In Vitro Production of Cucurbitacins From Trichosanthes cucumerina L. var. cucumerina

The object ives of this study were to investigate the effect of growth regulators in callus induction, increase of biomass and to the yield more of cucurbitacin and cucurbitacin-E in leaf explants of Trichosanthes cucumerina L. var. cucumerina. The explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins like 2,4-dich lorophenoxy acetic acid (2,4-D), [alpha]-naphthalene acetic acid (NAA), Indole butyric acid (IBA), Indole acetic acid (IAA), cytokinins like Kinetin (kn) and Benzyl adenine (BA). The different concentrations and combinations of BAP + IBA, 2, 4-D + BAP and 2, 4-D + kn increased the callus of fresh weight and dry weight. Among all concentrations and combinations, the best results revealed that leaf-derived callus cultured on 2,4-D (3.0mg -l ) + kin (1.0 mg -l ) produced the highest total cucurbitacins content with an optimum y ield 4.9% w/w and cucurbitacin-E 2.75% w/w at third week.


Introduction
The plant Trichosanthes cucumerina L. var. cucumerina (in English Chinese cucumber or wild snake gourd) belongs to family cucurbitaceae. The fru it is known to contain many form of cucurbitacins, (1,2,3) and due to its bitterness the fruit is been using in many ayurvedic preparations (4) and also in Indian fo lk medicine to cure jaundice (5), to reduce congestion on congestive cardiac failure (6).
These days many people are interested in the beneficial effects of food on health, and cucurbitacins have been studied because of the wide range of biological activit ies they exhibit in living beings. They are predominantly found in the Cucurbitaceae and several other families of the plant kingdom. A nu mber o f co mpounds of this group have been investigated for their cytotoxic (7), hepatoprotective (8), cardiovascular (9), antidiabetic (10) Antibacterial (11), anti-inflammatory (12)(13) and antio xidant activity of cucurbitacins B and I and the glucosides of cucurbitacin I and L (14). Additionally, several studies indicated that different cucurbitacin species inhibit the proliferat ion of cancer cells through different mechanis ms (15)(16)(17)(18)(19) The members of cucurbitaceae has gained increasing attention as a natural insecticide and its activ ity has been evaluated against many economically impo rtant insect species. Cucurbitta spp. are deterrent, antifeedant, growthregulating and fertilityreducing properties on insects (20)(21) Also, it is used as an abortifacient, cathartic, purgative and vermifuse, and for the treatment of fever, cancer, amenorrhea, jaundice, leukemia, rheumat ism, tu mour and as an insect repellant (22).The content of cucurbitacins in various organs of Trichosanthes cucumerina L. var. cucumerina has been investigated (23).
The present study was carried out to develop an efficient protocol for callus induction, proliferat ion, total cucurbitacins and cucurbitacin-E accu mulation under the effect of different growth hormones with leaf exp lant of Trichosanthes cucumerina L. var. cucumerina to study various pharmacological effects.

Materials and Methods
Quantitati ve determinati on of cucurbi tacins from in vitro callus culture:

Plant material
Trichosanthes cucumerina L. var. cucumerina seeds were obtained fro m mature fru its collected from Khanapur forest Bhalki taluka, Bidar District India. The collected seed materials were botanically authenticated by the Botany department, Gulbarga Un iversity, Gulbarga (Voucher No. HGUG-804). Also, the plant was confirmed with authenticated herbariu ms at the Centre for Eco logical Studies, IISc, Bangalore, and Botanical Survey of India, Pune. The col-lected seeds preserved in amber bottles under normal lab conditions until used.

Media preparation
The basal mediu m described by Murashige and Skoog (MS) (24) was used. Deferent concentrations of plant growth regulators were added to the MS mediu m. The media were sterilized by autoclaving at 121℃ for 15 min.

Callus induction
The seeds were surface sterilized with 2% mercuric chloride for 1 min. then washed thrice with distilled water and soaked for 24 hrs in a beaker. The sterilized seeds were used for germination on MS hormone free med iu m. After few days, the seedlings were excised to yield exp lants for callus production. The init iated callus was then maintained on MS mediu m supplemented with BAP 1.0mg -l and IBA 0.5mg -l at 24±2℃ in continuous light (2400 lu x) and maintained by transferring approximately 1 g of callus every 4 weeks.

Callus propagation
First experiment Approximately 1 g o f init iated callus material was cu ltured, on different med ia, to select the best plant growth regulator comb ination.
Second experiment 1-1.5 g aliquots of callus were cu ltured in conical flask containing 100 ml MS mediu m supplemented with Kn/2,4-D co mbinations with hormone concentrations of 0.0, 0.5, 1.0, 2.0 and 3.0 mg -l to determine the best callus proliferat ion and yield of cucurbitacins and cucurbitacin E.

Fresh and dry weight measurement
For the first experiment the callus samples were sacrificed on week 3, wh ile fo r the second one, samples were collected at weekly intervals for a maximu m of 5 weeks. After obtaining the fresh weights, the samples were then dried at 40℃ and the dry weights obtained after 24 h.

Determinati on of cucurbi tacins Solvents and reagents
Absolute ethanol, petroleum ether 30-40℃, ch loroform and phosphomolybdic acid (all at analar grade). A cucu rbitacin E reference standard was used.

Sample solutions
For total cucurbitacin assay, dried callus material (100-200 mg per sample) was ext racted with absolute ethanol (5ml) for 2 h, after centrifugation (2000 rp m for 3 min), the s upernatant was mixed with an equal volu me of petroleu m ether, the precip itate obtained was filtered and d issolved in absolute ethanol (5ml), and then reduced to a volume of 2 ml as above.

Reference solution
The reference standard cucurbitacin E was dissolved in ethanol and serial dilutions (0.01-1.0 mg/ ml) were prepared.

Assey
All samples (100μl, in duplicate), together with various concentrations of cucurbitacin E standard as per (25) at room temperature. The absorbance was measured at 492 n m after 5 min on a MTP reader STATFAX2100, USA. The results were exp ressed as w/w% calcu lated fro m dry callus weight and then analyzed statistically by ANOVA .

Results and Discussion
Quantitati ve determination cucurbi tacins from in vitro callus culture: Experiment 1 with mixed PGRs grid Bio mass accumulation: Table-1 indicated that BAP was the best cytokinin in combination with IBA as regards callus accumu lation. Calluses on these plant growth regulators were friable and white in co lour, while with kinetin and 2,4-D, these were relatively hard and brown in colour, and showed a slow rate of accumu lation. Secondary metabolite accu mulat ion: On other hand 2,4-D and Kn gave the most significant Cu and CuE accumu lation, especially when co mpared to IBA and BAP (Tab le-2). Overall, an inverse proportionality was observed between callus weight and cucurbitacin yield. This was clearly shown in the comb ination of 2,4-D and IBA with BA P, having dry weights of 187 and 258 mg , respectively, and corresponding Cu contents of 0.359 and 0.256% w/ w. similar results were observed in Ecballium elaterium (26).   It is evident that the co mbination with the highest callus accumulat ion proved to be the 0.5mg -l 2,4-D as co mpared to the rest. The accumulation of secondary metabolites was best at week 3 with a decline in Cu and Cu E at week 4. The 1.0mg -l Kn concentration in combination with different 2,4-D concentrations gave optimu m metabolite accu mulation 0.52-5.0 % w/w at week 3, (Table-3, Fig-1). Increase in the lone concentration of 2,4-D and Kn showed increase in the significant accu mulation of Cu and Cu E, wh ile there was a decline with an increase in 2,4-D and Kn concentration. The rate of production of CuE fro m the Kn 1.0mg -l + 2,4-D 3.0mg -l treat ment was approximately one and half times higher than that from Kn 2.0mg -l and 2,4-D 3.0mg -l . But, at moderate concentrations 2,4-D inhibits Cu accumu lation, as occurs with other metabolites (27). In the case of Cu E, significant yields were obtained with higher 2,4-D concentrations. These results are in agreement with the findings of Halaweish and Tallamy , (28). They confirmed that MS-mediu m supplemented by 2 mg -1 2,4-D in co mbination with 1 mg -1 kin produced the highest biomass of callus tissues when induced fro m the rootless seedlings explants Cucurbita andreana. The optimu m mediu m for callus induction of Eremochloa ophiuroides (Munro) was MS media supplemented with 2,4-D at 1.0 mg -1 (29).
The leaf explants of T. cucumerina L. var. cucumerina demonstrated better callus induction and also proved to synthesize total cucurbitacins and cucurbitacin-E in undif-ferentiated callus. Using of 2, 4-D in co mbination with kn was found to be the best treatment for cucurbitacins production and accumulations of total cucurbitacins and cucurbitacin-E in callus tissues. Enhancement of callus induction and accumulation of cucurbitacins were higher than those obtained from the in vivo grown plant parts. This study suggested that in vitro secondary metabolites production by T. cucumerina L. var. cucumerina callus cultures could be considered an appropriate alternative method to whole plant ext raction.