Effect of dikegulac on biomass and alkaloid production in <i>Catharanthus roseus</i>(L)<i> </i>G. Don under <i>tin vitro </i>condition

Indian Journal of Experimental Biology Vol. 37, June 1999, pp. 594-598

Effect of dikegulac on biomass and alkaloid production in Catharanthus roseus(L) G. Don under tin vitro condition

S Choudhury & K Gupta*

Botany Department, Burdwan University, Burdwan 713 104, India

Received 25 September 1998; revised 22 February 1999

Ca11us culture of Catharanthus roseus in LS medium was treated with different concentrations of sodium dikegulac (DK). Maximum growth was obtained in cultures with 50 )lglml after 25 days and in 100 )lglml after 30 days of every culture .cycle whereas 150 and 200 )lglml exhibited an increase in biomass only in second and first culture cycle respectively, followed by a decline. There was an increase in total alkaloid content, RNA, amino acid, soluble carbohydrate and tryptophan levels at 50 )lglml of dikegulac over control. Dikegulac at lower concentration (50 )lg/ml) seems to be a potent chemical for higher biomass and alkaloid production in long term treatment of C. rose us under tissue culture.

Catharanthus roseus (L) G. Don contains over 100 alkaloids, some with a remarkable pharmacological activity'. The most important of these components are anti-leukemic alkaloids, vincristine and vinblastine, and the antihypertensive alkaloids ajmalicine and serpentine. Low yield of antileukemic alkaloids in the plant and high market price of a1kaloids has encouraged intense research for alternative methods for the production of these alkaloids e.g. synthesis or semisynthesis² and cell and tissue culture^3. However, under in vitro conditions, the most valuable components, the dimeric alkaloids vincristine and vinblastine, have so far only been detected in callus or organ culture of C. roseus⁴. Over the years, considerable effort has been made to optimise the culture medium for growth and production of indole alkaloid by plant growth regulators⁵. Several organic compounds have also been added to the culture medium in order to enhance the availability of alkaloid precursors. Two different appr.oaches have been employed for the purpose - viz. (1) direct introduction of precursors into the culture medium, or (2) by addition of compounds interfering with precursor metabolism.

Dikegulac (sodium 2,3 :4,6-di-O-isopropylidene-2- keto-L- gulonate) is a plant growth regulator which affects plant development, internal hormone balance and modulates carbohydrate and protein metabolism⁶.

Membrane integrity is greatly influenced by

*C;orrespondent author

dikegulac application ⁷. Considering its overall regulatory property, an effort has been made presently to evaluate the effect of dikegulac under tissue culture system on growth and alkaloid production in C.

roseus.

Materials and Methods

Healthy seeds of Catharanthus roseus (L) G. Don were harvested and soaked in 0.5% tween-SO solution for 10 min and then washed for 30 min in running tap water. Futher, the seeds were surface sterilised in 3%

sodium hypo-chlorite solution for 15 min and washed subsequently 3-5 times in sterile distilled water. After sterilisation, isolated seeds were placed on Murashige and Skoog⁸ agar medium without vitamins, glycine and growth regulators in 250 ml conical flasks for gennination. From aseptically raised seedlings, cotyledonary leaf lamina were excised as explants and placed on Linsmaier and Skoog⁹ basal agar (O.S%) nutrient medium. The medium was supplemented with NAA (0.2 mg/L), BAP (2 mgIL) and sucrose (3%). Callus was initiated in this medium and subGultured on LS medium with the same hormonal composition for 6 months. Thereafter for experimental purposes, different concentrations of sodium dikegulac (50, 100, 150, 200 and 250 )lg/ml) were added to the basal medium to Si.lJdy the effect of these concentrations on growth and total alkaloid content over control. All media were adjusted to pH 5.S before sterilisation at 121°C for 15 min. Unless

CHOUDHURY & GUPTA: DIKEGULAC & ALKALOID PRODUCTION IN CATHARANTHUS 595

stated otherwise, cultures were subcultured at every 28 day interval. Cultures were incubated at 28°±2°C in dark.

All the experiments were carried out three times with 8 replica in each treatment. Growth was estimated by measuring the total fresh weight and dry weight (60°C for 48hr). Relative leakage ratio (RLR) of the UV absorbing compounds was measured following the method of Redmann et -aLlo. Injury index was measured using the method of Sullivan ".

Extraction and estimation of RNA was done according to Cherryl2. Amino acid¹³ and soluble carbohydrate I ⁴ content were also measured. Total tryptophan content (free

-+

conjugated) was estimated following the method of Mertz et atf5.

Total alkaloids were extracted and measured following the methods of Kuteny et aL. 16 with modifications as suggested by Godoy-Hernandez and Loyola-Vargas 17. Control and treated dry callus of equal quantities were extracted with ethanol. Alcohol from the extract was evaporated. The residue was resuspended in H2S04 (2.5%) and washed several times with ethyl acetate. This was concentrated and washed with chloroform. The chloroform phase was concentrated, dried and resuspended in ethanol and stored for total alkaloid determination. Total alkaloid content was determined by measuring absorbance of the sample at A = 280 nm in spectrophotometer (Beckman DU-64). Calibration curves were prepared- with ajmaline, ajmalicine, vincristine and vinblastine obtained from Sigma (U.S.A.). For quantitative determination of specific alkaloids, HPLC analyses were. made. HPLC system consisted of Waters M-510 pumps coupled to a Waters Automated Gradient controller. The sample was introduced via a Waters U6K loop (20 J.l1) injector. A Waters™ 486 Tunable Absorbance. Detector and associated Waters 746 Data Module were used for detection. A Water Sep- pak CIS Cartidge column [0.5 mm (id) x 4.2 mm] and a Waters Nova Pak C 18 [3.9 mm (id) x 150 mm]

reversed phase steel column were used. Gradient system was followed as described by NaaranLahti et aL. 18 with slight modification as - methanol- acetonitrile· - (0.025 M) ammonium acetate at an initial ratio of 13:32:55 changing to 19:46:35. Buffer was adjusted to pH 6.8. The initial flow rate was I rnJ/min and after 3 min, it was increased to 1.5 rnJ/min up to 5 min and then increased to 2 mVmin up to 10 min and continued up to 15 min. UV detection wavelength was 280 nm.

Results and Discussion

In consecutive culture cycles, it was noted that growth (both fresh and dry weight) increased gradually and was maximum at third and fourth culture cycle (Table I). In sodium dikegulac, growth was almost completely inhibited at 250 IlglrnJ on first culture (Table 2). An identical growth rate was noted in 200 IlglrnJ and in control in the first cycle, but at the second culture cycle considerable inhibition was observed. Using 150 J.lg/ml of dikegulac, increased biomass accumulation was noticed in the first two subculturings, but inhibition occurred in the third culture cycle. On the other hand, the lower concentration of 100 and 50 Ilglml increased the biomass production in consecutive three cycle and the subsequent culture cycles. Growth kinetics at 100 and 50 Ilglml were not significantly modified up to 15 days, but at the end of culture cycle, significant difference was observed (Fig. la). Higher biomass

Table I- Effect of subculturing on growth of C. roseus callus at the 30th day of culture cycle

r

Val ues are mean of 20 replicates]

Subculturing Fresh weight Dry weight stage (gil S ml medium) (giiS mI medium)

I 1.67 0.1

2 2.13 0.11

3 2.S6 0.13

4 2.69 0.13

L S. D. at S% 0.34 0.02

Table 2- Effect of different concentrations of dikegulac on growth of C. rosells callus in subsequent subculturing at the

30th day of culture cycle.

[Values are mean or20 replicates]

Concentration Subculturing Fresh weight Dry weight of dikegulac stage (gil S mI (gl15 mI

(Ilg/ml) medium) medium)

0 2.69 0.129

2.45 0.133

50 2 2.67 0.142

3 3.)8 0.180

2.29 0.090

100 2 2.82 0.125

3 2.87 0.126

2.40 0.106

150 2 3.26 0.127

3 0.10 0.010

200 2.79 0.135

2 0.10 0.010

250 0.10 0.010

L S. D. at5% 0.264 0.032

596 INDIAN J EXP SIOL, JUNE 1999

accumulation was observed on 25th day in 50 Ilg/ml and on 30th day in lOOllg/ml.

Total alkaloid content on per g dry weight basis was, however, increased by 128 % over control in 50 llg/mJ of dikegulac (Fig. Ib), but a significant inhibition was noted at 100 Ilg/ml of dikegulac.

Oikegulac was thus found to be a potent chemical at lower concentration for biomass and alkaloid production. Modification in growth kinetics, visible on subsequent subculturing, were due to altered cell metabolism that were induced during different culture cycles by long duration treatment.

Ultrastructural changes have been observed with long term treatment of elicitors on Papaver bracteatum cell cultures^J9. The long-term treatment could act on the cell wall structure, the ionic equilibrium or the membrane permeability that found expression at the

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10 15 20 25 30 35

TIME INTERVAL (Days)

TYPE OF TREATMENT

Fig.l-Drowth kinetlcs-(A) alkaloid production (% of control);

& (B) of cultures of C roseus callus with different concentrations

of dikegulac.[100 Jlg/ml Co.-), 50 Jlg/ml (-.-) and control (-e-)].

same time in the change of cell water content, visible only upon subculturing, and ^In the alkaloid

d . 20

pro uctIOn .

From HPLC analysis, when the chromatogram was compared with respect to known alkaloids like ajrnaline, vincristine and ajmalicine in the callus of both retardant treated and untreated sample, ajmaline was detected in substantially higher amount at both the concentrations of OK (Fig. 2). When semi- quantitative analysis of ajmaline was carried out, it was found that 0.135 and 0.2 I 2 mg' per g of callus dry weight was present in 50 and 100 llg/mJ of OK respectively. In addition to this, a number of predominating peaks were also present, but were not identified with reference to standard alkaloid (Table 3). The distribution of different indole alkaloids may be related to the nature of each

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A B c

Fig. 2-HPLC-UY (280 nm) chromatogram of C roseus callus alkaloids at different concentrations of dikegulac. (A) Control, (B) 50 Jlg/mJ of dikegulac and (C) 100 JlglmJ of dikegulac. [Chromatographic details are in Table 3].

CHOUDHURY & GUPTA: DIKEGULAC & ALKAWID PRODUCTION IN CATHARANTHUS

597

Table 3-Effect of different concentrations of dikegulac on HPLC separation of different indole alkaloids in Catharanthus roseus callus culture

Types of treatment No. of Area (%)of some unidentified and known peaks'

(~glmI) predominant

peaks

I 2

Control 3 44.049 53.18

(1,04) ( 1.28)

50 Dikegulac 7 19.887 30.479

( 1.09) ( 1.28)

I {)() Dikegulac 4 27.667 42.256

(1.04) ( 1.28) Number in parentheses indicate retention time.

*denotes ajmaline.

compound. Indeed, ajmalicine is a weak basic alkaloid (pk

=

^6.3) that can diffuse across plant cell membrane and released into culture mediuni²⁰ and due to this reason; it may not be detected in callus tissue. Higher rates of retardant application cause inhibition of cell division . The physiological reason for this effect IS probably an altered membrane

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TIME INTERVAL (Days)

Fig. 3-Time course of metaboiite level of C. roseus callus culture. (A) RNA (mglg dry weight); (B) amino acid (mglg dry weight), (C) soluble carbohydrate (mglg dry w~ight) and (D) tryptophan (mglg dry weight). [50 ~glmI (-_-) of dikegulac and control (-e-)].

3 2.78 (1.85)

13.847*

(1.52) 21.997*

( 1.51)

4

8.012 ( 1.65) 8.08 (4.28)

5

1.303 (2.15)

6

3.078 (3.53)

7

23.394 (4.26)

functioning.Dikegulac affects the integrity of plasmalemma²¹. Zilkah and Gressel⁷ reported that at lower concentrations, dikegulac inhibits cell division, but has a least effect on plasmamembrane.At higher concentrations, plasmalemma bursts, leading to disintegration of protoplasts. Many enzymes of indole alkaloid biosynthesis are membrane-bound and for e"xerting the activity, need membrane integrit/².

In the present work, an inhibition of alkaloid production by dikegulac at 100 Ilglrnl appears to be the reason for its efficacy because the relative leakage ratio and the injury index value was much higher indicating damage of the active side of the membrane (Table 4). It has been reported²⁵ that bisindole alkaloids and vindoline accumulate only in green tissues, and are not found in root or cell suspension cultures²³. The regulation of vindoline biosynthesis by light has been shown to be mediated by phytochrome²⁴ . Meijer et al. 25 have reported that alkaloid metabolism seems to be restricted to certain tissues and is modulated by different developmental and environmental mechanisms.

Alkaloid biosynthesis is increased by feeding of precursors^26. The level of direct precursor i.e. soluble carbohydrate and tryptophan (mother amino acid) of indole alkaloid biosynthesis was significantly high in dikegulac (50 Ilglrnl) over control throughout the culture cycle (Fig. 3). RNA and total amino acid pool was also higher in treated tissues than that of control (Fig. 3). Dikegulac modifies the internal hormone balance, mainly of IAA, GA and cytokinin²⁷ and stimulates carbohydrate²⁸ and tryptophan production²⁸ in intact plants. It has been reported that cytokinin and BA stimulate alkaloid synthesis in cell cultures of C. roseus and these changes perhaps modify the physical properties (fluidity and/or direct

598 INDIAN J EXP BIOL, JUNE 1999

Table 4-Effect of different concentrations of dikegulac on membrane permeability in terms of relative leakage ratio (abs

at 280 nm) and on injury index of C. roseus.

[Values are mean of 8 replicates]

Concentrations of Relative leakage ratio Injury index dikegulac (~g/ml) (absorbance at (%)

280 nm)

Control . 0.492

50 0.499 1.3

100 0.607 21.79

L. S. D. at 5% 0.006 1.48

lipid protein binding), resulting in enhanced actIvItIes of some membrane-bound enzymes permitting (directly or not) thereby an optimal

functioning of the indole alkaloid pathwa/^9.

Addition of tryptophan to culture medium also increases the alkaloid production in Cathara'nthus tissue^22. Therefore, the results obtained upon addition of 50 !lglml of dikegulac on long-term treatment, suggest that this compound acts as a positive modulator of the indole alkaloid biosynthesis in Catharanthus sp because of higher precursor availability along with intact membrane system and normal enzyme activity.

Acknowledgement

The authors are indebted to the University Grants Commission, New Delhi, for financial assistance and to SAP (UGC) and the University of Burdwan for providing necessary research facilities. The authors thankfully acknowledge Dr R MAGG. Ltd., Dielsdorf, Switzerland, for supplying the chemical, dikegulac as a gift. Constructive suggestions of Prof.

S Thakur are acknowledged.

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