Ecballium elaterium: an in vitro source of cucurbitacins

Fitoterapia 72 Ž2001. 46᎐53

Ecballium elaterium: an in vitro source of cucurbitacins Everaldo G. AttardU , Anthony Scicluna-Spiteri Institute of Agriculture, Uni¨ ersity of Malta, Msida, MSD06, Malta Received 14 December 1999; accepted in revised form 11 July 2000

Abstract Biomass and secondary metabolite accumulation were assayed on a wide range of auxins, cytokinins and their combinations using Ecballium elaterium callus tissue. The best combination was that consisting of ␣-naphthalene acetic acid ŽNAA. and benzylamino purine ŽBAP. Ž1 mgrl of medium, each.. NAA and BAP were set up in a grid to determine concentrations that produced the best callus as regards fresh and dry weights and yield of cucurbitacins, particularly cucurbitacin E. The best combination for callus proliferation consisted of 5 mgrl NAA and 5 mgrl BAP, reaching a peak at week 5. For cucurbitacin production, an optimum yield was obtained at week 3 with 5 mgrl NAA. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Ecballium elaterium; Cell culture; Cucurbitacins

1. Introduction Ecballium elaterium ŽL.. A. Rich. ŽCucurbitaceae ., also known as the ‘squirting cucumber’, is a wild medicinal plant found abundantly in the Mediterranean region. The content of cucurbitacins ŽCu. in different organs of E. elaterium has been investigated by various authors w1᎐3x. Since tissue culture of this plant had never been attempted before, we initiated its micropropagation with the aim of obtaining in culture these tetracyclic triterpenoids which exhibited various pharmacological effects w4᎐8x. U

Corresponding author. E-mail address: [email protected] ŽE.G. Attard.. 0367-326Xr00r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 0 . 0 0 2 5 6 - 2

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In this paper, we have reported on the effects of different plant growth regulators ŽPGRs., on callus biomass accumulation and secondary metabolite production, particularly cucurbitacin E ŽCuE.. The PGRs used included indole-3acetic acid ŽIAA., indole-3-butyric acid ŽEBA., ␣-naphthalene acetic acid ŽNAA., 2,4-dichlorophenoxyacetic acid Ž2,4-D., kinetin ŽKi. and benzylamino purine ŽBAP..

2. Experimental 2.1. Plant material E. elaterium seeds were obtained from immature fruits collected in December 1997, from Marsascala ŽEzSi. and University campus ŽEuSi., Msida. The seeds were identified at the Institute of Agriculture, University of Malta where a voucher specimen was deposited. The fruits were surface sterilised with 70% EtOH for 30 s, then soaked in 10% NaClO solution for 20 min, and finally rinsed in three changes of sterile distilled water. 2.2. Media preparation The basal medium described by Murashige and Skoog ŽMS. was used w9x. PGRs were added to the MS medium in the concentrations indicated in Tables 1 and 3. The media were sterilised by autoclaving at 121⬚C for 15 min. 2.3. Callus induction The sterilised fruits were excised under aseptic conditions. The seeds Ž n s 39. were removed carefully and then germinated on MS medium. After 3 weeks, the seedlings were excised to yield explants for callus production. The initiated callus was then maintained on MS medium supplemented with NAArBAP Ž0.1 and 1 mgrl, respectively. at 24 " 1⬚C in continuous light Ž2400 lux. and maintained by transferring approximately 1 g of callus every 4 weeks. The two best seeds Žstrains . with a high callus turnover, strains 3 ŽEzBSi3. and 14 ŽEuASi14., were selected for further experiments. 2.4. Callus propagation For the first experiment, strain 3 was used. Approximately 1 g of initiated callus material was cultured, in quadruples, on different media Ž n s 15., to select the best PGR combination Žsee Table 1 for the matrix of PGRs used.. For the second experiment, strain 14 was used. Approximately 1᎐1.5-g aliquots of callus were cultured, in five pairs Ž10 grids., in 100-ml jars containing 20 ml MS medium supplemented with NAArBAP combinations Ž n s 25. with hormone concentrations of 0, 0.01, 0.1, 1 and 5 mgr1 to determine the best callus proliferation and yield of CurCuE.

48

None

None Ki BAP

2,4-D

IBA

IAA

NAA

F

D

F

D

F

D

F

D

F

D

1766 " 99 1211 " 70 U 2232 " 165

110 " 6 101 " 10 U 203 " 6

1477 " 87 1478 " 172 1521 " 140

112 " 6 108 " 12 133 " 5

1209 " 122 988 " 103 1427 " 21

76 " 5 80 " 9 127 " 5

1045 " 18 1357 " 82 1115 " 20

79 " 9 96 " 3 99 " 5

1288 " 41 1106 " 89 U 3779 " 87

221 " 75 87 " 7 U 218 " 2

U

a BAP, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Ki, kinetin; NAA, ␣-naphthalene acetic acid; F, fresh weight Žmg.; D, dry weight Žmg. winitial weight of callus: 1000 mgx. Values are mean " S.E.M Ž n s 4, per treatment .. U P - 0.001 Žd.f.s 45. vs. control; Bonferroni t-test.

E.G. Attard, A. Scicluna-Spiteri r Fitoterapia 72 (2001) 46᎐53

Table 1 The effects of plant growth regulators Ž1 mgrml of medium. on growth of Ecballium elaterium callus after 3 weeks in culture for strain EzBSi 3 a

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2.5. Fresh and dry weight measurement For the first experiment the callus samples Ž n s 60. were sacrificed on week 3, while for the second one, samples Ž n s 50. were collected at weekly intervals for a maximum of 5 weeks. After obtaining the fresh weights, the samples were then dried at 40⬚C and the dry weights obtained after 24 h. 2.6. Determination of cucurbitacins 2.6.1. Sol¨ ents and reagents Absolute ethanol, petroleum ether Ž30᎐40⬚C., chloroform and phosphomolybdic acid Žall at ‘AnalaR grade’. were purchased from BDH, Dorset, UK. A cucurbitacin E reference standard was obtained from Prof David Lavie, The Weizmann Institute of Science, Rehovot, Israel. 2.6.2. Sample solutions Ž1. For total Cu assay, dried callus material Ž100᎐220 mg per sample. was extracted with absolute ethanol Ž5 ml. for 2 h; after centrifugation Ž2000 rev.rmin, 3 min., the supernatant was reduced to 2 ml on a water bath Ž40⬚C.. Ž2. For CuE assay, dried callus was extracted with chloroform Ž5 ml. for 2 h; after centrifugation, the supernatant was mixed with an equal volume of petroleum ether; the precipitate obtained was filtered and dissolved in absolute ethanol Ž5 ml., and then reduced to a volume of 2 ml as above. 2.6.3. Reference solution The reference standard CuE was dissolved in ethanol and serial dilutions Ž0.0174᎐1.113 mgrml. were prepared. 2.6.4. Assay All samples Ž100 ␮l, in duplicate., together with various concentrations of CuE standard, were mixed with 100 ␮l of a 2% solution of phosphomolybdic acid in absolute ethanol w10x at room temperature, using a 96-well plate ŽNUNC, Denmark. format. The absorbance was measured at 492 nm after 5 min on a MTP reader ŽSTATFAX 2100, USA.. The results were worked out as wrw%, calculated from dry callus weight and then analysed statistically by ANOVA and the Bonferroni post-hoc test for equality of means, and then ANCOVA and pairwise t-test for parallelism between treatments on the trends obtained.

3. Results and discussion 3.1. Experiment 1: mixed PGR grid 3.1.1. Biomass accumulation The results reported in Table 1 indicated that BAP was the best cytokinin, on its

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Table 2 The effects of plant growth regulators Ž1 mgrml of medium. on the production of Cu and CuE Ž% wrw, on dry weight. after 3 weeks for strain EzBSi 3 a None None 2,4-D IBA IAA NAA

Cu CuE Cu CuE Cu CuE Cu CuE Cu CuE

0.450 " 0.004 0.290 " 0.002 0.526 " 0.011 0.229 " 0.001 0.436 " 0.030 0.340 " 0.002 0.486 " 0.014 0.322 " 0.001 0.394 " 0.004 0.079 " 0.001

Ki

BAP U

0.564 " 0.005 0.390 " 0.020 U 0.584 " 0.005 0.218 " 0.006 0.431 " 0.002 0.335 " 0.014 U 0.305 " 0.001 0.292 " 0.003 0.515 " 0.014 U 0.373 " 0.009

U

0.186 " 0.006 U 0.149 " 0.001 U 0.216 " 0.003 U 0.198 " 0.001 U 0.265 " 0.004 0.227 " 0.002 0.373 " 0.013 U 0.369 " 0.009 U 0.171 " 0.003 U 0.034 " 0.001

a BAP, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Ki, kinetin acetic acid; NAA, ␣-naphthalene; Cu, cucurbitacins; CuE, cururbitacin E. Values are mean " S.E.M Ž n s 2, per treatment .. U P - 0.001 Žd.f.s 15. vs. control; Bonferroni t-test.

own and in combination with NAA ŽBonferroni t-test, P - 0.0001, n s 60., as regards callus accumulation. Calluses on these PGRs were friable and white in colour, while with Ki and 2,4-D, these were relatively hard and brown in colour, and showed a slow rate of accumulation. 3.1.2. Secondary metabolite accumulation On the other hand, 2,4-D and Ki gave the most significant Cu and CuE accumulation, especially when compared to NAA and BAP ŽTable 2.. Overall, an inverse proportionality was observed between callus weight and terpenoid yield. This was clearly shown in the combination of 2,4-D and NAA with BAP, having dry weights of 133 and 218 mg, respectively, and corresponding Cu contents of 0.216 and 0.171% Žwrw.. 3.2. Experiment 2: NAA r BAP grid 3.2.1. Biomass accumulation Exponential fresh and dry callus accumulation continued until week 5. Media containing 5 mgrl BAP showed a significantly high rate of callus proliferation and mean accumulation Ž P - 0.0240, d.f.s 49. as compared to the rest. 3.2.2. Secondary metabolite accumulation Cu and CuE accumulation reached a peak at week 3 ŽTable 3. showing a decline thereafter. Media containing 5 mgrl NAA produced the best results with a Cu content of 3.064% Žwrw. and a CuE content of 2.970% Žwrw. calculated on dry weight basis. In fact, a significant Cu accumulation was observed with NAA

NAA Žmgrl. 0 BAP Žmgrl.

a

0 0.01 0.1 1 5

0.01 U

2.0615 " 0.0655 U 2.2955 " 0.0265 U 1.893 " 0.0590 U 1.861 " 0.0120 U 2.0505 " 0.0235

U

2.3575 " 0.0425 U 2.453 " 0.0260 U 1.9125 " 0.0165 U 1.8545 " 0.0115 U 1.9365 " 0.0435

0.1

1

5

2.807 " 0.0500 2.9905 " 0.0485 2.661 " 0.0260 U 1.937 " 0.0200 U 1.8535 " 0.0105

2.654 " 0.0420 3.0225 " 0.0175 2.739 " 0.0010 UU 2.666 " 0.0620 U 2.143 " 0.043

3.0635 " 0.0845 2.913 " 0.0360 UU 2.9635 " 0.0115 UU 2.562 " 0.0490 U 2.578 " 0.0470

UU

BAP, benzylaminopurine; NAA, ␣-naphthalene acetic acid. Values are mean " S.E.M Ž n s 2, per treatment .. Statistically different from 5 mgrl NAA Ž t-test, P - 0.0228, d.f.s 49.. UU Statistically different within a horizontal trend Ž t-test, P - 0.0175, d.f.s 49. for the mean secondary metabolite accumulation over the 3-week period. U

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Table 3 The variation of total cucurbitacins content Ž% wrw dry weight. with different combinations of plant growth regulators at week 3 for strain EuASi 14 a

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Fig. 1. The pattern of growth-linked accumulation of cucurbitacins ŽCu. and cucurbitacin E ŽCuE. in relation to accumulation of strain EuASi 14 callus cell cultures wtreatment: ␣-naphthalene acetic acid Ž5 mgrml. q benzylamino purine Ž0.01 mgrml.x.

concentrations higher than 0.1 mgrl, while there was a decline with an increase in BAP concentration. The rate of production for the 0.1-mgrl NAA treatment was approximately two times higher than for the 0.1-mgrl NAA and 5 mgrl BAP. A statistical difference in the mean Cu and CuE metabolism Ž P - 0.0236, d.f.s 99. indicated that, at high BAP concentrations, the metabolism of Cu was shifted away from the production of CuE resulting in inferior CuE content. 3.3. Conclusions E. elaterium was shown to synthesise Cu in undifferentiated callus. The biosynthesis of CuE in the total cucurbitacin content was found to be very high. In the mixed PGR grid with strain EzBSi 3, the yield of Cu in callus tissues Ž0.167᎐0.589% wrw. was higher than that obtained from different organs of the intact plant Ž0.0026᎐0.340% wrw., but inferior to that obtained from fruits Ž2.204᎐0.409% wrw. w2x. However, the Cu yield from the NAArBAP combinations was between 1.854 and 3.064% wrw, for the strain EuASi 14. The results from the NAArBAP grid, allowed us to conclude that BAP played a role in callus proliferation, but not in Cu production. Also, callus production was proportionally related to cucurbitacin production, until the biomass reached a maximum rate of accumulation ŽFig. 1.. In fact, this is termed as growth-linked transient accumulation of secondary metabolites w11x, as observed in other studies, especially with Phytolacca americana w12x and Chenopodium rubrum w13x.

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Acknowledgements The authors are grateful to the Ministry of Agriculture and Fisheries for providing the necessary facilities and support at the Micropropagation Centre, Lija, Malta. Finally, one of the authors, Everaldo Attard, would like to thank Prof David Lavie, The Weizmann Institute of Science, Rehovot, Israel, for the receipt of the gift sample of CuE.

References w1x w2x w3x w4x w5x w6x w7x w8x w9x w10x w11x

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