Studies on Banyan (Ficus benghalensis L.): Characterization of fruit and callus induction

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1 Journal of Scientific & Industrial Research Vol. 72, Sept - Oct 2013, pp Studies on Banyan (Ficus benghalensis L.): Characterization of fruit and callus induction H Bandekar, N Nagavekar and S S Lele* Food Engineering & Technology Department, Institute of Chemical Technology (ICT), Mumbai , India Received 19 March 2013; revised 09 May 2013; accepted 29 May 2013 This study explores fruits of Banyan (Ficus benghalensis L.) as a possible source of nutrition and may be introduced as a novel food source. Proximate analysis of fruit gave: moisture, 71.02±0.2 (wet basis); ash, 2.83±0.25; crude fat, 8.66±0.57; fibre, 17.08±0.05; protein, 4.83±0.11; and carbohydrate, 66.63±0.55%. Anti-nutritional factors identified were: tannins, 1.88±0.76; oxalates, 0.01±0.00; cyanogenic glucosides, 0.006±0.00; alkaloids, 9.7±0.07; and saponins, 4.25±0.25%. Total polyphenols (276±0.84 mg/g GAE) were determined by Folin Ciocalteau method. Antioxidant DPPH scavenging activity with IC ±0.16 ug/ml was recorded. An attempt towards initiating callus culture on MS medium from nodal segments of this plant using hormones BAP and NAA for anthocyanin production has been done. Keywords: Alternate foods, Anthocyanin, Anti-nutritional factors, Antioxidants, Callus culture, Ficus benghalensis L. Introduction Fruits provide adequate supplies of minerals, fat, protein, carbohydrate and fiber 1. Anti nutritional factors interact with proteins, vitamins, and several minerals of human and animal nutrition thereby restricting their bioavailability 2. Banyan (Ficus benghalensis L.; Family, Moraceae), commonly known as Indian banyan tree, bears cheery sized bright red fruits that are derived from a specially adapted type of inflorescence 3 called as synconium (Fig. 1). Bright red colour of fruits of banyan tree indicates presence of anthocyanins, which have been reported in common edible fig, F. carica 4. Explants of F. benghalensis can be used as a source of callus culture for in vitro production of anthocyanins. Manipulation of nutrient medium improves yield of metabolites in plant cell cultures 5. Anthocyanins have high antioxidant activity, anticancer properties and potential health effects against mild cardiovascular disorders, neurological diseases, diabetes and inflammation 6. This study presents proximate composition and anti nutritional factors of banyan fruits, besides total polyphenolic content, anti oxidant activity and anthocyanin content. Optimization for extraction of anthocyanin pigment was performed. An attempt towards developing *Author for correspondence dr.smita.lele@gmail.com a callus culture for in vitro production of anthocyanin using explants of F. benghalensis was undertaken. Experimental Section Sample Preparation for Fruit Studies and Plant Tissue Culture Fruits were obtained from ICT, Mumbai campus, and washed twice with tap water to remove surface flora and dirt, followed by wash by distilled water. Excess water was removed by a tissue paper. Inedible portions of fruits were removed. Fruits were then ground in a mixer and dried in a tray dryer at 65 o C for 2h. Dried powder was ground again in mixer and sieved to obtain uniformly sized particles. Powder obtained was stored in air tight plastic bags in desiccators until analysis. For tissue culture, F. benghalensis explants (nodal segments, leaf discs) obtained from ICT Mumbai campus wer e washed thor oughly to remove sur face contaminants, again washed by few drops of commercial detergent for 5 min, followed by another wash using Tween 20 (Atlas Chemie), as detergent was given for 3-5 min. Plant material was washed with distilled water and stored in a clean petri plate. Under sterile conditions, explants were transferred to a sterile bottle, 70% alcohol was added, and swirled for 2 min. Alcohol was decanted and plant material was washed with distilled water. A next wash of 4% sodium hypochlorite solution was given for 5 min, followed by removal of sterilizing agents with

2 554 J SCI IND RES VOL 72 SEPT - OCT 2013 medium. Explants obtained on sterilization were used for inoculation. Segments (5 mm long) were excised and cultured in sterile tubes containing 15ml MS media. Cultures were maintained at 25±2 o C with a 16 h light, 8 h dark photo period. Fig. 1 Fruits of banyan (Ficus benghalensis L.) a thorough wash of distilled water. Explants were then used for inoculation. Proximate Composition and Anti Nutrients Proximate composition analysis of dry samples for ash, crude protein, crude fat, crude fibre and total carbohydrate were carried out in triplicates according to the standard methods of analysis 7. Energy value was calculated using Atwater factors of 4, 9, and 4 for protein, fat and carbohydrate, respectively 8. Quantitative determination of oxalates was done by volumetric method 9. Tannins were estimated by spectrophotometric method using Folin Denis agent. Cyanogenic glucosides were determined as per AOAC method 7. Phytates were estimated spectrophotometricaly by extraction in trichloro acetic acid (TCA) and precipitation as ferric salts 10. Saponins were estimated by gravimetric method 11. Alkaloids were estimated gravimetrically by alkaline precipitation method 12. Total Polyphenol Content, Antioxidant Activity and Total Anthocyanins Anthocyanins and polyphenols are predominantly found in peels, hence peels were used for assays. Total polyphenol was determined spectrophotometry, using gallic acid as standard by Folin Ciocalteau method. Total phenolic acid content is expressed as mg gallic acid equivalents (GAE)/g. Antioxidant activity of dried peels and standard was assessed on the basis of radical scavenging effect of stable 1, 1, diphenyl-2,- picrylhrdrazyl (DPPH) free radical activity. Total anthocyanin estimation was done by ph differential method 7 using acidified (0.1%) solvents. Explant Selection Leaf discs and nodal segments (1-2 cm) were used for initiation of callus cultures on Murashige and Skoog Media Selection Murashige & Skoog medium (MS) (HiMedia Modification No. 3) was used for plant tissue culture studies. According to optimized protocol (Bandekar H. et al, ICT Mumbai, unpublished data), 3% sucrose (HiMedia) concentration and 0.1 mg/ml L- glutamine (Merck) were added to MS medium. Napthalene acetic acid (NAA) (0.1 mg/l) and 6- benzyl aminopurine (BAP) (2 mg/l) hormones were supplemented in the medium. Bacteriological agar (0.35%), gelrite (0.12%) (Gelrite Gelan Gum, sigma), was used for solid phase cultures. Controls were maintained at all stages of experimentation. Medium ph was adjusted to 5.8. Chemicals and reagents for analysis were procured from S.D. Fine Chemicals, Pvt. Ltd., unless mentioned. All assays were done in triplicates. Results and Discussion Proximate Composition and Anti Nutritional Factors Proximate composition of banyan fruit gave (dry basis): moisture, 71.02±0.2 (wet basis); ash, 2.83±0.25; crude fat, 8.66±0.57; fibre, 17.08±0.05; protein, 4.83±0.11; and carbohydrate, 66.63±0.55%. Thus fruits are rich in ash content (minerals), carbohydrates, crude fibres and are a good source of energy. High mineral content aids in better digestibility by improving enzyme activity. Crude fibre consumption contributes to an increase in fecal bulk thus increased rate of intestinal transit. Total metabolizable energy ( kcal/ 100 g) can be used as a very good source for human nutrition. Edible variety [F. carica (anjeer)] is reported 13 to contain: ash, 1.86; carbohydrates, 63.87; fibre, 9.8; and protein, 3.3%. Thus suitable processing of banyan fruit can provide a high energy edible product. Anti nutrients in banyan fruits were found as follows: tannins, 1.88±0.76; oxalates, 0.01±0.00; cyanogenic glucosides, 0.006±0.00; alkaloids, 9.7±0.07; and saponins, 4.25±0.25%. Fruits contain high amount of saponins, alkaloids and tannins. Other anti nutrients namely oxalates (toxic above 4-5 g) 14, phytates (toxic above mg/g over long period consumption) 15 and cyanogenic glucosides (toxic above 3.7 mg/kg body weight) 15 are present in moderate amount and under acceptable limits.

3 555 Fig. 2 Solvent selection for anthocyanin extraction Fig. 3 Optimization of time period for anthocyanin extraction Saponins reduce uptake of certain nutrients like glucose and cholesterols in the gut through intra-lumenal physicochemical interaction, besides inhibit activities of digestive enzymes like trypsin and chymotrypsin. Alkaloids are often toxic to humans having dramatic physiological and neurological effects 13,16. Tannins are responsible for decrease in feed intake, growth rate, feed efficiency, net metabolizable energy and protein digestibility in experimental animals 17. Cyanogenic glucosides inhibit metal ions Fe ++, Cu ++ and Mn ++ inhibiting vital processes like Electron transport chain 18. High oxalate foods exert a negative effect on calcium and iron absorption, and interfere with carbohydrate mechanism particularly by succinic dehydrogenase inhibition 14. Phytic acid has ability to bind minerals, proteins, and starch. Proximate analysis data and study of anti nutrients suggest that this fruit could be a potential food source if consumption of anti nutritional factors is monitored by blanching, cooking, pressure cooking, soaking, fermentation etc. Total Polyphenol Content and Free Radical Scavenging Activity of Fruit Polyphenols content in fruit was 276±0.84 mg/g GAE. As antioxidants, polyphenols may protect cell constituents against oxidative damage and, therefore, limit risk of various degenerative diseases associated to oxidative stress. Polyphenols help in chelating metal ions (copper and iron), which act as pro oxidants. Antioxidant DPPH scavenging activity with IC ± 0.16 ug/ ml was recorded. Free radical scavenging activity is indicative of polyphenolic content of the source. Optimization for Extraction of Anthocyanin from Fruit Peels Extraction of anthocyanins from peels of fruits is better when acidified solvents are used. Anthocyanins were best extracted in acidified (0.1%) methanol as a solvent (Fig. 2). Extraction of anthocyanins at different time periods (½, 1, 1½, 2, 2½ and 3 h) was found to be maximum at 1.5 h (Fig. 3). Temperature studies for extraction of anthocyanins (Fig. 4) indicate that maximum

4 556 J SCI IND RES VOL 72 SEPT - OCT 2013 Fig. 4 Optimization of temperature for anthocyanin extraction Fig. 5 Callus induction in MS medium (3% sucrose, 0.1 mg/ml L glutamine, 2 mg/l BAP, 0.1 mg/l NAA) after different weeks (3, 6 and 10) extraction was obtained at 37 C and lower and higher temperatures affected yield of pigment. Moisture% was accounted for in these experiments by drying an equal amount of peels till constant weight and accounting for weight reduction. All estimations were in triplicates and controls were maintained. Explant Selection and Callus Induction, Proliferation and Maturation Callus induction was seen when nodal segments were used as explants. Leaf disks did not give favourable results. Initiation of callus was observed within 2-3 weeks after inoculation of explants (Fig. 5). Obtained callus was allowed to mature for 6 weeks before sub-culturing. Conclusions Banyan fruits were found to be rich in mineral content, fibres and carbohydrates. A possible food product may be developed by decreasing concentration of anti nutrients (saponins, alkaloids and tannins) to acceptable limits using soaking, blanching, cooking etc. Calli induced from nodal explants can be used to explore their potential for anthocyanin production, thus resulting to an alternate in vitro source of natural pigments to be used in various food and dye industrial applications. Acknowledgment Authors thank Department of Biotechnology and University Grants Commission- Special Assistance Program, India for providing financial support for the project. References 1 Nkafamiya I I, Manji A J, Modibbo U U and Umaru H A, Biochemical evaluation of Cassipourea congoensis (Tunti) and Nuclea latifolia (Luzzi) fruits, Afr J Biotechnol, 6 (2006) Malik I O, Babiker E E, Yousif N E and El Tinay A H, In vitro availability of minerals of some tropical and citrus fruits as influenced by anti nutritional factors, Nahrung/Food, 48 (2004),

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