INTRODUCTION:

Nymphaeae pubescens willd (Nymphaceae) is a aquatic fresh water rhizomatous herbs, leaves long petioled, peltate, orbicular, sagittate when young, 15-25x12-20 cm, pubescent below, sharply dentate, venation radiatingly reticulate: flowers white or pink, solitary, 4-15 cm cross, slightly fragrant, arising from the rhizomes; berries spongy; seeds minute¹. Leaves rising above, submerged in or usually floating on the water, found in the over India in ponds and ditches. The hairy water lily is known as Shapla in Bengali, Kokaa in Hindi and Kumuda in Sanskrit. The leaves of this plant have fuzzy or hairy undersides and the stems are covered by the same hairs as well, hence the name "pubescens" or "hairy" of the species. This is not a characteristic that is apparent when looking at the plant from above the water though. This species of water lily has quite a few artificially raised varieties, in addition to many natural hybrids. Nymphaea pubescens is known under a number of different synonyms, the most common of which is Nymphaea rubra for the reddish variant known under the commercial name Red water lily, which often has also purplish leaves. The powdered roots are used for piles as a demulcent, also for dysentery, dyspepsia and cardiac tonic activity. Flowers cardiotonic used for the preparation of Ghillad and Gulkand and Seeds are used as a cooling medicine in cutaneous diseases². The roots were useful in the treatment of diabetes³_. Decoction of its roots is employed in dysuria, haemorrhoids⁴.

Leaves are applied topically in erysipelas, whereas the macerated leaves are used as lotion in eruptive fevers. The objective of the present work is to study the macro, micro morphological feature, phytochemical, fluorescence and powder character of the flower part of the plant.

MATERIAL AMD METHOD:

Collection and specimens:

The plant specimens for the proposed study were collected around Thiruvarur and Nagapattinum Districts of Tamilnadu in July first week. The flower was taken authenticated by G.V.S. Murthy. Joint Director, Botanical survey of India, Southern circle, Coimbatore (No.BSI/SC/5/23/07-08/Tech 360). The required samples of different organs were cut and removed from the plant and fixed in FAA (formalin 5ml+ Acetic acid 5 ml + 70% ethyl alcohol 90 ml). After 24 hours of fixing, the specimens were dehydrated with graded series of tertiary-butyl alcohol^5.Infiltration of the specimens was carried by gradual addition of paraffin was (melting point 58-60c) until tertiary-butyl alcohol solution attained super saturation. The specimens were cast into paraffin blocks.

Sectioning:

The paraffin embedded specimens were sectioned with the help of rotary Microtome. The thickness of the sections was 10-12 micro meter. The sections were stained with toluidine blue .The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet colour to the mucilage and blue to the protein bodies. Macro, micro morphological feature, and powder characters of the flower part of the plant were studied. Photographs of different magnification were taken with Nikon Labphot 2 microscopic unit.

Table-1: Preliminary phytochemical tests for flower parts of Nymphaeae pubescens

S. No.	Test	Powdered flower	Alcohol extract
1.	Alkaloids	+	+
2.	carbohydrate	+	+
3.	Glycosides	+	+
4.	Phytosterols	+	+
5.	Fixed oil and fat	-	-
6.	Saponins	-	-
7.	Tannin and phenolic compounds	+	+
8.	Protein and amino acid	-	-
9.	Mucilage	-	-
10.	Flavanoids	+	-
11.	Lignins	-	-

( +)- Present ( -)- Absent

Figure 1. Microscopic features of the Peduncle

Col-Colenchyma, Ep- Epidermis, Pa –Parenchyma, OVB- Original Variable Bundle, SC -Seed Coat

Figure 2. Microscopic features of the Anther filament

Mi -Middle portion, Ma-Marginal portion, Ae -Aerenchyma,

Figure 3. Microscopic features of the Anther

PC –Peri Carp, PC -Pollen Chamber, PG -Pollen Grains,

Figure 4. Microscopic features of the Fruit-Ovary

VB - Vascular Bundle, PC –Peri Carp,

Figure 5. Microscopic features of the Trichosclereids

TSc - Tricho Sclereid, Tr - Trichome,

Figure 6. Microscopic features of the Trichosclereids

BC -Basal Cell,

Figure 7. Microscopic features of the calcium oxalate crystals

BSc -Branchy Sclereid, SG -Starch Grains,

Table.3: Physico-chemical parameters of the flower part of Nymphaeae pubescens

S. No.	Parameter	Mean value (%w/w)(n=3) ±S.D
1.	Total ash%	5.92±0.60
2.	Water soluble ash%	6.35±0.31
3.	Acid insoluble ash%	0.63±0.002
4.	Sulphated ash%	8.32±0.07

Macroscopic and Microscopic Analysis:

The macroscopic and microscopy of the flowers were studied according to the method of Brain and Turner⁶. For the microscopical studies, cross sections were prepared and stained as per the procedure of Johansen⁷. Quantitative microscopy was done as per the procedure given by standard procedures ^8-10.

Preliminary phytochemical, fluorescence and physicochemical parameter analysis:

All parameters are applied on only flowers Physico – chemical analysis i.e. percentage of ash values and extractive values were performed according to the official methods prescribed (Indian pharmacopoeia)¹¹. Fluorescence analysis was carried out according to the method of Chase and Pratt^12.Preliminary phytochemical screening was carried out by using standard procedures^13,14 .

RESULT AND DISCUSSION:

Macroscopic features:

Flowers small, 7.5-10 cm across, sepals oblong-acute. Stamens 6 or indefinite; all free and inserted on fleshy torus surrounding or rarely enveloping the gynoeciums. Carpels 3 or more, free or united or sunk in the pits of the torus; stigmas of the distinct carpels terminal and peltate, of many carpelled ovaries as many as the carpels, adnate to the upper surface; ovules anatropous or orthotropus; few and pendent from the submit of the cell or many, covering the walls of the cells. Fruits of distinct indehiscent carpels or many- carpelled fleshy or spongy. Seeds arilled or not; albumen floury, fleshy or rarely 0; embryo enclosed in the enlarged persistent embryo-sac.

Table.4: Extractive values of the flower part of Nymphaeae pubescens

S. No.	Nature of the extract	Extractive value (in %)
1.	Alcohol soluble extractive	30.66 ±1.2
2.	Water soluble extractive	38.27±1.5

Figure 8. Microscopic features of the calcium oxalate crystals

BSc -Branchy Sclereid, Cr –Crystals.

Microscopic features of the flower:

Peduncle:

It is a single layered epidermis covered externally by a thin striated cuticle and has a large number of hydropotens. 3-5 layered collenchyma and the rest is parenchyma cells. Air canals are vertically distributed in the parenchymatous zone. Four at the centre around this eight smaller and other eight which is much smaller than the other two. Mucilage hairs, trichosclereids with calcium oxalate crystals as small prism impregnated in their walls. The vascular bundles are either circular or ovate in shape. They have a few paralled rows of circular, wide, thin walled xylem elements and dense layer of small phloem elements. The metaxylem elements are also present. (Figure 1)

Anther filament:

The filament of the stamen is flat, thin along the margin and fairly thick in the middle. The middle portion is 350 micrometer thick and the marginal part 100 micrometer thick. The epidermal layers are thick with vertically rectangular cells. The ground tissue is aerenchymatous comprising of small circular, four layer of air. Chambers separated by thin uniseriate partitions. Small vascular bundles are seen in horizontal median line. (Figure2).

Anther:

The anther is dithaecous, and four chambered. It is Plano convex with flat adaxial side. The anther dehisces longitudinally along the median part of the anther lobes. The anther is 2.5 mm is horizontal plane and 900 micrometer in vertical plane. The anther wall endothecium’s consists of a single layer of elongated cells with annular thickenings. The endothecium’s is 50 micrometer thick. The pollen grains are circular with smooth exine. The pollen is 30 micrometer. They are librated through the opening called stomium (Figure3).

Fruit-ovary:

The fruit is berry with thick fleshy pericarp. It is mutichambered with several thick partitions bearing several seeds on superficial placentation. The central portion has swollen placenta and circular axis. The seeds have smooth, thin testa and ovate embryo. (Figure 4).

Powder analysis of the flower part:

Powdered preparation of the flower shows the following components. Small fragments of lamina having epidermal trichomes and dense network of trichosclereids (Figure5,6).the separated sclereids shows varying shapes, long, slender cells, sparingly branched cells and highly lobed horned cells. The sclereid has echinate surface, thick lignified walls and narrow lumen. Wide lobed sclereids with wide lumen are seen in the ground tissue of the rhizome, these sclereid have minute granular calcium oxalate crystals of prismatic type on the surface (figure7, 8). The trichomes of nonglandular or covering type are seen in abundance in the powder. The trichome is three-celled, uniseriate and unbranched. The basal, two cells are short and rectangular the third cell is elongated into long, tapering pointed end. The walls are thick, smooth and lignified. The trichome is 850 micrometer long and 100 micrometer wide.

Preliminary photochemical, fluorescence, physicochemical parameters analysis:

Preliminary photochemical results showed the presence or absence of certain phytochemicals in the drug. Alcoholic extract showed positive result for the presence of alkaloids, carbohydrates, glycosides, phytosterols, tannins and phenolic compounds and negative result for fixed oil, saponins, protein and aminoacids,mucilage, flavanoids and lignins.whereas the powdered flower gave positive result for alkaloids, carbohydrates, glycosides, phytosterols, tannins phenolic compounds and flavanoids.(Table 1)

The results of fluorescence analysis have been presented in Table 2. Quantitative standards revealed that the total ash, water soluble ash content and sulphated ash content was 5.92%, 6.35% and 8.32% respectively. Negligible amount of acid insoluble ash matter (0.63%) was present in the flower. (Table3)The alcoholic extractive value and water soluble extractive value was 30.66% and 38.27 %.( Table5).

CONCLUSION:

In conclusion, the present study on pharmacognostical characters of Nymphaeae pubescens will be providing useful information with regard to its correct identity and help to differentiate from the closely related other species of Nymphaeae .The parameters observed and reported will be of use to the future workers in selecting the correct herbal specimen

REFERENCES:

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6. Brain KR and Turner TD. The Practical Evaluation of Phytopharmaceuticals, Wright – Scientechnica, Bristol. 1975: pp.4 -9.

7. Johansen DA. Plant Microtechnique, McGraw Hill, New York, 1940; pp.182.

8. Wallis TE. Text book of Pharmacognosy fifth edition, CBS publishers, New Delhi, India, 1985; pp.652.

9. Trease GE. A text book of Pharmacognosy, Bailliere, Tindall, London, 1961: pp. 34.

10. Evans WC. Trease and Evan’s Pharmacognosy, Fourteenth edition, WB Saunders Co., Ltd., London, 1996: pp. 1 -40.

11. Indian Pharmacopoeia, 4th edn., Vol. II, Government of India, Ministry of Health and Welfare, Controller of Publications, New Delhi, 1996: pp. A53-A54.

12. Chase CR and Pratt RJ. Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. J. Am. Pharmacol. Assoc. 1949: pp38, 32.

13. Kokate CK. Practical Pharmacognosy, 1st edn, Vallabh Prakashan, New Delhi, 1986: pp. 15-30.

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Received on 30.08.2009 Modified on 25.10.2009

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 210-213

Reagents	Powdered flower
Reagents	Day light	UV light
Powder	Pale purple colour	Greenish purple colour
Powder + 1N Hcl	Reddish pink colour	Dark green
Powder + 1N NaOH (aqueous)	Yellowish green	Dark green
Powder + 1N NaOH (alcoholic)	Yellowish blue	Dark green
Powder + 50% HNO₃	Reddish brown	Dark green
Powder + 50% H₂SO₄	Light green	Light green
Powder + Methanol	Pinkish blue	Dark blue
Powder + NH₄	Buff colour	Dark brown
Powder + Iodine	Pink yellow	Dark brown
Powder + Fecl₃	Dark green	Dark green