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APPRAISAL OF SECONDARY METABOLITES IN IN VITRO CULTURES OF Citrullus colocynthis (L.) SCHARD.

Byline: Hina Tanveer, Safdar Ali and Zaheer-ud-Din-Khan

ABSTRACT : Plant cell culture systems represent a potential renewable source of valuable medicinal compounds, flavors, fragrances, and colorants, which cannot be produced by microbial cells or chemical synthesis. The present research work was carried out for the analysis of secondary metabolites in callus culture of Citrullus colocynthis (L.) Schard. In vitro grown seedlings were used as explant source. Murashige and Skoog (MS) medium was used as culture medium supplemented with different concentrations and combinations of 2,4-D + Kin mg/L and BAP + NAA mg/L. The combination of BAP 2 mg/L and NAA 3 mg/L was commonly successful in inducing callus in leaf as well as in internode explants while NAA in individual application did not produced good results for callus induction. Laboratory grown seedling parts and the relevant callus cultures raised from different explants were subjected to analysis for secondary metabolites through GC-MS.

Among secondary metabolites determined, the common secondary metabolites present in leaf and internode tissues were Toluene, a-sphinosterol, Tetradecane, Pentadecane, Hexadecane, Octadecane, Hentriactone, and 9,12-Octadecadienoicacid[7,7]. Majority of the compounds were detected in quantities less than 1% in seedling tissues as well as in respective calluses. From the present study, it was observed that regardless of the similar compounds found, many metabolites were different in leaf and internode tissues and in respective calluses.

INTRODUCTION

Citrullus, an important genus of the family contains medicinally very important species. Citrullus colocynthis (L.) Schard commonly called "Tuma" also known as bitter cucumber, bitter apple, egusi or vine of sodom [1]. It is native of warmer parts of Asia, Syria, Egypt and Morocco for purpose of export. It occurs in the subcontinent including Pakistan and is seen growing wild in the warm and arid sandy tracts [2].

The plant secondary metabolites, important for the human consumption as food and as medicinal compounds used in the pharmaceutical industry require special attention. If the naturally occurring compounds of medicinal importance are produced in in vitro cultures, the problem of provision of raw materials for the extraction of bioactive compounds can be solved at door step. Plant parts containing important compounds such as polyphenols, anthocyanins, flavonols and vitamins etc., the main source of natural antioxidants can be cultured on artificial medium as renewable source of required chemical compounds [3]. Different chemical compounds present in the plant material can be detected and analysed. This study was undertaken to analyze and compare secondary metabolites in in vitro cultures of C. colocynthis and seedling tissues.

MATERIALS AND METHODS

Plant Material and Seed Germination

The seeds procured from market, were decoated and surface sterilized by treating with 2.5% NaOCl (Sodium hypochlorite) solution for aseptic germination. In vitro raised seedlings were used as the explant source for callus induction.

Medium Preparation

For medium preparation measured quantities of stock solutions of MS medium [4] were mixed/L for preparation of MS working medium as per requirement. Sucrose was dissolved at the rate of 3.0% (w/v). Calculated quantities of growth regulators (mg/L) for a particular medium were added. The final volume of the medium was made up to the mark by adding double distilled water. pH of the medium was adjusted at 5.8. The medium was jelled with 0.8% agar. The medium was dispensed in appropriate volumes into the culture vessels for sterilization.

Preparation and Incubation of Explant

Leaf, internode and cotyledon from in vitro grown seedlings, excised under aseptic conditions, were transferred to the culture vessels containing sterilized media. After culturing the explant, culture vessels containing explants were placed in growth room at temperature of 25@C +- 3oC with light period 16 h/ day cycle and 2500 to 3000 lux light intensity.

Subculturing of Callus Culture

To avoid the deficiency of nutrients, callus cultures were transferred aseptically to fresh medium after every 15 days.

Callus Index

Callus index (CI) was calculated by the following formula [5].

CI=100n x G/N (n = Number of explants initiating callus,

G=Visual callus rating of initiated explants, N = Total number of explants planted)

ANALYSIS OF SECONDARY METABOLITES BY GC-MS

The calli biomass was crushed (if compact in nature) and ground by using a porcelain pestle mortar. The homogenous slurry was diluted and semisolid mass and solvent mixture was separated by filtration. The solvent mixture was distilled in water bath in a round bottom flask to obtain essential oil. The combined solvent extract was dried over anhydrous sodium sulphate (Na2SO4) and the solvent was distilled off, leaving the essential oil as a residue in the flask. The essential oil was subjected to GC-MS.

For GC-MS analysis, sample of steam distilled oil was injected and run under the following conditions: Capillaray Column of HP-5ms 5% nonpolar, column at Inlet temperature 200degC and detection Temperature 250degC. Helium gas used as Carrier Gas with flow rate1.0 ml/min. Column Oven Programing, 50-230min @ 5oC/m.

RESULTS

Seed germination

During seed germination, the seeds were subjected to different soaking periods such as 1, 2, 3, 4 and 12 hours. No germination was observed in seeds with soaking time periods from 1 to 4 hours while seed germination was observed only in seeds soaked for 12 hours. Five seeds per petriplate were employed, while amount of water used to moist the cotton pad per petriplate ranged from 10 to 25ml with the interval of 5ml. No germination was observed in petriplates having cotton pads soaked with 15, 20 and 25ml of water. Germination was observed in petriplates moistened with 10ml of water.

Callus induction

Different parameters studied were callus initiation, callus induction %age, duration of callus induction, callus index, callus colour and callus texture. Callogenesis was observed in leaf and internode explants of C. colocynthis while cotyledon failed to respond. Time duration for callus initiation from leaf of C. colocynthis was 3 to 8 days. Callus formation initiated at the margins. In MN5 callus induction was 33% where as callus index was 33 and embryogenic green callus was formed from leaf explants (Table 1). In MBN2,3 (Table 1) callus induction was 100% where as callus index was 300 and embryogenic brownish green callus was formed from leaf as shown in Fig 2. Good results for callus induction were observed in MBN2,3. Time duration for callus induction from internode of C. colocynthis was also 3-8 days.

In MN5 callus induction was 33% where as callus index was 33 and embryogenic green callus was formed from internode explants (Table 1). In MBN2,3 callus induction was 66% where as callus index was 200 and embryogenic light green callus was induced on internode(Table1).

Table 1: Effect of different concentrations of BAP + NAA mg/L for the callus induction from leaf of C. colocynthis

Explant###Medium###PGRs###No of explant###CI###VCR###CI###Initiation###Characteristics

Type###Code###(BAP/NAA###producing###%age###Duration###of Callus

###mg/L)###callus###(Days)

Leaf###MN5###-/5###1###33###1###33###8###Green, E

-###MBN2,3###2/3###3###100###4###300###4###Brownish green, E

Internode###MN5###-/5###1###33###1###33###8###Green, E

-###MBN2,3###2/3###2###66###4###200###3###Light green, E

GC-MS ANALYSIS OF IN VITRO GROWN SEEDLING AND CALLUS CULTURES

Different compounds, as secondary metabolites from leaf and leaf callus cultures under the influence of different PGRs, were detected in different quantities from different sources, (Table 2). Compounds more than 10% were Toluene (63.515), Benzene1,3-dimethyl (12.589), Undecane (52.29) and Aniline, N-methyl (13.964), compounds from 1 to 10% were Ethylbenzene (2.771) and o-Xylene (3.097) whereas compounds less than 1% were Nonane (0.426), Nonane (0.168), 2-Propenoic acid (0.372), Undecane (0.082), Dodecane (0.074), Tridecane (0.077), Tetradecane (0.069), Pentadecane (0.045), Hexadecane (0.054) and Octadecane (0.031) detected in callus cultures under the influence of MBN2,3 medium. The same trend was found in internode and internode derived cultures (Table 3), the compounds detected are can be classified into three categories according their percentage of occurrence.

Table 2: Percentage composition of secondary metabolites from seedling-leaf and leaf callus under different concentrations of PGRs.

Sr. # Names of compounds detected###Retention Time###Retention Time###Retention Time

###through GC-MS###(min) /Average %age###(min)/Average %age###(min)/Average %age

###of compounds in###of compounds in###of compounds in

###leaf tissue###leaf callus (MBN0,5)###leaf callus (MBN2,3)

1###Toluene###3.89/1.692###3.59/20.4###3.74/63.51

2###Nonane###4.82/1.184###4.87/.127###4.12/0.426

3###Benzene 1,3-dimethyl###5.99/0.649###-###6.43/12.58

4###Ethylbenzene###6.10/0.237###6.09/3.93###6.09/2.77

5###o-Xylene###-###7.22/3.081###7.58/3.097

6###2-Propanoic acid, butyl ester###7.89/0.424###7.76/0.619###7.75/0.372

7###3-Cyclohexene-1-01,4-methyl-1

###-[1-methylethyl]###-###10/2.111###-

8###a-Sphinosterol###12.03/0.250###-###-

9###Undecane###12.25/15.348###12.04/52.29###12.24/0.82

10###Dodecane###13.65/0.120###13.65/0.120###13.65/0.074

11###Tridecane###-###14.60/0.118###13.60/0.077

12###Aniline, N-methyle###-###-###14/13.96

13###2,6octadiene,-1-01,3,7

###-dimethyl-acetate,###14.99/0.858###-###-

14###Tetradecane###15.36/0.173###15.36/0.77###15.36/0.069

15###Pentadecane###16.02/1.692###16.02/0.079###16.02/0.045

16###Hexadecanes###16.61/0.240###16.61/0.043###16.61/0.054

17###Heptadecane###-###17.17/0.053###-

18###Octadecane###17.69/0.984###17.69/0.01###17.69/0.031

19###Hentriactone###19.89/0.280###-###-

20###1,2 Benzenedicarboxylic

###acid,diisoctylether###-###-###21.43/1.667

21###9,12-Octadecadienoicacid[7,7]###22.21/70.893###-###-

22###n-Hexadecanoic acid###26.02/0.187###-###-

Table 3: Percentage composition of secondary metabolites from seedling-internode and internode callus under different concentrations of PGRs.

Sr. # Names of compounds detected###Retention Time###Retention Time###Retention Time

###through GC-MS###(min) /Average %age###(min)/Average %age###(min)/Average %age

###of compounds in###of compounds in###of compounds in

###Internode###Internode callus###Internode callus

###(MBN0,5)###(MBN2,3)

1###Toluene###3.92/0.162###3.39/13.96###3.59/31.85

2###Nonane###-###4.86/0.426###4.86/0.127

3###Ethyle benzene###-###6.09/14.707###-

4###Benzene 1,3-dimethyl###-###6.42/3.93###-

5###b-Phellandrene###6.73/0.495###-###-

6###o-Xylene###7.53/5.434###7.58/2.77###7.52/1457

7###2-Propanoic acid, butyl ester###-###7.76/1.866###7.66/6.19

8###Iso-orientin###870/0.237###-###-

9###6-C-p-hydroxybenzoylvitexin###9.26/0.424###-###-

10###8-C-p-hydroxybenzoylvitexin###9.68/0.437###-###-

11###Citrullol###11.19/0.583###11.99/23.097###-

12###Undecane###-###12.25/0.372###12.25/0.111

13###Elatericin B###11.47/1.776###-###-

14###Dodecane###-###13.65/0.082###13.65/ 0.12

15###a-sphinosterol###12.03/0.858###-###-

16###Tridecane###-###14.60/0.194###14.60/0.118

17###Tetradecane###15.420.341###15.36/0.077###15.36/0.077

18###Pentadecane###16.13/1.184###16.02/0.069###16.02/0.069

19###aElaterin###-###14.10/44.29###-

20###Hexadecane###16.61/0.108###16.61/0.045###16.61/0.043

21###Octadecane###17.69/2.623###-###-

22###Heptadecane###-###17.17/0.054###-

23###Hentriactone###19.89/0.240###-###-

24###9,12-Octadecadienoicacid[7,7]###22.01/1.221###-###-

25###Elaterin###25.99/0.280###-###-

26###n-Hexadecanoic acid###26.42/82.585###-###-

DISCUSSION

Effect of different combinations of PGRs on in vitro cultures is well pronounced. In application of PGRs to raise in vitro cultures, the combined effect of different concentrations of BAP/NAA was prominent such as medium code MBN2,3 containing BAP/NAA in a ratio of 2:3 mg/L, showed quick response to initiate embryogenic callus in shorter time duration with callus index of 300 from leaf explant (Table 1). The same was observed for internode explant with callus index of 200. The callus from leaf explant was brownish green and yellowish green from internode. The similar results have been described by many workers [6][7], they found that the highest percentage of callus cultures was derived from explants grown on BAP/NAA combinations than BAP/NAA alone. In MN,5 (NAA 5mg/L) callus induction recorded was 33% with callus index of 33. The embryogenic green callus was generated from both leaf and internode explant.

These results were supported by Attard and Spiteri who demonstrated that media containing 5 mg/L NAA did not support to induce high percentage of callus in Ecballium elaterium [8].

Leaves, roots, flowers and fruits consist of complex cellular tissues containing compartmentalized phytochemicals. Cell, callus and organ cultures of plants are capable of producing most types of secondary compounds, including phenylpropanoids, polyketides, terpenoids, alkaloids, and carbohydrates, and offer excellent materials for biosynthetic studies because they consist of populations of actively growing cells [9]. The analysis of in vitro cultures through GC-MS revealed the presence of flavonoids, steroids, alkaloids and saponins. Presence of flavonoids has been reported from many plant species like Lycium barbarum [10], Passia palmer [11], Cassia angustifolia [12], Jatropha curcus L. [13]. The presence of compounds such as iso-vitexin, iso-orientin and iso-orientin 3'-methyl ether, 8-C-p- hydroxybenzoylisovitexin 4'-O-glucoside was also detected. As described by Matooq et. al., they identified six flavone C-glycosides [14].

Similary Hatam et al., also reported the presence of glycosides 2-O-b-d-glucopyranosyl in C. colocynthis [15]. The tables 3 and 4 show the similarities and differences as regards the presence of secondary metabolites in seedling leaf and internode tissue and leaf and internode calluses respectively. Many metabolites from seedling tissue and its respective callus were different. The differences in secondary metabolites in leaf and leaf callus and internode and internode callus are summarized in Figs. 2 to 5. Mulabagal and Tsay, observed differences in content of the induced callus among the six source organs [3]. Analysis by HPLC revealed that both stem-node and microtuber derived suspension cells contained diosgenin. The content of microtuber derived cell suspension culture contained 3.2% diosgenin per gram dry-weight, where as, the stem-node derived cultures contained only 0.3%. Callus from microtuber showed yellowish color and embryogenic in appearance and contained the highest diosgenin content (3.3-3.5%).

The amount of diosgenin obtained from tuber derived cell suspension was high and comparable with that found in the intact tuber [16]. Gamborg et al., also suggested that the metabolites produced in seedling tissue may be different from the metabolites produced in respective callus [17].

The kinds and concentrations of PGRs also influence the production of secondary metabolites. According to Mulabagal and Tsay and Asrafi et al., for establishing a rapidly growing and finely dispersed cell suspension culture, the best medium composition was 1/2 strength of MS medium supplemented with 1 mg/L 2,4-D, 0.1 mg/L kinetin, and 3% sucrose. All the cultures were routinely sub cultured at an interval of 14 days [3][18]. It was found that MS basal media was the best among the media tested on imperatorin production. When influence of auxins on imperatorin production was investigated, deletion of auxins from the medium was beneficial to imperatorin production and addition of BA (0.5-1 mg/L) promoted synthesis of imperatorin in suspension cells.

As was observed in this study that callus cultures from leaf explant raised on MS medium supplemented with NAA at a concentration of 5 mg/L were lacking the compounds like Benzene 1,3-dimethyl and Aniline, N-methyl (Table 2), but on reducing the amount of auxin and addition of BAP (BAP/NAA, 2/3 mg/L) resulted in the production of the said compounds. The concentration of these compounds increased manifold in the callus cultures than the leaf tissue. The results were found vice versa in the internode derived callus cultures where the same combination of PGRs supplemented in the same way retarded the production of some compounds which were being produced in the internode callus cultures influenced by NAA (5 mg/L). From the comparative analysis of secondary metabolites, it is concluded that most of the secondary metabolites from seedling tissue were present in their respective calluses.

From the present study, it was also observed that many of the metabolites from seedling tissues and their respective calluses can vary under the influence of different combinations PGRs.

REFERNCES

[1] Davis and C. Parke. Manual of therapeutics, 262-266, 1909.

[2] Anonymous. Hamdard Pharmacopoeia of Eastern Medicine, Hamdard National Foundation, Pakistan 2nd Impression. P. 373, 1970.

[3] Mulabagal V. and H-S. Tsay. Plant Cell Cultures - An Alternative and Efficient Source for the Production of Biologically Important Secondary Metabolites. Int. J. Appl. Sci. Eng., 2 (1): 29-48, 2004.

[4] Murashige, T. and F. Skoog. Arevised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plants, 15:437-496, 1962.

[5] Khosh, M. and K. Singh. Callus induction and culture of roses. Sci. Hort., 17: 361, 1982.

[6] Nabi, S. A., M. M. Rashid, M. A. Amin and M.G. Rasul. Organogenesis in teasle gourd (Momordica dioica Roxb.). Plant Tissue Cult., 12: 173-180, 2002.

[7] Duangporn, P. and P. Siripong. Effect of auxin and cytokinin on phyllanthusol A production by callus cultures of Phyllanthus acidus skeels. Amer-Eurasian J. Agri. Environ. Sci., 5 (2): 258-263, 2009.

[8] Attard, E. and Scicluna-Spiteri, A. Ecballium elaterium: An in vitro Source of Cucurbitacins. Fitoterapia. 72 : 46-53, . 2001.

[9]Towers G. H. Neil and S. Ellis Human Medicinal Agents from Plants Vancouver, British Columbia V6T 1Z4, Canada, ACS Symposium Series, Vol. 534, pp 56-78, 1993.

[10]Harsh, M. L., T. N. Nag and S. Jain. Arid zone plants of Rajasthan a source of antimicrobials. Com. Phys. Eco., 8: 129-131, 1983.

[11]Ulubelen A., J. J. Mabry, G. Dellamonicas. and J. Chopin. Flavonoids of Passiflora plamer. J. Nat. Prod., 47: 384-385, 1984.

[12] Goswami A. and A. Reddi. Antimicrobial activity of flavonoids of medicinally important plant Cassia angustifolia in vivo and in vitro. J. Phyto. Res., 17: 179- 181. 2004.

[13]Saxena S., R. Sharma, S. A. B. A. Rajore. 2005. Isolation and identification of flavonoid "Vitexin" from Jatropha curcas L. J. Plant Sci. Res., 21: 116-117.

[14]Matooq, G. T., S. H. E. Sharkawy, M. S. Afifi and J. P. N. Rosazza. C-p hydroxybenzoylglycoflavones from C. colocynthis. Phytochem., 44(1): 187 190, 1997.

[15] Hatam, N. A. R., D. A. Whiting and N. J. Yousif. Cucurbitacin glycosides from C. colocynthis. Phytochem., 28(4): 1268-1271, 1989.

[17] Gamborg, O. L., F. Constabel, T. L. Rue, R. A. Miller and W. Steck. The influence of hormones on secondary metabolite formation in plant cell cultures. Colloq. Int., 193: 335, 1971..

[18] Asrafi, S., M. R. Mofid, M. Otroshi, M. Ebrahimi, M. Khosroshahli, Effects of plant growth regulators on the callogenesis and Taxol production in cell suspension of Taxus baccata L. Trakia J of Sciences, 8 (2) 36-43m, 2010.

[16] Chen, A. H. Study on application of diosgenin-I. Analysis of diosgenin constituent of plants from Taiwan. Science Monthly, 43: 79-85, 1985..
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Author:Tanveer, Hina; Ali, Safdar; Zaheer-ud-Din-Khan
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Date:Dec 31, 2011
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