Hence the present investigation includes morphological and microscopy of leaf, determination of physico-chemical constants and preliminary phytochemical screening of the different extracts of Citrus paradisi var. foster.

MATERIAL AND METHODS:

Plant material:

The leaves of Citrus paradisi var. foster were procured and identified from a cultivated source: Punjab Agricultural University Regional Centre at Abohar (Punjab, India) in the month of March 2007.

Chemicals and instruments:

α- naphthol, chloral hydrate, ferric chloride, gelatin, iodine, nitric acid, picric acid, potassium iodide, sodium hydroxide, sulphuric acid, vanillin, toluidine blue, tertiary butyl alcohol, ethyl alcohol, acetic acid, formalin, chloral hydrate, ethanol, hexane, petroleum ether, glycerin, Camera Lucida, drawing sheet, glass slides, cover slips, watch glass. Photographic of different magnification were taken with Nikon Labhot 2 Microscopic unit.

Macroscopic and microscopic analysis:

The macroscopic and microscopy of the plant studied were according to the method of Brain and Turner¹³. The cross sections were prepared and stained as per the procedure of Evans¹⁴.

Physico-chemical analysis:

Physico-chemical values such as the percentage of ash values and extractive values were performed according to official methods prescribed Indian Pharmacopoeia, 1996 and the Who guidelines on quality control methods for medicinal plant materials¹⁵.

Preliminary phytochemical screening:

Preliminary phytochemical screening was carried out by using standard procedure described by Kokate and Harborne^16-17.

RESULTS AND DISCUSSION:

Macroscopic characters:

Organoleptic features of the leaves of Citrus paradisi var. foster were observed (Table 1)

Table 1 Organoleptic character of leaves

Organoleptic Character	Citrus paradisi var. foster
Type	Simple Leaf
Colour	Upper surface: green Lower surface: Light green
Odour	Characteristic
Taste	Astringent, Bitter
Size	9 -11cm in length, 4-6 cm in width
Shape	Ovate
Petiole	Narrow Winged
Margin	Serrate
Apex	Obtuse
Surface	Glabrous

Microscopy:

This leaf is bifacial with prominent midrib and distinct palisade spongy mesophyll differentiation.

Midrib: is very thick and biconvex in sectional view. It is 1.25 mm thick vertically & 800 micro meter horizontally. The epidermal layer of midrib is thin with small papillae cells. Cuticle is less prominent. The ground tissue around the vascular cylinder is 100 micro meters wide with 10-12 layers of compact thin walled parenchyma cells. Wide secretary cavities are seen in the upper and lower portions of the ground tissue. The cavities are 100-120 micro meter wide. The cells in the periphery of midrib are smaller and become wider towards the interior. The vascular cylinder is wide and prominent. It has elliptical xylem cylinder enclosing a narrow core of parenchyma cells. Xylem consists of long radial rows of vessels and fibres. The vessels are narrow and thick walled and are up to 20 micro meter wide. Xylem is surrounded by a wide band of phloem which is unsheathed by thick, discontinuous cylinder of perivascular fibres (Figure 1)

Figure 1 T.S of mid rib of citrus paradisi var. foster

Ep – Epidermis, GT – Ground tissue, Ph- Phloem, X- Xylem.

Lamina: The lamina is dorsiventral with smooth surfaces. It is 350 micro meter thick. The adaxial epidermis is 15 micro meters thick; the cells are squarish and thick walled. The abaxial epidermis is comparatively thin and darker with less prominent cuticle. It consists of three layers of palisade cells which are vertically long and compact. The palisade zone is 80 micro meters in height. The fourth layer beneath the palisade zone is wider and rectangular resembling the palisade cells. The spongy mesophyll tissue is 12-14 layered. The cells are large, lobed and interconnected forming aerenchymatous tissue. Small lateral bundles of the veins are situated in the upper spongy parenchyma tissue; they are up to 70 micro meters wide. They are collateral with small cluster of xylem and a nest of phloem. A single layer of parenchymatous bundle sheath is seen around the vascular bundle (Figure 2). Crystalliferous idioblasts are also seen beneath the adaxial epidermis. These cells have dense mucilage with druses type of cells.

Figure 2: T.S. of Lamina citrus paradisi var. foster

AbE – Abaxial epidermis, AdE – Adaxial epidermis, LV- Lateral vein, PM – Palisade mesophyll, SM- Spongy mesophyll.

Powder microscopy:

Stomata: The stomata are cyclocytic type; each stomata is surrounded by 6-8 rectangular cells. These may be one or two circles of cells. The guard cells are 20-25 micro meter long and 15 micro meters thick. The epidermal cells are polyhedral with thin straight walls. (Figure 3)

Figure 3: Abaxial epidermis with stomata of citrus paradisi var. foster

Ep- Epidermal cells, St- Stomata

Secretory Cavity: The secretory cavity is schizogenous type; it is wide and circular, measuring 180 micro meter in diameter. It is surrounded by 10-15 narrow spindal shaped epithelial cells. (Figure 4)

Figure 4 – Secretory Canal of the mesophyll tissue of citrus paradisi var. foster

MT- Mesophyll tissue, EC- Epithelial cells, SC- Secretory Cavity

Crystals: Calcium oxalate crystals are abundant in the leaf and midrib. The crystals are mostly prismatic type. In the lamina the crystals occur in the palisade as well as spongy parenchyma. In the midrib, this occurs adjoining the perivascular cylinder and in the phloem cylinder .The crystals in the phloem are smaller than those outside fiber cylinder. The crystals are up to 40 micro meters thick. (Figure 5)

Figure 5 – Prismatic Crystals in the midrib of citrus paradisi var. foster

Cr- Crystals, PVF- Perivascular fibres, X – xylem, Ph – Phloem, GT- Ground tissue

Physico-chemical constants:

The Physico-chemical constants of powdered leaves of Citrus paradisi var. foster were evaluated. (Table 2)

Table 2 Physical parameters of powdered leaves of Citrus paradisi var. foster

Physical parameter	*Citrus paradisi* **var. foster % (air dried drug)**
Volatile oil content	0.8
Ethanol-soluble extractive	7.6
Water soluble extractive	9.8
Total ash	6.5
Acid insoluble ash	0.7
Loss on drying	7.3

Preparation of leaf extract:

Various extracts of the powdered leaves (100 gm) of Citrus paradisi var. foster were prepared. Yields of extracts have been obtained.

Phytochemical Screening:

Phytochemical screening showed the presence of proteins, amino acids and flavonoids in the methanol soluble extract of Citrus paradisi var. foster (Table 3)

Table 3 Phytochemical screening of leaf extracts of Citrus paradisi var. foster

Extracts (with % yield w/w)

Plant Constituent

Petroleum Ether Extract (0.879)

Chloroform Extract (0.870)

Methanol Extract

(0.796)

Aqueous Extract

(0.976)

Test/Reagent Used

1)Alkaloids

Hager’s reagent

Wagner’s reagent

Mayer’s reagent

Dragendorff’s reagent

─

2) Carbohydrate

Molisch’s reagent

Fehling’s solution

Benedict’s reagent

─

3)Protiens and aminoacids

Ninhydrin reagent

Biuret test

Millon’s test

─

4) Phytosterols

Liebermann-Burchard’s test

─

5)Phenolic compounds and tannins

Ferric Chloride solution

Lead acetate test

─

6) Saponins

Foam test

─

7) Triterpenoids

Liebermann-Burchard’s test

─

8) Flavonoids

Lead acetate test

Sodium hydroxide test

Magnesium ribbon test

Ammonia test

─

CONCLUSION:

As there is no pharmacognostical work on record of this traditionally much valued drug, the present work was taken up with a view to lay down standards, which could be useful to detect the authenticity of this medicinally useful plant. Macro and micro morphological standards discussed here can be considered as identifying parameters to authenticate the drug.

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Received on 19.12.2009

Accepted on 22.02.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 140-143