Microscopic Evaluation of Leaves of Memecylon umbellatum Burm

Objective. Aim of present work is to perform the microscopic evaluation and physicochemical analysis and to explore the morphology parameters ofMemecylon umbellatum Burm leaves.Methods. Fresh, dried and desiccated powdered leaf samples were studied for their morphology, microscopy, organoleptic characters, and an assortment of other WHO recommended methods for standardisation. Results. The microscopy revealed the dorsiventral nature of the leaf. Midrib showed presence of nonlignified phloem, lignified xylem with well-defined xylem fibers, vessels, and parenchyma. Presence of Phloecentric vascular bundles surrounded by endodermis and crystal sheath. Well-defined patches of collenchyma were observed above and below the vascular bundles in the midrib area. Trichomes are mostly absent and stomata (anomocytic) were observed on both epidermal surfaces. Conclusions. It can be concluded that the microscopic analysis and pharmacognostic parameters can serve as tool for developing standards for proper authentication, quality, and purity ofMemecylon umbellatum Burm leaves.


Introduction
Memecylon umbellatum Burm (family: Melastomataceae) is a small evergreen shrub or tree which grows up to 8-14 m tall having young tree branches and bears numerous umbellate cymes. The plant is known as "Anjani" in Sanskrit, Anakkayavu in Malayalam, and "Ironwood tree" in English. It is distributed mostly in coastal regions of the Deccan peninsula, the eastern and southern part of India all along the Western Ghats and in the Andaman islands [1,2]. It is also found distributed in Orissa, Assam, Sylhet, Tenasserim, Ceylon, Malay, Peninsula-Malay, and Archipelago [3]. Different extracts of Memecylon umbellatum Burm Inflorescences [4] and bark [5] have been evaluated for its antimicrobial potential. Estimation of total content of tannin [6] and seasonal variation of tannin content in different parts has also been carried out [7]. Estimation of sugars and minerals in healthy and infected parts has also been carried out [8]. Different root extract have been reported to possess antioxidant activity [9], while leaves of Memecylon umbellatum have also been evaluated for their antimicrobial activity [10]. Use of leaves was also reported in snakebite [11]. The seeds are used to cure cough and sedative [12]. The leaf powder has antidiabetic potential [13]. Leaves are used to treat eye troubles, gonorrhea, leucorrhea, wounds [14], and skin diseases [15]. It also has an antioxidant property [16]. The leaves are reported to possess antiviral activity [17][18][19]. Wound healing activity of ethanolic extract of the leaves has also been reported [20]. Plant contains a wide variety of phytoconstituents such as umbellactone, -amyrin, Oleanolic acid, ursolic acid, sitosterol and organic acids [21][22][23].
As aforesaid, M. umbellatum has ample pharmacological activities and has the potential to cure various diseases. As this plant has special importance to humankind in their daily routine life it can be a good source of revenue. So to earn more money adulteration of this plant with various species of genus Memecylon like M. dasyanthum, M. flavescens, and M. kunstleri may take place. So it has become necessary to set the standard parameters for proper authentication of this plant or its part. So the aim of present study is to explore the morphological and microscopical parameters of Memecylon umbellatum Burm leaves for its proper authentication so that it cannot be easily adulterated. Microscopy (T.S) and powder characters of leaf drug were done using compound microscope (Inco-Ambala), inbuilt light microscope (Metzer-Metzer Optical Instrument, Mathura), and photographs were taken using photographic microscope (Motic-Image-2003). Quantitative microscopical measurements were made using eye piece, stage micrometer (Erma-Japan), and camera lucida (Prism type-Swift-Ivis). All the reagents, solvents, and chemicals used are of A.R. grade (Merck, Loba, Qualigens) purchased from local supplier.

Anatomy of Leaf.
The leaf sample was studied microscopically by taking transverse section (T.S.) through midrib with small portion of lamina and thin section was double stained with hematoxylin and saffranin and observed under compound microscope and photos were taken by using photographic microscope. The powder sample was also mounted in different reagent and cellular diagnostic and diagnostic cell inclusions were observed. Quantitatively, the diameter of starch grains, length of fibers, and diameter of vessels were determined using stage and ocular micrometers while leaf constants were determined using Camera Lucida.

Diameter of Starch Grains.
Eyepiece micrometer was calibrated using stage micrometer to determine the value for one division of eyepiece micrometer (3.152 at high power and 14.87 at low power). Powder sample was gently spread over clean glass slide with the help of muslin cloth, warmed near the flame with 2-4 drops of clearing solution (chloral hydrate) for 2-3 times. Slide was stained with few drops of N/50 Iodine solution, excess stain was removed and cover slip was placed with 1-2 drops of glycerin water solution. Faint blue colored starch grains were observed and none of divisions of eyepiece micrometer occupied by individual starch grain were noted. In all group of 25 starch grains were counted and such four groups were analyzed from different slides. From the data obtained, average diameter of starch grains was determined using formula where = number of grains and = number of divisions.

Length and Diameter of Fibers and Vessels.
The slide was prepared with fine powder sample of leaf, cleared with clearing solution, and stained with phloroglucinol and conc. hydrochloric acid (1 : 1). Pink colored fibers and dark rosy pink colored vessels were selected and their length and diameter were determined in similar fashion as that of diameter of starch grains using calibrated eyepiece micrometer.

Determination of Leaf
Average values were determined and results were expressed per sq.mm.
(ii) Vein-Islet and Vein Termination Numbers. The leaf portion between midrib and margin was macerated in concentrated chloral hydrate solution for 24 h and decolorized with bleaching solution (5% calcium chloro-hypochlorite). The cleared lamina portion was mounted on glass slide and vein islet and vein terminations were traced on black sheet between 0.5 mm square using low power (5x). The process was repeated and values were determined per sq.mm of leaf area between midrib and margin.
(iii) Palisade Ratio. The leaf sample was disintegrated with caustic potash and conc. HCl by boiling on water bath, further bleached with bleaching solution, and then clarified with chloral hydrate solution. Cleared leaf surface was then mounted on glass slide. Four sets of contagious epidermal cells were traced using Camera Lucida and by lowering the objective spherical palisade cells were traced. The average numbers of palisade cells were calculated and palisade ratio was determined.

Powder Analysis.
Fine sample powder was mounted on clean glass slide and clarified with clearing solution. The powder sample was then treated with different chemical  Fats and oils Sudan red III and IV 7 Mucilage Ruthenium red reagents. Stained samples were then mounted in glycerin water fluid and observed for identification of diagnostic characters. Different chemical reagents used for identification of diagnostic cellular characters and cell inclusions are as given in Table 1.

Powder Characters.
Powder sample of leaf showed presence of anomocytic stomata, prism and rosette type calcium oxalate crystals, aleurone and starch grains, brownish matter, upper and lower epidermis, xylem fibers, xylem vessels and palisade tissue as shown in Figures 9 and 10.  Vein-termination number 7-9 7 P a l i s a d er a t i o 8 -1 1

Conclusion
The detailed study of Pharmacognostic parameters of Memecylon umbellatum Burm leaves setup the standards which could be beneficial and serves as diagnostic tool for proper authentication of this medicinally important plant.