SAPONINS, GLYCOSIDES AND FLAVONOIDS IN CELLS AND TISSUES OF BALANITES AEGYPTIACA CULTURED ON SOLID AND LIQUID CULTURE MEDIA

Objectives: The main objective of the present study is to obtain callus and cell suspension culture from Balanitesa egyptiaca sterile plantlets grown in vitro and to compare growth and the biosynthetic potential of saponins, flavonoids and glycosides by callus and cell suspension culture Balanitisa egyptiaca. Methods: Callus was induced from the mother plants on MS culture media supplemented with 2.0 mg/l BA + 2.0 mg/l 2,4-D with and without agar gelling. Total saponins, glycosides and flavonoids were estimated in both types of cultures over a period extending from 1 to 5 weeks to compare the productivity of such secondary metabolites in callus and cell suspension cultures. Results: The results obtained indicated that both calli and cell suspension cultures were able to synthesize the target active ingredients and that cell suspension culture was superior to the callus culture in the biosynthesis and accumulation processes. By the end of the incubation period, the amount of total saponins in cell suspension culture reached up 51.97±0.26 dry biomass compared to 35.02 ±0.06 mg/g in callus culture. The amount of total flavonoids in cell suspension culture reached up 10.88±0.24 dry biomass compared to 6.40±0.02 mg/g in callus culture and of total glycosides reached up 6.11±0.25 dry biomass compared to 5.06 ±0.05 mg/g in callus culture. Conclusions: The results obtained in this study may indicate the promising role that plant cell culture will play in the future in phytopharmaceutical industry.


INTRODUCTION
Balanitesa egyptiaca, which is known as desert palm or heglig 1 , is a multipurpose evergreen spiny tree species that can be used as a fodder, a source of wood for charcoal industry, a timber, a fuel wood and a raw material for many other purposes 2 . More recently, the oil of the seeds of this tree is being used in many industries such as soap, shampoo, cream, herbal medicine and even the production of biodiesel 3,4,5, 6 . This plant synthesizes many secondary active metabolites like saponins, flavonoids and glycosides and this may explain the many pharmacological effects of the plant 7 , which were reported by many researchers 8,9,10 . Saponins of Balanitesa egyptiaca (diosgenin and yamogenin) are used in the partial synthesis of steroidal drugs 11 . Plant cell culture based product is a modern perspective biotechnological application of plant tissue culture. Cell culture systems are preferable over conventional whole plant cultivation in production of phytopharmaceuticals to avoid the adverse effects of external factors on secondary metabolites biosynthesis; the cultured cells are not threatened by the attacks of microorganisms or insects; cells of any plant even rare or endangered ones can be cultured in vitro; regulation of secondary metabolite production decreases costs and improves productivity 12 . The aim of the present study is to compare growth and the biosynthetic potential of saponins, flavonoids and glycosides by callus and cell suspension culture Balanitisa egyptiaca.

Phytochemical analysis of callus and cell suspension cultures Extraction:
Calli and cell suspension cultures were harvested washed and excessive water was removed. The harvest of both types was then dried in shade. After constant weights were attained, calli and cell suspensions were grinded into fine powder. One gram of dry fine calli and cell suspension powders was soaked in pure methanol (150 ml) separately at room temperature for 72 hours with stirring from time to time. After that, filtration was carried out and the residue was washed with three successive rinses (100 ml) of pure methanol. The filtrate and washings were combined and evaporated to 10 ml. The obtained extract was used for determination of total saponins, flavonoids and glycosides.

Determination of total saponins
For total saponins estimation, 0.5 ml of methanolic extract for each of callus and cell suspension cultures extracts and 0.5 ml of o.5 % p-anisaldehyde reagent were mixed and kept for 10 minute. Later, 2ml of 50% sulphuric acid was added, and then tubes were vortexed. Then kept in water bath with constant tempura of 60 0 C for 10 minute then cooled and the absorbance of the developed yellow color was measured at 435 nm. The amount of saponins was calculated as saponin equivalent from the calibration curve of standard saponin 100 -1000 µ/ml 14 .

Determination of total flavonoids
Total flavonoid content of the methanol extracts of both callus and cell suspension cultures were determined by Aluminum chloride method. To 0.5 ml aliquots of the extracts and standard solution (0.01-1.0 mg/ml), 2ml of distilled water and 0.15 ml of sodium nitrite (5% NaNO 2 , w/v) were added and mixed then left to stand 6 minutes. Then 0.15 ml of (10% AlCl 3 , w/v) solution was added and mixed well in a tube. Allowed to stand for further 6 min and after that 2 ml of sodium hydroxide (4% NaOH, w/v) solution were added, mix and the final volume of each was completed to 5 ml distilled water. After thorough mixing the solutions, allowed to stand for another 15 min. The absorbance of each mixture was determined at 510 nm against the same mixture without callus extract as a blank 15.

Determination of total glycosides
Glycosides determination was done using Baljet reagent [95 ml of Picric acid (1%) with 5 ml of sodium hydroxide (10%)]. Eight ml of callus extract were transferred to a 100 ml flask; 60 ml of H 2 O and 8ml of 12.5% lead acetate were added, mixed and filtered. Fifty ml of H 2 O and 8 ml of the filtrate was transferred into another 100 ml flask and 8 ml of 47% Na 2 HPO 4 were added, mixed and completed the volume with distilled water and filtered twice. Ten ml of purified filtrate were transferred into clean flask and treated with 10 ml Baljet reagent. This was allowed to stand for one hour at room temperature for complete color development. The color intensity was measured calorimetrically at 495 nm against the blank of 10 ml distilled water and 10 ml Baljet reagent incubated for one hour at the same conditions 16.

Data analysis
Data represent mean ± standard deviation of 3 different values. Statistical significance was determined using two-way ANOVA: and p<0.0001; post hoc test: Tukey using Graph Pad Prism 7 software. In the estimation of contents different character is showing significant different and the same character is showing no significant different.

RESULTS AND DISCUSSION
Results of the present study (Table 1) show that with respect to callus culture, the amount of saponins increased from 1.51±0,02 mg/g dry callus biomass after one week of growth to 30.02±0.06 mg/g after 5 weeks of growth. With respect to cell suspension and microcalli developed on the liquid media, the amount of saponins increased from18.28±0.42mg/g dry biomass after one week of growth up to 51.97±0.26 mg/g dry biomass.

Figure 1: Effect of incubation period of calli and cell suspension culture of B. aegyticaca on content of total Saponin (mg/g)
The results obtained may indicate that both callus and cell suspension cultures of B. aegyptiaca were able to synthesize saponins but with different potentialities; The accumulation of saponins increased with the increase of age of both cultures from 1 to 5 weeks of incubation and in general, the cell suspension culture was much more active in accumulating saponins. The differences between callus and cell suspension culture in the biosynthesis or accumulation of saponins was statistically significant in all stages of growth. Callus and cell suspension cultures can be seen in Figure 1. Results of the present study ( Table 1) may show that the amount of total flavonoids estimated on the basis of the dry B. egyptiaca biomass depended on both age and type of the culture. After one week of incubation, the amount of flavonoids in the callus culture was1.08±0.03 mg/g compared to 1.51±0.02mg/g dry weight in microcalli and cell suspension culture.   Table 1), there were almost no significant differences between callus and cell suspension culture regardless of the general increase in the amount of glycosides determined from the first to the fifth weeks. The amount estimated in the first week was 1.30±0.03in callus culture compared to 1.57±0.36 mg/g in the cell suspension culture. By the end of the fifth week of incybation, the amount recorded in callus culture was 5.06±0.05 compared to 6.11 ±0.25mg/g dry weight recorded in the cell and microcalli suspension culture. The results obtained in this study may agree with many of the most recent papers on many other plants. Plant tissue culture techniques being independent of climatic and geographical conditions will provide an incessant, sustainable, economical and viable production of secondary metabolites 17 . Cell suspension culture of Spilanthe sacmella Murr used successfully to produce scopoletin 18 . It has been mentioned that Callus induction and multiplication have been extensively used in Product Development and Manufacturing Center "PDMC" in vitro production. It is an efficient approach to produce PDMCs in large scale when compared to other techniques, mainly because the in vitro callus induction is a straightforward and rapid system of cell multiplication 19 . It has been reported that the factors that induce callogenesis are well studied and that callus cultures are used for production of secondary metabolites on commercial level already for some decades 20 and stated also that medicinal plant cell suspension cultures (MPCSC) with plant cell totipotency can be regarded as a promising alternative for production of secondary metabolites 21 .

CONCLUSION
Callus was initiated from B. aegypiaca plantlets growing in vitro in our lab. Both types of cultures were able to synthesize and accumulate antioxidative secondary metabolites (saponines, flavonoids and glycosides). The increase in the age of the culture was accompanied by an increase in the accumulation of such metabolites from the second to the fifth weeks of growth. In general cell suspension culture was more active in accumulation of the targeted metabolites. The present study represents additional evidence that plant cell culture will play a major role in the future of industry of phytopharmaceuticals.