Production of lignans in Forsythia intermedia cell cultures

Phytochemlstry,Vol 29, No 6, pp 1861-1866,1990. Prmted m Great Bntaln

PRODUCTION

003l-9422/90 $3 00+ 0.00 0 1990PergamonPressplc

OF LIGNANS IN FORSYTHIA CULTURES

INTERMEDIA

CELL

MAIADA M. A. RAHMAN, PAUL M. DEWICK, DAVID E. JACKSON and JOHN A. LUCAS Departments of Pharmaceutical Sciences and Botany, Umversity of Nottingham, Nottmgham NG7 2RD, U.K (Recewed 18 September 1989)

Key Word Index--Forsythia intermeduz;Oleaceae; tissue cultures, hgnans.

Abstract-Callus and cell suspension cultures of Forsythia intermedza have been established and their capacity to produce lignans studied. Lignan production m both callus and cell suspension was markedly dependent on the culture medium used and not on the source of the original explant, although lignan patterns m the explants are different. Cell lines were established which synthesized either matairesinol 4’-0-glucoside or epipmoresinol 4’-0-glucoside as the major lignans. Cell suspension cultures rapidly metabohzed exogenous hgnans without producing detectable hgnan transformation products.

INTRODUCTION

Analysis of leaf and stem tissues of Forsythia intermedia has demonstrated that these are a rich source of dibenzylbutyrolactone and furofuran lignans [ 11. Analysis identified them as arctigemn (5), matairesinol(3), phillygenin (9) and epipinoresinol (7), together with their 0-glucosides arctiin (arctigenin 4’-0-glucoside, 6), matairesmol 4’-0glucoside (4), phillyrm (phillygenin 4-0-glucoside, 10) and epipinoresinol 4’-0-glucoside (8). The lignan contents varied with season and also with tissue type, so that the furofuran derivatives epipinoresinol/epipinoresinol glucoside predominated in leaves, whilst the dibenzylbutyrolactones matairesinol/matairesinol glucoside represented the major lignan material in stems. In young shoot tips, however, arctigemn/arctim were the main lignan constituents isolated. Biosynthetlcally, these lignans are likely to be derived from a common pathway, which subsequently branches mto furofuran or dibenzylbutyrolactone systems. This communication describes the establishment of callus and cell suspension cultures from F. intermedla tissues and production of lignans m these cultures. The lignan profiles appear to depend on culture conditions and this has allowed the establishment of cultures giving either furofuran or dibenzylbutyrolactone lignans as predominant metabolites, thus substantiatmg the proposed biosynthetic relationship between the two classes of lignans. Some of the results have been presented earlier in abstract form [2]. RESULTS AND DISCUSSION Callus initiation and growth

Callus initiation from leaf, stem and shoot-tip explants was assessed on different media based on Murashige and Skoog (MS) salts [3]. No callus formation occurred on media lacking growth hormones (MSO, see Experimental). MS salts supplemented with 2,4-D (Ml) sup-

ported callus growth without regeneration. Media containing NAA and BAP (MSPl) initiated callus growth but also regeneration of plantlets from leaf, stem and shoot tip eiplants In media where BAP was replaced by kinetin (MS2NAA and MS), proliferation of callus with regeneration of roots was observed. The best callus response was obtained on M8 medium, in which the concentration of MS salts is reduced to one-third, where callus was initiated from explants within 14 days of culture. Maintenance and subculture of callus on this medium led to a reduction in, and ultimately complete loss of, root generation. M8 was also superior to White’s [4] and Gamborg’s B5 [5] media for callus maintenance, while Heller’s medium [6] did not support callus growth. Lignan production m callus cultures

For lignan analyses, calluses were divided into two halves, one for extraction, the other for subculture. The tissue was freeze-dried and extracted with methanol, this extract being analysed by HPLC using the procedure developed earlier for separation of the lignan mixtures [ 11. Alternatively, enzymic hydrolysis of the crude methanolic extract using /?-D-glucosidase was employed. HPLC analysis then quantified lignans in terms of the aglycones [l]. Llgnans were identified by comparison with authentic material isolated from intact plant tissues

111.

In preliminary experiments, callus was initiated from leaf explants and maintained on Ml medium. Analysis of this callus showed that epipinoresinol4’-0-glucoside was the predominant hgnan produced. Other Forsythia hgnans were absent, or could be detected in minor quantities only. Callus maintained on other media, namely MS, B5 and White’s, yielded matairesinol-4’-0-glucoside as the major product. Lignan analyses (in terms of total aglycones quantified after fi-glucosidase treatment) for call1 cultured on various media maintained under different growth conditions are given in Table 1. Thus,

1861

M M A RAHMAN et al

1862

Table Culture medmm Ml MS

B5

White’s

1. Llgnan

Condltlons’

productlon

Matalresmol

2 3 1 2 3 1 2 3

*l five-week-old t Llgnan content tr = trace

derived

from Forsythia

110 65 78 58 II 2 63 1s 32 7 430

rntermedm leaf

Yield (mg/g dry cell wt)t Phlllygenm Eplpmoresmol Arctlgenm tr

62

3

I

m callus cultures

tr

tr tr tr tr

tr 12

callus grown m dark, 2 12-week-old callus grown m dark measured m terms of aglycone after enzymlc hydrolysis

matairesinol (as the glucoside) is the sole product from calli maintained on M8 medium, except under condtttons (1) when a trace amount of arctigenin was also detected. Call1 grown on B5 medium produced trace amounts of other lignans as well as matairesmol, whtle call] cultured on White’s medium produced significant quantities of arctigenin and phillygenin m addition to matairesinol and traces of epipinoresmol. In contrast, epipmoresmol was produced in significant quantities only usmg Ml medium; it then formed the bulk of the lignans, only traces of phdlygenm bemg detected Lignan yields were highest from calli maintained on White’s medium, especially when cultures were grown m the light. However, this medium supported only very slow growth of callus. White’s medium in general is deficient m salts, especially phosphate and nitrate, compared to other media Comparison of secondary metabolite and biomass yields on the media tested is consistent with the concept that secondary metabolism becomes important only after the growth phase when nutrients are consumed. However, the slow growth rate on White’s medium, coupled with the observed syntheses of predominantly a single hgnan m other media, made M8 and Ml the media of choice for further studies. Calli mitiated and grown in different media produce hgnan patterns quite different from those of the origmal explants [l]. Accordingly, this change was momtored during subculture. Leaf and stem explants from young Forsyth shoots were divided mto two parts, one for analysis and the other for culture. Callus was initiated and mamtamed on M8 medmm either under constant illummation, or in the dark Calli were subcultured onto fresh medium every 28 days and maintained under the same condittons, with samples removed for analysis at every subculture. Ltgnans were analysed m terms of aglycones quantified after /I-glucosidase hydrolysis of the crude extract. The results (Fig. 1) showed pronounced but surprisingly similar changes m hgnan patterns m each case. Leaf explant material contains high amounts of arctigemn, medium quantities of epipinoresinol and phdlygemn, but very low contents of matairesinol. On culture, there was a complete loss of epipinoresmol, a decrease to msigmficant contents for phillygenm and arctigenm content also fell to minimum values. In contrast, matairesinol production

tr tr 35 tr tr

Callus consistency brown, friable white, friable white, friable green or white, frtable white, friable white, fnable green, hard white, hard white, hard green, hard

3 12-week-old

callus grown

m hght

increased rapidly over three to four subcultures to a maximum and subsequently declined. Explants from young stems contamed much more matatresinol than leaf explants, but arctigenin agam predominated; epipinoresmol was the minor lignan constituent. Calli from stems showed essentially the same pattern of change m lignans as did the leaf call1 Thus, there was a complete loss of eptpmoresinol, a decrease to insignificant contents for phillygenm and arcttgenm, but a large increase m matairesinol production, dechnmg after three to four subcultures Light grown and dark grown cultures were not markedly different, though stem cultures grown in the dark contamed no detectable amounts of arctigenm, phillygemn or eptpinoresinol. Although matairesmol 1s present in different amounts m the dried leaf and stem material Cl], and m low concentration m the young growth of Forsyth plants used for explants, production of this hgnan m culture was independent of the ortgm of the explant and reached even higher contents (ca 18 mgg- 1 dry wt) than the maximum recorded for dry stem material (ca 15 mg g- ’ dry wt) [ 1). The hgnan profile in the callus appears to be dependent only on the culture medium employed. Despite the subsequent decrease in matairesmol, none of the other hgnans increased again, and it is presumably catabohzed or directed towards synthesis of hgnin

Lignan production in cell suspensron cultures Call1 cultured on Ml or M8 media were used to initiate cell suspensions three weeks after subculture, transferring to equivalent liquid media and culturing under constant tllumination. Cell cultures in Ml liquid medium were dark brown and contained large clumps of cells, whereas M8 medium gave green or white cells of a more dispersed nature Analysis of the harvested cells showed they were still capable of lignan synthesis, but the amount of lignans produced was usually less than the amount produced in the parent callus (Table 2). Cultures grown in Ml hqmd medium gave epipmoresmol4’-0-glucoside in yields of up to 3 mgg- ’ dry wt, dependmg on cell lure used Cells cultured m M8 liqmd medium were better lignan producers, giving mainly matauesinol 4’-0-glucoside, with typical yields of 10 mg g- 1 dry wt

Llgnans

from Forsythia

26 24 22 20 18 16 14 -

1863

cultures

Matalresmol Plnllygenm n Eplpmoresmol 0 Arctlgerun q l

M .

F

12 IO 8642OExplant

I

I

I

I

I

1

2

3

4

5

18 16 14 12 IO 8642O-

I

Explant

I

I

Explant

1

I

I

I

I

2

3

4

5

Number of subcultures

Number of subcultures

26 r Cc) 24 22 20 18 16 14 12 - . 10 86- . 42_& O- t

I 1

26cd) 24 22 20 18 -

‘c-o_..-.

n Matsuresmol l Plullygenm . Eplpmoresmol o Arctlgenm

.-.-.-.-.

I

I

I

I

2

3

4

5

Explant

1

2

3

4

5

Number of subcultures

Number of subcultures

Fig. 1. Effects of subculturmg on hgnan production m Forsythta mtermedm callus cultures (M8 medium). A. Lightgrown callus derived from leaf explants, B hght-grown callus derived from stem explants; C. dark-grown callus derived from leaf explants; D dark-grown callus derived from stem explants.

Table

2 Comparison of hgnan productIon m Forsythra mtermedm callus and cell suspension cultures derived from them

1dry cell wt)

Culture

Medmm

Llgnans present (mg g-

Callus

Ml

Eptpinoresmol4’-0-glucoslde Phlllyrm (1.8) Eplpmoresmol (1 3) Eplpmoresmol 4’-0-glucoslde

(7 3)

Callus Cell suspension

Ml

Eplpmoresmol None detected

(2)

Callus Cell suspension

MS

Matalresmol 4’-0-glucoside Matalresmol4’-0-glucoslde

Cell suspension

Lignan production rate was assayed in a matairesinol 4’-0-glucoside producing cell line and related to cell growth rate (Fig. 2). Aliquots of lCday-old cells were subcultured into eight replicate flasks. Growth rate was monitored every three to six days on the basis of packed cell volume and dry cell weight, and cell viability was assessed by staining with the vita1 dye fluorescein diacet-

4’-0-glucoslde

(1.4)

(18) (10)

ate (FDA). Analysis for lignans was carried out on both cells and culture filtrates, but under normal conditions, lignans were not excreted into the medium until the culture was m the late stationary phase, when trace amounts could be detected. The cell suspensions gave a sigmoid curve with a lag phase of five to six days and an exponential phase of nine to 15 days (Fig. 2). The

1864

M M A

RAHMAN

et al

Table 3 Effect of subculturmg on matatresmol productron m Forsythia cell cultures 80 2 3

70 -

2 2

60-

d ;

50 -

Callus Subculture Subculture Subculture Subculture Subculture Subculture

0

-

40.

B 3 5

30-

= -s

20 -

“u a”

IO-

@

o-

Matatresmol (mgg-’ dry cell wt)

Culture*

1 2 3 4 5 6

132 44 67 80 79 70 61

*Subculture pertod 3 weeks

-I i 0

0

Days

Ftg 2. Growth pattern and hgnan productton m Forsytha mtermedla call suspenston culture (M8 medmm)

matairestnol content, measured as total aglycone after hydrolysis, increased in parallel with the increase m biomass durmg the exponential phase. The percentage of Productton of viable cells decreased wtth time matairesinol contmued to increase, reachmg a maximum at the stationary phase, day 27, after which levels started to decrease, though minor quantities of epipmoresinol were then observed. No released matairesinol could be detected in the medmm except for trace amounts at day 40. The effects of subculturmg on lignan productton were studied by transferring an aliquot of suspended cells to fresh M8 medium every three weeks. The rest of the cells were extracted and matairesmol quantified after enzymic hydrolyses (Table 3). The first generatton of the cell suspenston culture showed low yields of matatresmol (ca 4 mgg-’ dry wt cells), although m subsequent generations levels increased to ca 8 mg g-t. However, quantities never reached those of the parent callus (ca 13 mgg-‘). For subsequent expenments, especially for metabohc studies, cell suspenston cultures were generally used only after a stabilization period of culture (three generations). However, there was a gradual decline in productton after four subcultures which made tt dtfficult to keep a high

yielding cell suspensron culture growing over long periods. To overcome this, the period between subcultures was extended to three months during which high yteldmg suspenstons could be maintained. Forsythra cells do not seem to be adversely affected by the long term maintenance in M8 hqurd medium, based on their reasonable viabthty (> 50% after three months), and healthy appearance (consistency and colour). It was found that productton of matanesinol 4’-Oglucoslde was optrmal when cells m suspension were subcultured using the same medium as they had been maintained m, namely M8 hqutd medrum (Table 4). When cells were transferred to Ml medmm, the amounts of matairesinol glucosrde decreased markedly, with less pronounced falls observed when the cells were transferred to White’s medium However, under both condmons, trace quantmes of arctlgenm (after hydrolysis) were observed. When the cultural condmons were changed by transferring from light to dark, the result was a decrease m productton to ca one-fifth the control value, wtth no other detectable hgnans produced. The ability of F rntermedza cell suspensions to synthestze these high quantities of matatresmol 4’-0-glucostde has been exploited to produce 14C-labelled matanesinol by feedmg [U-14C]phenylalanine to cell suspenston cultures [7]. The labelled precursor was added to a 16-dayold culture, correspondmg to the begmmng of the stationary phase, and allowed to metabolize for three days This gave a good yteld of matau-esmol with high mcorporatton of label and of sufficient spectfic acttvtty to be used m further feeding expenments [7]_

Table 4 Influence of change of medium on matanesmol productton m suspenston cultures of Forsytha rntermedla Medmm Old

New

Matanesmol content (mgg-’ dry wt cells)*

M8 M8 M8 M8 (Lrght)

M8 Ml White’s M8 (Dark)

85 15 52 17

Other hgnans

tr arctrgenm tr arctigemn

* Lrgnan content measured m terms of aglycone after enzymtc hydrolyses tr = trace

Llgnans

Scheme

Table

1. Proposed

5 Metabohsm

from Forsythra

txosynthetlc

pathway

of Forsythm hgnans

cultures

to hgnans

added

1865

m Forsythia mtermedra

to suspension

cultures

Plant cells

Cell line

Culture medium

Volume of culture (ml)

Incubation time (days)

Lignan

Forsythza IntermedIa

A

M8

7 7 7 1 2 3 1

Arctlgenin (4) Phdlygemn (4) Epqunoresmol Epqnnoresmol Eplpmoresinol Epipmoresmol Epipinoresmol

(4) (10) (10) (10) (10)

1

Epqxnoresmol

(56)

Forsythia mtermedm

B

M8

Forsythia mtermedla Solanum dulcamara

C

MSPl

25 25 25 25 25 25 25

D

MSPI

65

Metabolic studies

Based on a structural analysis of the lignans found in F. intermedza [l], a biosynthetic sequence (Scheme 1) may be proposed. The two groups, furofuran and dibenzylbutyrolactone hgnans, are envisaged as arising from a common quinonemethide intermediate (2), derived by phenohc oxldative coupling of two phenylpropane units, here postulated to be comferyl alcohol (1) units. The furofuran hgnans are produced from 2 by nucleophilic additions, whilst the dlbenzylbutyrolactone derlvatlves reqmre reduction and formation of the lactone ring. The overall effect of culturing F. intermedza cells appears to be channelhng of biosynthesis to the production of epipinoresmol or matalresmol glucosldes as the predominant lignans. This effect was noted in both callus and

added (mg)

Lignan

recovered

(mg)

Epipmoresinol (medium, 2; cells, 5 3)

suspension cultures. Thus, m the postulated pathway, one of the routes from the quinonemethide (2) is blocked, or highly inhibited in each case, resulting in formation of either dlbenzylbutyrolactones or furofurans as the predominant lignans. The biosynthetic pathways to phillygenin or arctigenin, presumably involving methylation of eplpinoresinol and matairesinol, respectively, are also blocked. However, glucosylation IS not impaired. These observations point to blocking of certain biosynthetic pathways in the cultured cells, presumably by repression or absence of critical enzymes. A maJor block after the common quinonemethide seems to exist m each culture. In addition, the capacity of the cultures for further methylatlon of the lignans seems to have disappeared, though glucosylatlon 1s not impaired. These

M M A RAHMAN et a/

1866

patterns of hgnan production m the various cell hnes are consistent wtth the btosynthetic pathway m the normal plant being as postulated m Scheme 1. If blocks m the pathway are relattvely early, then it may be posstble to detect methylation and/or glucosylation of hgnan aglycones by feedmg experiments m the appropriate cell lines Accordmgly, each of the hgnan aglycones arcttgenm, phtllygenm and eptpmoresmol, dissolved in ethanol or DMSO, was fed to a lCday-old culture producing matairesmol4’-0-glucoside. The latter solvent was better tolerated by the cells, ethanol causmg some browning and reducmg the growth rate Both cells and medmm were analysed for lignans after seven days Neither cells nor medtum contained hgnans other than matatresmol 4’-0glucoside (Table 5) Over shorter mcubatton periods, eptpmoresmol was found to rapidly dtsappear, bemg catabohzed within the first day When a different cell hne, this ttme an unproducttve one, was used, eptpmoresmol was agam found to disappear after only one day Thts seems to Indicate that Forsythia cells are capable of metabohzmg exogenously fed hgnans normally produced by the plant. However, a cell suspenston culture of Solanum dulcamara was also shown to rapidly metabohze large amounts of eptpmoresmol, with 87% of the hgnan disappearmg wtthm one day This seems to suggest that plant cell suspension cultures may have high capacity for catabohzmg lignans, whether the ortgmal plant synthesizes them or not Although the subsequent fate of the added hgnan 1s not known, there ts a high probabthty it may be mcorporated mto hgnm EXPERIMENTAL TKWZ culture

media.

Murashige and Skoog medium was purchased (Flow Laboratories) m powder form and modified by the addition

of other components

as follows

MSO, no addltlons,

MSPI, NAA (2 0 mg I- ‘), BAP (0 5 mg I ‘), MS2NAA, cavern hydrolysate (500 mgl-I), NAA (2 0 mgl-‘), kmetm (0 2 mgl-‘), M8, one-third MS salts concn plus casem hydrolysate (500 mgl-I), NAA (20 mgl-I), kmetm (0 2 mgl-‘), thidmme (9 mgl- ‘), Ml, 2,4-D (1 mgl- ‘) In each case, the sucrose content was 30 gl-‘, pH was adjusted to 5 8 Gamborg’s B5 medmm was also purchased m powder form (Flow Laboratories), White’s [4] and Heller’s [6] media were prepd according to ht Sucrose contents were 20 g I- ’ and pH was adJusted to 5 5 Media were prepd m hquld form, or sohdlfied with 08% wjv agar The cell suspension culture of Solanum dulcumara (kindly provided by Dr P K. Chand, Botany Department) was cultivated and mdmtakned m hquld MSPI medmm Preparatum of explants Young shoots of garden-grown F cntermedm were used as explant source material After excision, shoots were kept under H,O prior to prepn and then washed brlefly with H,O Shoots were sepd mto leaf and stem material, then surface sterlhzed m 2 5 or 5% NaOCl soln, respectively, for 15 mm followed by at least 3 washes m sterile dust H,O Any damaged tissue was removed and explants (ca 1 cm length/ diameter) were transferred to sterilized medmm Callug rnrtlatron and culture Explants were placed on the surface of 50 ml agar-sohdlfied medium m Powder Round Jars, or on 25 ml sohddied medium m 9 cm Petri dishes Petri dishes were sealed with Nescofilm Cultures were mamtamed either m

the dark, or under 3000 lux constant dlumrnatlon provided by dayhght fluorescent tubes All call1 were kept at 25 k 1” and were routmely subcultured onto fr medmm every 4 weeks Suspension culture wutmtm and mamtenante Callus (2.5 g) was suspended m hquld medium (65 ml) in a 250 ml comcal flask and agitated on a rotary shaker at 80 rpm under constant light (2000 lux) at 25 & 1 Fourteen days after mltlatlon, the cell suspension was subcultured by tramferrrng IS ml ahquots of suspended cells to Rasks contammg fr culture medium (50 ml) Growth measurements Fr wts were measured directly and dry wts after freeze drymg of tissue Packed cell vol was measured after ca 30 mms sedlmentatlon of a suspension culture m a measurrng cylinder Cell vlablhty was dsse\sed by the fluorescem dlacetate stain method [8] L~ynan analyrrc Callus cultures were freeze-dried. then powdered m a mortar Samples (50 mg) were extracted by stirring with MeOH (3 5 ml) m 5 ml Redctlvlals at 70’ for I hr The mlxts were then filtered, the residue washed with further MeOH and the combmed crude extracts evapd to dryness Enzymlc hydrolySIS, where appropriate, was conducted by addmg a soln (2 ml, 2 mg ml- ’ in 0 1 M NaOAc buffer. pH 5) of b-o-glucosidase (Sigma) and stirring at 37 for 24hr The hydrolysate was dll extracted with CH,CI, with H,O (IO ml), then (3 x 10 ml) The combined extracts were evapd to dryness and analysed by HPLC usmg a Spherlsorb S5 ODS? column (250 x 46 mm) with d precolumn of the same packing material, MeOH-H,O (9 11) with a flow rate of 0 84 ml mm ’ with UV detectlon at 280 nm Peak areas were determined by integration. with calibration usmg ref solns of authentic hgnans [I] R,s (mm) were as follows eplpmore\mol3’-0-glucosldejmatalresmol 4’-0-glucoslde, ca 8, arctnn, c (I 13, phlllqrm, ca 15, matalresmol, ca 22, eplpmoresmol, cu 25, arctlgenm, ca 35, phdlygemn, ca 48 Cell suspension cultures were filtered under suction and the cells freeze-dried, powdered and cxtd ds abobe Residual medium was also extd with CH,CI, (3 x 10 ml), the combined extracts evapd to dryness, \ubJected to enrqmic hydrolysis and analysed as above Feedmy experiments m cell sucpencron cultures Llgnans (ca 4 mg) [1] were dissolved rn EtOH (2 5 ml), then filter sterlhzed and added to d 14-day-old culture (25 ml) An alternative, and preferred procedure, was to dissolve the ltgnans m DMSO (ca 0 7 ml) and add them to the culture wlthout sterlhzatlon The culture was returned to the shaker and grown on under the same condltlons for a further period (13 days) Cells and media were separately extracted and analyacd for hgnans as above REFERENCES Rahman,

M M A, Dewick, P M, Jackson, J A (1990) Phytochemlstry 29, 1971 Rahman, M , Dewtck. P M , Jackson, D E (1986) J Pharm Pharmac 38. 15P Murashlge, T and Skoog, F (1962) Ph~~rol White, P R (1954) The Cult~tarum ofAmmal Ronald Press, New York Gamborg, 0 M , Mrller, R A and OJlma, K Ret 50, 151 Heller, R (1954) Ann Bm/ 30, 261 Rahman, M M A, Dewlck, P M . Jackson, J A (1990) Phytochemlitrr 29, 1841 Wldholm. J (1972) S/urn Techno/ 47, 186

D E and Lucas, and Lucas, J A Plant

15, 473

and Plant Cells.

(1968) Eup CelI

D E and Lucas,