formatton (Ftg. 5). Since sodium chloride did not affect the formation of

1374 Table

T

1 Constituents Colchtcum

Constituent IBA Kmetm Sucrose KNO, NH,NO, Na,SO, CaCI, 2H,O KH,PO, FeNaEDTA CoCl, C&O, H,BO, Kl MgSO, 7H,O MnSO, 4H20 Na,MoO, 2H,O ZnSO, 7H,O Inosltol

Nlcotmlc acid Thtamme HCI PyrldoxmeHCI Glycme

of the tissue culture medmm autumnale (COL medium) mgll 01 01 30 000 1900 1650 2840 120 170 36 7 0025 0 025 62 0 83 370 22 3 0 25 86 100

05 01 05 20

HAYASHI

used for

mM ~_ 0 0005 0 0005 88 18 8 206 20 30 1 25 01 00001 000016 01 0005 15 0 1 10 003 056

0 004 0 0003 0 0025 0 027 _

et al

callus mto test liquid medmm, small pieces of the stock callus were transferred to the same basal hquld medium (50 ml) with no hormones m 200 ml Erlenmeyer flasks for 3 days to adapt cells to hquld medrum Culture condltmns Murashlge & Skoog [lo] me&urn contalrung 0 5 PM IBA and 0 5 PM kmetm was used as the starting hquld medmm Ca 125 mg fr wt of suspension-cultured callus was maculated mto a 50 ml Erlenmeyer flask contammg 10 ml of test medium, after which the callus was cultured at 25” on a rotary shaker (100 cpm) m the dark for 4 week Determmatmn of content of colchwne alkalmds Fresh callus tissues (2c-30 mg) were extracted with 100 ~1 of ethanol at 80 for 30 mm The mlxt was cooled and centrifuged at 17000 rpm for 20 mm and the supernatant (10~1) analysed by HPLC on a LlChrosorb RP-18 (5 pm) column L16] Analysis was carried out at room temp usmg MeCN--MeOH-2OmM K,PO, (pH 62) (7 1 12) A UV momtor operated at 350 nm was used as detector Alkaloid content was expressed as pg.‘g fr vv( Spectral analyvc MS were measured with d direct sample inlet system at 220“ Iomzatlon energy was mamtamed at 70 eV, the lomzmg current at 3OOpA Acknowledgements~~-We thank Dr T Furuya (Kltasato UmversIty, Japan) for his valuable advice and Dr H Flores (LouIslana State Wmverslty, U S A), for stlmulatmg dlscusslon durmg the final preparation of this paper We also thank Director K Tahara of this laboratory for his guidance and encouragement and MS M Furuya for performmg the MS

REFERENCES

colchlcme alkaloids, the effect was probably due to sulphate Ions We have not identified demecolcme, which 1s an Important component of the alkaloids found m Intact Col&cum tissues By HPLC analysis of callus extracts, the peak which corresponded to demecolcme was obtained only occaslonally when high levels of colchlcme accumulation were obtamed The formation of demecolcme probably requires high levels of colchlcme accumulation which may control the biosynthetic pathway between demecolcme and colchlcme However, the amount of colchlcme m our callus cultures (30-40 pg/gfr wt) IS usually ten times lower than that m Intact corms (240-400 pg/gfr wt) Light lrradlatlon also did not mcrease the formation of alkaloids, although hght may stimulate the metabohsm of phenols which are mvolved m the blosynthesls of colchlcme [ 141 Further studies on the productlon of colchlcme alkaloids examme the regulatlon of colchlcme blosynthesls [ 151 EXPERIMENTAL

Cultured callus Callus tissues were derived from flower shoots of C autumn& cultured on Murashlge & Skoog [lo] basal agar medium contammg lo-’ M 2,4-D and 10m6 M kmetm These tissues were transferred to the same basal medium contammg 0 5 1tM IBA and 0 5 PM krnetm every 2 months All cultures were mamtamed at 25” m the dark Before moculatlon of the

I

2 3 4

9 10 I1 12

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