INTRODUCTION:

People living in rural areas from their personal experience know that these traditional remedies are valuable source of natural products to maintain human health. Herbal medicines are in great demand in both developed and developing countries as a source of primary health care owing to their attributes having wide biological and medicinal activities, high safety margins and lesser costs. Mesua ferrea Linn. belonging to the family Guttiferea is known in Hindi as Nagkesara¹ It is medium sized evergreen tree upto 13 mm tall, often buttressed at the base with the trunk up to 90 cm in diameter and found In India, Srilanka, Southern Nepal, Burma, Thailand, Malayasia, And Sumatara Regions.

Leaves about 3 to 5 inches long, it is simple, narro, ovate or egg shaped. It is dark green in colour, with a whitish underside, the new growing leaves are red in colour then slowly they turn into yellow colour. Flowers are about 4 to 7.5 cm in diameter with four white petals and a centre of numerous yellow stamens. Fruits are oblong in shape; they are about 2.5 to 5.0 cm long with persistent calyx. Seeds 1 to 4 in number, dark brown in colour, cotyledon is fleshy and oily^2,3. Nagkesar is a very famous Ayurvedic herb used in treating fever, vomiting, urinary tract disorders, vaginal affections and migraine etc. It is one among Chaturjata group of herbs. It is used as in powder form along with other spices, and put into many herbal jams including Chyawanprash and also used to preparation of several compound formulation viz. Candanabalalaksadi Taila, Kumaryasava, Nagakesaradi Curna, Kankasava, Eladi curna, Jatiphaladi curna and Karpuradi churna, Pushyanug Curna, Suparipak and Pradaranashak curna^4-8etc. The purpose of this standardization involves the safe, proper selection and handling of crude materials, ensure quality, safety, efficacy and stability of finished products. And another there are no reports of systematic pharmacognostic study of stamen of this plant. Keeping this aim into consideration, the present study was designed to scientific evaluation of Nagakesara stamen. The study includes macro and microscopic characters, powder microscopic characteristics, High Performance Thin Layer Chromatography fingerprints, preliminary phytochemical screening and physicochemical parameters. The information generated by this particulars study provides relevant pharmacognostical and physicochemical data needed for proper identification and authentication of Nagakesara stamens.

MATERIAL AND METHODS:

Collection and processing of plant material:

The fresh plant stamens of Mesua ferrea was collected from the, Chennai in the month of August. Samples were authenticated ^9-11 by Botanist Dr. Manoj Tripathi of Ayurveda Sadan (Research Laboratory), Deendayal Research Institute Chitrakoot. The voucher specimen (AD/AS/110/2015) prepared as per standard procedure¹² and maintained in the herbarium of Department of Pharmacognosy, Ayurveda Sadan (Research Laboratory), Deendayal Research Institute Chitrakoot for further reference. Fresh material was used for anatomical studies whereas shade dried material was powdered in electric grinder for physico-chemical study, phytochemical investigation and development of High Performance Thin Layer Chromatography fingerprint profile.

Macroscopic study:

Macroscopic or organoleptic characters like appearance, colour, odour and taste were evaluated.

Powder microscopic study:

The dried stamens were subjected to powdered and completely passes through 355 μm IS Sieve (old sieve number 44) and not less than 50% pass on through 180 μm IS Sieve (old sieve number 85). About 2 g of powder washed thoroughly with potable water, pour out the water without loss of material. Mounted a small portion in glycerine, warmed a few mg with chloral hydrate solution, wash and mounted in glycerin. Treat a few mg with iodine solution and mount in glycerine, about 1 g of powder warmed over water bath with Chloral hydrate solution till brown fumes appear, cool and wash with water thoroughly and mount a small portion in glycerin and seen under microscope at 40X x 10X magnification of the Trinocular Research Microscope^13-15 .

Physico-chemical parameters:

Physico-chemical parameters such as moisture content (loss on drying at 105⁰C ), water soluble extractive value, alcohol soluble extractive value, total ash value, acid insoluble ash value and water soluble ash were calculated^16,17.

Preliminary phytochemical studies:

Preliminary phytochemical tests were carried out on ethanolic and water extract for the presence\absence of phyto-constituents like alkaloids, flavanoids, steroids, terpenoids, tannins, resins, carbohydrates, proteins and saponins^18,19.

High Performance Thin Layer Chromatography (HPTLC):

For High performance thin layer chromatography, the powdered stamens 5 gm of sample was extracted with 100 ml of ethanol overnight, filtered and concentrated. It was applied by spotting extracted sample on pre-coated silica-gel aluminium plate 60 F₂₅₄ (5x10 cm with 0.2 mm layer thickness Merk Germany) using Camag Linomat -5 sample applicator and a 100 μl Hamilton syringe. The samples, in the form of bands of length 6 mm, were spotted 15 mm from the bottom, 15 mm from left margin the plate and 10 mm part. Plates were developed using mobile phase consisting of Hexane: Ethyl acetate (7:3 v\v). Linear ascending development was carried out in 10x10cm twin through glass chamber equilibrated with mobile phase. The optimized chamber saturation time for mobile phase was 30 min. at room temperature. The length of chromatogram run was 8 cm. 20 ml of the mobile phase. Subsequent to the development, Thin Layer Chromatography plate was dried with the help of Hot Air Oven. The peak area for samples and standard were recorded with Camera photo documentation system Camag Reprostar3. Visualization of spot was made before and after derivatization (with 5% Methanolic - sulphuric acid reagent) at 254nm, 366nm and day light with Win cat software and R_f values noted^20,21.

RESULTS AND DISCUSSION:

Macroscopic characters:

The stamens consist of anther, connective and filament, filament united at base forming a fleshy basal sheath; stamen 0.80 cm to 2 cm long; anther lobe about 0.6 cm long, basifixed, linear, containing pollen grains, filament 0.8 cm to 1.4 cm long, filiform, slender, Pollen grains. (Figures.1 and 2).

Powder microscopic study:

Mesua ferrea stamens powder colour is golden brown, taste astringent and odour characteristics. Under microscope examined powder shows fragments of beaded endothecium cells, abundant pollen grains in different shape oblate to sub-oblate; exine surface reticulate, thickness 1-2 µm are 3-5 with average size 35 X 45 µm (range 28 to 45. X 35 to 55 µm, epidermal cells of anther, vessel with spiral and annular thickening, different types of trichomes (unicellular, multicellular uniseriate, stelate trichome of anther lobe and unicellular multiseriate trichome of filament and parenchymatous cells (Figures 3 to 8).

Figure-1. Nagakesara, dried flowers

Figure-2. Nagakesara,


Figure-3 endothecium cell	Figure-4 Pollen grains	Figure-5 Epidermal cell of anther


Figure-6 Vessels with spiral and annular cells thickening	Figure-7 Trichomes	Figure-8 Parenchymatous

Physico-chemical analysis:

The physico-chemical parameters such as extractive values are useful for the determination of exhausted or adulterated drug; ash values of the drug gave an idea of the earthy matter or the inorganic composition and other impurities present along with the drug. Physico-chemical results of the drug are given in (Table 1).

Table 1: Physico-chemical analysis of the Mesua ferrea Linn.

S. No.	Parameters	Results
1	Foreign matter	1.5%
2	Loss on drying at 105⁰C	6.4%
3	Ethanol-soluble extractive	18%
4	Water- soluble extractive	22 %
5	Total ash value	6%
6	Acid-insoluble ash value	1.95%

Preliminary phyto-chemical investigation:

Qualitative phyto-constituents were screened in the extracts taken in water and ethyl alcohol. Phyto-chemical results of the drug are given in (Table 2).

Table 2:Phytochemical analysis of Mesua ferrea Linn. Stamens

S. No.	Name of phyto-constituents	Result
1	Alkaloids	Present
2	Carbohydrates	Present
3	Protein	Present
4	Saponin	Present
5	Steroids	Absent
6	Resin	Absent
7	Tannin	Present
8	Flavonoid	Absent

High performance thin layer chromatography:

High performance thin layer chromatography (HPTLC) study of the ethanolic extract two spots of the sample extracts applied in the Thin Layer Chromatography plate. Major spots R_fvalues with colour were recorded under, 254nm, 366nm, after derivatization 366nm. Chromatogram profile and R_fvalues are given (Figure.9 -11 and Table -3).

The macroscopic, microscopic and powder microscopic distinguished characters have been established to identify Mesua ferrea stamen. The pharmacognostic and physicochemical parameters can be used for checking the adulteration and purity of this drug. High performance thin layer chromatography finger print profile helps in identification of various phytochemical constituents present in the crude drug thereby substantiating and authenticating of crude drug. The High performance thin layer chromatography profile also helps to identify and isolate’s important phyto-constituents. These finding could be helpful in identification and authentication.

T₁ T₂

Figure-9. HPTLC fingerprint profile At 366nm before derivatization

Figure-10. HPTLC fingerprint profile At 366nm after derivatization

Figure-11. HPTLC fingerprint profile At daylight After derivatization

Table 3 : Rf values of HPTLC finger prints profile of test solution of Mesua ferrea Linn. Stamens

R_fvalues	*Test solution of Mesua ferrea* Linn. Stamens**
R_fvalues	At 366 nm(before derivatization)	At 366nm (after derivatization)	Under day light (after derivatization)
R_f1	0.04(light pink )	0. 06 (light pink)	0.32(light black)
R_f2	0.08(dark pink)	0.18( light brown)	0.54(brown)
R_f3	0.32(brown)	0.32(yellow)	0.60 (brown)
R_f4	0.34( reddish brown )	0. 34( sky blue)	0.70(green)
R_f5	0.60(dark red)	0.60(yellow)	0.80(dark brown)
R_f6	0.64(fluorescence)	0.70(sky yellow)	-
R_f7	0.70(blue)	0.80(yellow)	-
R_f8	0.80(dark red)	-	-

CONCLUSION:

Mesua ferrea has numerous uses in traditional medicine to treat several ailment viz. in treating fever, vomiting, urinary tract disorders, vaginal affections and migraine etc. It is one among Chaturjata group of herbs. It is used as in powder form along with other spices, and put into many herbal jams including Chyawanprash and also used to preparation of several compound formulation viz. Candanabalalaksadi Taila, Kumaryasava, Nagakesaradi Curna, Kankasava, Eladi curna, Jatiphaladi curna and Karpuradi churna, Pushyanug Curna, Suparipak and Pradaranashak curna etc.. Due to its wide therapeutic importance it is worthwhile to standardize it for use as drug. The present study reveals pharmacognostical standardization of drug Mesua ferrea, which would be of immense value in botanical identification and authentication of plant drug may help us in preventing its adulteration.

ACKNOWLEDGEMENT:

The authors are grateful to the Shri Abhay Mahajan, Organizing Secretary Deendayal Research Institute, Chitrakoot, Satna (M.P.) for providing necessary facilities.

REFERENCE:

1. Shome U, Mehrotra S. Sharma HP, Pharmacognostic study of flower of Mesua ferrea Linn., Proc Indian Acad Sci (Plnt Sci) 1982 ; (91) 211-226.

2. Anonymous, The Ayurvedic Pharmacopoeia of India. Part-I, Volume-II,Government of India, Ministry of Health and Family Welfare, Deptt. of ISM and H., The Controller of Publications Civil Lines, Delhi, 1999.

3. D. P. Gupta, The Herbs, Published by Smt. Prem Lata Gupta. Indore, 2008, 295-296.

4. Sahu Alakh N, Hemalatha S, Sairam K phyto-pharmacological review of Mesua ferrea Linn .International Journal of Phytopharmacology, 2014 ; 5(1) 6-14.

5. Raju MS, Srimannarayana G, Rao NVS Structure of Mesuanic acid Indian J Chem 1974 12 884-886.

6. Raju MS, Srimannarayana G, Rao NVS, Structure of Mesuaferrone- A , a new biflavone from the stamens of Mesua ferrea Linn. Indian J Chem 1978; 16B: 167-168.

7. Kakrani HK, Nair GV, Dennis TJ, Jagdale MH, Antimicrobial and anthelmintic activity of essential oil of Mesua ferrea Linn. Indian Drugs 1984; ( 21) 261-262.

8. Nishteswar K, and Hemadri K, Dravyaguna-Vijnana Published by Chaukhambha Sanskrit Pratishthan New Delhi 2011 ;13-14

9. Jain SK and Rao RR, A hand book of field and herbarium methods, Today and tomarrow printers and publications New Delhi 1977.

10. Kirtikar KR and Basu BD, Indian Medicinal Plants. Bishen Singh Mahendra Pal Singh, Dehra Dun 1935, Vols. 1(4) (2^nd ed.).

11. Verma DM, Balakrishnan NP and Dixit RD, Flora of Madhya Pradesh Botanical Survey of India, Calcatta 1993; Vol. (I).

12. Jain SK, Dictionary of Indian Folk Medicine and Ethnobotany. Deep Publications, New Delhi, 1991.

13. Evans WC, Trease and Evans Pharmacognosy, 15^th ed., (Saunders, London, 2003, 545-547.

14. Brain KR and Turner TD, The practical Evaluation of Phytopharmaceuticals (Wright-Sciencetechnica, Bristol, 1975, 4-9.

15. Kokate CK, Practical Pharmacognosy Is ted., Vallabh Prakashan, New Delhi 2006.

16. Anonymous, The Ayurvedic Pharmacopoeia of India. Part-I, Volume-III,Government of India, Ministry of Health and Family Welfare, Deptt. of ISM and H., The Controller of Publications Civil Lines, Delhi, 2000.

17. Anonymous, Protocol for Testing of Ayurvedic, Siddha and Unani Medicines. Government of India, Department of AYUSH, Ministry of Health and Family Welfare, Pharmacopoeial Laboratory for Indian Medicines Ghaziabad, 2007.

18. Tripathi M and Sikarwar RLS, Pharmacognostic Studies on Plaksa (Ficus virens Ait.) Stem Bark, Indian Journal of Natural Products and Resources 2015; 6(1) 27-32.

19. Tripathi M, Sikarwar RLS, Tiwari A and Dwivedi N., Pharmacognostical identification of ingredients in Laghulai curna, Indian Journal of Traditional Knowledge, 2015;14(4) 531-536.

20. Ansari SH, Essentials of Pharmacognosy Ist edition, Birla publications Pvt. Ltd, New Delhi, 2006.

21. Choudhary N, Siddiqui MB and Khatoon S, Pharmacognostic evaluation of Tinospora cordifolia Willd.) Miers and identification of biomarkers, Indian J Tradit Knowle, 2014 ; 13(3) 543-540.

Received on 19.05.2017 Modified on 03.06.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(3): 147-151.

DOI: 10.5958/0975-4385.2017.00027.9