1. INTRODUCTION:

Plants have been the major source of drugs in Indian system of medicine and other ancient systems in the world. Earliest description of curative properties of medicinal plants is found in Rig-Veda, Charaka Samhita and Sushrusha Samhita give extensive description on various medicinal herbs (Kirtikar et al., 1989). Information on medicinal plants in India has been systematically organized (Eds et al., 1976). India has an ancient heritage of traditional medicine. The Materia Medica of India provides a great deal of information on the folklore practices and traditional aspects of therapeutically important natural products. Indian traditional medicines based on various systems including Ayurveda, Siddha, Unani and Homeopathy. The evaluation of these drugs is primarily based on phytochemical, pharmacological and allied approaches including various instrumental techniques such as chromatography, microscopy and others.

With the emerging worldwide interest in adopting and studying traditional systems and exploiting their potential based on different health care systems, the evaluation of the rich heritage of traditional medicine is essential. In this regard, on such plant is Ficus bengalensis Linn. syn. Ficus banyana Oken. (Family-Moraceae). The plant is a large evergreen tree distributed all over India from sub himalayan region and in the deciduous forest of Deccan and south India. It is a grown in gardens and road sides for shades (Anonymous, 1999, Parrotta et al., 2001). It is a member of four sacred trees Nalpamara (Ksirivrksas) meant to be planted around the home and temples. It is found throughout the year, grows in evergreen except in dry localities where it is a leafless for a short time. It is hardy and drought-resistant; it withstands mild frost. It is epiphytic when young. Fruit of Ficus species (family-Moraceae) viz. Ficus benghalensis Linn. commonly known as ‘Chinese banyan or the banyan tree or Bargad’ respectively has been most widely used in traditional medicinal system of all over world including India (Figure 1). Ficus species are native to India, Nepal, Sri Lanka, Southwest China and Indochina also found throughout the plains of India upto 170 m altitude in the Himalayan region (Anonymous, 2000, Sharma et al., 2001, Gupta, 2008, Sharma, 2009).

Figure 1. Photograph of Ficus benghalensis fruit

Ficus benghalensis ia a major source of drugs in medicine and other ancient systems in the world. Herbalism is atraditional medicine or folk medicine practice based on the use of plants and plant extracts (Acharya and Shrivastava, 2008).This tree is considered to be sacred tree in India. The plant is a large evergreen tree distributed all over India from sub Himalayan region and in the deciduous forest of Deccan and south India. It is a grown in gardens and road sides for shades. The fruits are used as astringent, haemostatic, anti-septic, anti-inflammatory, antioxidant and anticancer agent and also in the treatment of diarrhoea, dysentery and in the treatment of skin diseases, ulcers, vaginal disorders, leucorrhoea, menorrhagia and deficient lactation. In the traditional system of medicine, the plant is used for various health problems and diseases (Khare, 2004, Joseph and Raj, 2011, Rawat et al., 2012).

Although the fruit of these species are important but very less studies has been reported so far on pharmacognostic and phytochemical parameters (Mishra and Tiwari, 2013). Hence this study was undertaken to develop comparative quality standards and HPTLC fingerprinting of fruit of these Ficus species and their evaluation. This may be useful to pharmaceutical industries for authentication of commercial sample and also to explore the possibility of using other species as complementary to each other.

MATERIALS AND METHODS:

Plant materials:

Ficus benghalensis (Indian banyan) fruit are collected from Arogyadham campus garden, Chitrakoot, Satna, Madhya Pradesh. They were washed with tap water, rinsed with distilled water and shade dried until the fracture is uniform and smooth. Then the dried fruit material was powdered. Then the final uniform powder was used for the extraction of active constituents of the fruit.

Preliminary Phytochemical Screening:

Phytochemical analysis of the extract was conducted following the procedure of Indian Pharmacopeia (Anonymous, 1996). By this analysis, the presence of several phytochemicals like alkaloid, flavonoid, tannin, saponin, resin, protein and carbohydrate were tested.

Physico-chemical analysis:

Air dried fruit material was used for the quantitative determination of loss on drying, total ash, acid insoluble ash, alcohol and water soluble extractive values according to standard procedure of Indian Pharmacopoeia and WHO/QCMMPM (Anonymous, 1996, Harbone, 1984).

Fluorescence analysis:

Fluorescence analysis of the fruit powder was treated with different chemicals and seen under the normal light and UV radiations at 254 and 365 nm wavelengths as per the standard procedure. The color development under the day light was also observed for the presence of various phytochemical compounds (Anonymous, 1998).

High Performance Thin Layer Chromatography (HPTLC):

For HPTLC, 5 g of coarsely fruit powdered in 250 ml stoppered conical flask & extracted with 100 ml ethanol for 24 hours by maceration technique with occasional shaking. The extract was extracted and volume was raised up to 100 ml in a volumetric flask. 25 ml of the extract was taken from the above stock solution and concentrated on a water bath to similarly, ethanol extracts were prepared for one sample of F. benghalensis L. as reference. TLC of extracts of all the samples and the reference ingredients was carried out on Silica Gel 60 F₂₅₄ precoated plates (0.2 mm thickness; from Merck India Limited). Camag Linomat 5 applicator was used for band application and Desaga Video documentation Unit 3 was used for documentation of fingerprinting. The mobile phase used was Toluene: Ethyl acetate (7:3). The plate was developed over a distance of 10 cm in a saturated development chamber (Twin trough chamber (10×10 cm with SS lid) and visualized under visible light, 254nm and 366nm. After spraying with 5% methanolic-sulphuric acid followed by heating at 110⁰C for 5-10 min (Kokate, 1994, Lohar, 2007, Anonymous, 2008).

RESULTS AND DISCUSSION:

The preliminary phytochemical screening of ethanol extracts showed the presence of flavonoids, carbohydrate, alkaloids, proteins and tannin presented in Table-1.

Table 1. Phytochemical screening of Ficus benghalensis

Phyto chemicals	Results
Flavonoid	+
Carbohydrate	+
Resin	-
Tannin	+
Alkaloid	+
Protein	+
Saponin	-

+ indicates presence whereas – indicates absence of phyto chemicals

Physico-chemical analysis:

Air dried fruit material was used for quantitative determination of phytochemical values. Loss on drying, total ash, acid insoluble ash, water soluble and alcohol soluble extractive were determined for five times as per WHO recommendations. (Table 2).

Table 2. Physico-chemical analysis of Ficus benghalensis fruit powder

Parameters	Result
Loss on drying at 105°C (%)	6.00
Total Ash (%)	6.22
Acid-insoluble ash (%)	1.20
Alcohol-soluble extractive (%)	18.20
Water-soluble extractive (%)	17.20

Fluorescence analysis:

The fluorescence analysis is a tool for the determination of constituents in the plant that gives a definite idea of the chemical nature. Thus the fluorescence analysis of the powder was carried out and data is presented in the (Table 3).

High Performance Thin Layer Chromatography (HPTLC):

The TLC plate were examine under ultra violet light at 366 nm; at visible for both before and after derivetisation with 5% methanolic- sulphuric acid reagent (Figures 2.1-2.3). The R_f values and colours of the bands obtained were recorded (Tables 4-6). It shows major spot under ultraviolet light (366 nm) R_f at 0.06 (blue), 0.39 (fluorescent), 0.55 (blue) and 0.95 (white). Spray the plate with 5% methanolic- sulphuric acid reagent followed by heating at 105⁰C for about 5 min and observe under ultraviolet light. It shows major R_f under ultraviolet light (366 nm) at R_f 0.06 (white), 0.19 (grey), 0.67 (white), 0.75 (white), 0.78 (yellow), 0.83 (fluorescent), 0.95(white) and observe under visible light 0.67(red), 0.74 (brick red), 0.78(pink), 0.83(brown), 0.95 (red).

Table 3. Fluorescence analysis of Ficus benghalensis fruit powder

Treatment	Colour of powder at different wavelength
Treatment	Day light	UV light at 254 nm	UV light at 366nm
Powder as such	Yellow green	Dark brown	Dark brown
Powder+ Distilled water	Yellow green	Dark brown	Lemon yellow
Powder + KOH	Grass green	Brown	Grass green
Powder + HCl	Light brown	Dark brown	Dark brown
Powder + H₂SO₄	Black	Black	Black
Powder + NaOH	Yellow green	Black	Brown
Powder + HNO₃	Yellow green	Grass green	Black
Powder + FeCl₃	Grass green	Dark brown	Dark brown
Powder + Ammonia solution	Grass green	Brown	Yellow green
Powder + Acetic acid	Grass green	Dark brown	Dark brown
Powder + Ethanol	Yellow green	Brown	Grass green
Powder + Methanol	Yellow green	Brown	Grass green

Table 4. R_fvalues in TLC finger prints of fruit of Ficus benghalensis at 366nm (before derivatization)

R_f value	*Ficus* *benghalensis*
R_f 1 (blue)	0.06
R_f 2 (fluorescent)	0.39
R_f 3 (blue)	0.55
R_f 4 (white)	0.95

Table 5. R_fvalues in TLC finger prints of fruit of Ficus benghalensis at 366 nm (after derivatization)

R_f value	*Ficus* *benghalensis*
R_f 1 (white)	0.06
R_f 2 (grey)	0.19
R_f 3 (white)	0.67
R_f 4 (white)	0.74
R_f 5 (yellow)	0.78
R_f 6 (fluorescent)	0.83
R_f 7 (white)	0.95

Table 6. R_fvalues in TLC finger prints of fruit of Ficus benghalensis at visible light (after derivatization)

R_f value	*Ficus* *benghalensis*
R_f 1 (red)	0.67
R_f 2 (brick red)	0.74
R_f 3 (pink)	0.78
R_f 4 (brown)	0.83
R_f 5 (red)	0.95

CONCLUSION:

There are over 400 different tribal and other ethnic groups in India which constitute about 7.5 % of India’s population. Tribal, rural and primitive societies have discovered solution for treatment of disease to almost all their needs and problems from the natural resources around them. Hence, in recent years, ethnomedicinal studies received much attention as this brings to light the numerous little known and unknown medicinal virtues especially of plant origin which needs evaluation on modern scientific lines such as phytochemical analysis and HPTLC fingerprinting. The present work was taken up with a view to lay down standards which will contribute significantly to quality control of these medicinally useful Ficus benghalensis specie. It also provides suitable criteria to differentiate the fruit of Ficus specie.

ACKNOWLEDGEMENT:

Authors are grateful to Dr. Bharat Pathak, General Secretary, Deendayal Research Institute, Chitrakoot, Satna (M.P.) for providing the infrastructure and support to conclude this type of research work successfully.

REFERENCES:

1. Kirtikar KR and Basu BD. Indian medicinal plants. Eds. E Blatter, Caius J. F., Lalit Maohan Basu, Allahabad, 2nd edn vol. II, 1989: 2389.

2. Eds SGV, Raina MK. and Sharma M. Medicinal Plants of India. Indian council of Medical Research, New Delhi: Vol. IV; 1976.

3. Anonymous. The Wealth of India . Volume-(F-G). In: A Dictionary of Indian Raw Materials and industrial products. CSIR, New Delhi, 4; 1999: 24-26.

4. Parrotta JA . Healing Plants of Peninsular India. USA: CABI Publishing, 2001: 517.

5. Anonymous. The Ayurvedic Pharmacopoeia of India. Ministry of Health & Family Welfare, Government of India, Department of ISM &H, The Controller of Publications, New Delhi, I (I); 2000: 88-89.

6. Sharma PC, Yelne MB and Dennis TJ. Database on Medicinal plant used in Ayurveda. Documentation and Publication Division, Central Council For Research in Ayurveda and Siddha, New Delhi, 2001:548-552.

7. Gupta DP. The Herbs, Habitate, morphology and pharmcognosy of medicinal plants. Published and Distributed by Smt. Prem Lata Gupta, Indore, Madhya Pradesh, 2008: 215-216.

8. Sharma PV. Dravyaguna – Vijnana (Vegetable Drugs). Published by Chaukhambha Bharti Academy, Varanasi, 2; 2009:664-666.

9. Acharya D and Shrivastava A. Indigenous herbalmedicine: tribal formulations and traditional herbal practices. 1st Edition, Avishkar publishers, Distributors, Jaipur, India, 2008: 13.

10. Joseph B and Raj SJ. An overview- ficus bengalensis Linn. International Journal of Pharmaceutical Sciences Review an Research, 6(1); 2011: 21-24.

11. Khare CP. Encyclopedia of Indian Medicinal Plants Springer publication, 2004: 216-217.

12. Rawat AKS, Tiwari SS, Srivastava A and Srivastava S. Comparative Botanical and Phytochemical Evaluation of Medicinally Important Stem Bark of Ficus species. Asian Pacific Journal of Tropical Disease, 2012: S34-S37.

13. Mishra R and Tiwari AK. Preliminary phytochemical screening and HPTLC fingerprinting of fruits of three Ficus species. Journal of Chemical and Pharmaceutical Research, 5(7); 2013:240-245.

14. Harbone JB. Phytochemical Methods: A guide to modern techniques of plant analysis, London: Chapman and Hall Ltd., 1984.

15. Anonymous. Indian Pharmacopoeia. Government of Indian, Ministry of Health and Human Welfare, New Delhi, India, Controller of publications, , 2; 1996: A53.

16. Anonymous. Quality control methods for medicial plant materials. WHO, Geneva. 1998.

17. Kokate CK. Practical Pharmacognosy. Vallabh Prakashan, New Delhi, India. 1994: 54.

18. Lohar DR. Protocol For Testing Ayurvedic, Siddha & Unani medicines. Govt of India, Department of AYUSH, Ministry of Health & Family Welfare, PLIM, Ghaziabad. 2007: 40-108. Anonymous. Quality Control Manual for Ayurvedic, Siddha & Unani medicines. Government of India, Department of AYUSH, Ministry of Health & Family Welfare, PLIM, Ghaziabad. 2008:1-99.

Received on 18.04.2014 Modified on 22.08.2014

Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 147-150