1. INTRODUCTION:
Pharmacognosy is the study of the structural, physical, chemical and sensory characters of crude drugs of plants, animals and mineral origin. The search for biologically active compounds from natural source has always been of great interest to researchers looking for new source of drugs useful in infectious diseases. Plants have played a vital role as the source of important therapeutic agents.
Aponogeton natans (Linn.) Engl. & Krause. belongs to family Aponogetonaceae family. The plant occurs in plains, in the ponds and marshy places in Asia, Australia, India and Srilanka. Leaf pastes are consumed with hot water to treat Cuts &Wounds [1].
Fresh tuber are ground into a paste and boiled with 200 ml of coconut oil and applied on hair before bath for three days to get rid of fungal infection [2].A perusal of existing reports reveals that the no detailed anatomical study had been done earlier. Hence, it is felt desirable to pursue a study on morphological and anatomical of Aponogeton natans (Linn.) Engl. & Krause. to supplement useful data in regard to its correct identity of this plant .
2. MATERIAL AND METHODS:
2.1. Collection and authentication of plant material
Fresh parts of Aponogeton natans (Linn.) Engl. & Krause.were collected from Salipur, Cuttack, Odisha, India which was identified and authenticated by Prof. P. Jayaraman, PARC Chennai. The voucher specimen was given the No. PARC/2008/97.
2.2. Preparation of the specimen
The plant specimens for the proposed study were collected from Salipur, Cuttack, Odisha. Care was taken to select the healthy plant and organs. The required samples of different organs were cut and removed from the plant and fixed in FAA (Formalin-5ml+ Acetic acid-5ml+ 70% Ethyl alcohol-90 ml). After 24 hour of fixing, the specimens were dehydrated with graded series of tertiary – butyl alcohol [3]. Infiltrations of the specimen were carried by gradual addition of paraffin wax (melting point 58-60˚C) until TBA (tertiary – butyl alcohol) solution attained super saturation. The specimen was cast into paraffin blocks.
2.3. Sectioning
The paraffin embedded specimens were sectioned with the help of Rotary microtome. The thickness of the section was 10-12 µm. dewaxing of the sections was done by customary procedure [4]. The section was stained with toludine blue. Since toludine blue is a polychromatic stain, the staining results were remarkably good and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose wall, blue to the lignified cell , dark colur to the suberin, violet to the mucilage, blue to the protein bodies etc. wherever necessary sections were also stained with safranin , fast green and Iodine potassium iodide (for starch).
For studying the stomatal morphology, venetion pattern and trichome distribution, paradermal sections (section taken parallel to the surface of leaf) as well as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey’s maceration fluid [3] were prepared. Glycerine mounted temporary preparations were made for macerated/ cleared materials. Powdered material of different parts were cleared with sodium hydroxide and mounted in glycerine medium after staining. Different cell component were studied and measured.
2.4. Photomicrographs
Microscopic descriptions of tissues were supplemented with the micrographs wherever necessary. Photographs of different magnification were taken with Nikon labphoto 2 microscopic unit. For the normal observation bright field was used. For the study of crystals, starch grains and lignified cells , polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifications of figures were indicated by the scale-bars. Descriptive terms of the anatomical features are as given in the standard anatomical books and research papers [5, 6].
3. RESULT AND DISCUSSION:
3.1. Macroscopical characters of the plant
The plant has globose tuber which is smooth brown. The leaves arise from upper part of the tuber and fibrous dense roots arise from the basal part. The leaves are oblong, chartaceous, glabrous, base subcordate margins entire. Petiole 6-40cm long. Spike cylindrical, dense, 2-7cm long; tepals: violet, two and clawed. Stamens: 3+3, free ; ovary: 3 carpels, free, ovules 8, style- terminal; Fruit globose, smooth, sharply beaked; seeds- terete, eight, outer seed coat winged.
Figure A (1, 2, 3): T.S of leaf through midrib with lamina
Figure B (1, 2): T.S of lamina
3.2. Anatomical characters of different parts of the plant
3.2.1. Anatomy of midrib
The midrib is flat on the adaxial side and broadly “V” – shaped on the abaxial side. The midrib is 1mm in vertical plane and 1.1mm in horizontal plane [Fig: A (1)] the palisade tissue is horizontally transcurrent on the adaxial part of the midrib. The palisade cells are cylindrical and loosely arranged [Fig: A (2, 3)].
Below the palisade region, the abaxial part consists of wide reticulation of circular air chambers. The air chambers are divided by thin filamentous partitions of one cell thicks. Smaller vascular bundles are located in the junctions of the filaments at the lower region of the abaxial part.
The vascular system of the midrib consists of three vascular bundles. The median bundle is larger and occupies center of the midrib; and the other two vascular bundle are lateral above the adaxial palisade cell; the median bundle is prominent and includes few xylem elements and a group of phloem elements [Fig:A (2,3)]. The vascular strand is surrounded by inner circle of smaller and outer circle of wider, dilated hyaline bundle sheath cell. The adaxial bundles are also similar to the median bundle. The abaxial epidermis is comparatively thin with narrowly oblong cells. The vascular bundle is of monocot type.
3.2.2. Anatomy of Lamina
The lamina is smooth and even in cross sectional view. It is dorsiventral, hypostomatic and hydromorphic. The lamina is 250µm thick. The adaxial epidermis is thin walled and the cells are squarish or rectangular. The mesophyll is differentiated into an adaxial band of palisade cells which are nearly 100 µm in height and occupy two third thickness of the lamina [Fig: B (1)]. The palisade region is followed by spongy mesophyll cell, which are circular to polygonal and less compact.
The vascular system of the lateral veins consists of smaller bundles situated in the spongy mesophyll region abutting the palisade cells. The vascular strand consists of a small group of xylem and phloem elements. The abaxial epidermis is comparatively thick with narrowly oblong cells [Fig: B (1)].
The leaf margin is semicircular in sectional view and it is as thick as lamina. The marginal part as distinct epidermal layer of small, squarish thick walled cell. There is sub epidermal layer of circular, thin walled parenchyma cells. The mesophyll tissues of the margin consist of two layered palisade zone and three or four layered spongy parenchyma cell [Fig: B (2)].
Figure C (1, 2): Anatomy of petiole
Figure D: Anatomy of petiole (wing with median portion)
3.2.3. Anatomy of the petiole
Basal part of the petiole is triangular in cross sectional view [Fig: C (1)]. The petiole has a flat adaxial side and a broad “V” shaped abaxial part. The two arms of the “V” extend upto the adaxial side and form two lateral wings. An epidermal layer of the petiole is thin; cells are small and squarish. Inner to the epidermal layer are two or three layers of compact, thin walled parenchyma cells. Inner to the epidermal region are wide reticulation of air chambers which are circular to polygonal and are divided by thin filamentous partitions of one cell thick and thin walled [Fig: C (2), D].
The vascular strand occurs along the periphery, at the ridges as well as in the interlinks of the partition filaments [Fig: C (2), D]. The vascular strands of the ridges are small, consisting of few xylem and phloem elements, encircled by thin walled, smaller bundle sheath cells [Fig:C (2)].
The vascular strands in the middle region are the layers consisting of xylem and phloem elements, ensheathed by thin walled bundle of sheath cells. Both the peripheral ridge bundle and the central bundles are similar [Fig: D].
Vascular bundle of the petiole has monocot type structure. It has one wide metaxylem element and two or three protoxylem elements; the xylem elements occur in “Y” shaped pattern. Phloem is located in between the two arms of “Y”. The vascular bundles have distinct sheath [Fig: C (2), D].
Fig E: Anatomy of rhizome- A sector enlarged
Fig F: Structure of the rhizome
3.2.4. Anatomy of the corm or bulb
The corm is short and thick and bears numerous lateral roots. In the cross section of the rhizome is circular in outline and measures 5mm in diameter.
The epidermis is replaced by four to five layers of tangentially oblong periderm tissue [Fig: 5; 6(1)]. Inner to this region is the ground tissue, which is circular to polygonal compact with abundant starch grains [Fig: E; F (2)]. The vascular bundles of the rhizome is scattered in the ground tissue. Vascular strand consist of one or two xylem elements and few phloem elements [Fig: F (2)].
Fig G: Powder microscopy of the leaf
Fig H: Powder microscopy of the rhizome
3.2.5. Powder microscopy of the leaf
In the powder of the leaf several characters such as stomatal type and venetion pattern were observed. Fragments of abaxial epidermis with stomata: The stomata are in parallel rows. The stomata are paracytic and the guard cells are narrowly elliptical with two lateral subsidiary cells. The epidermal cells are rectangular and have straight, thick cell walls [Fig: G (1)].
Venetion pattern: cleared fragment lamina showing a thick central midrib with two slightly thin parallel additional midribs close to the central one. There are two lateral veins one running along with middle part and the other along the submarginal part of the lamina. All these veins run parallel to each other. The parallel major veins are interlinked by thin horizontal veins forming a ladder like pattern. Less prominent veinlets are seen forming reticulate pattern in the area between the horizontal veins. The vein islets form wide rectangular vein islets; no vein terminations are evident [Fig: G (2, 3)].
3.2.6. Powder microscopy of the rhizome or corm
Rhizome powder viewed under polarized light microscope. Starch grains are seen in abundance in the ground tissue. Starch grains appear bright against dark background under polarized light [Fig: 8(1)]. The starch grains are circular and concentric. The hilum is in the centre. The starch grains vary in size. The larger starch grains are up to 20µm in diameter [Fig: H (2)].
4. DISCUSSION:
The present study on anatomical characters of Aponogeton natans (Linn.) Engl. & Krause.will be providing useful information in regard to its correct identity and help to differentiate from the closely related other species of Aponogeton. The characters like flat midrib on the adaxial side with broadly “V” – shaped on the abaxial side and abaxial epidermis is comparatively thick with narrowly oblong cells are the characteristic features of Aponogeton natans(Linn.) Engl. & Krause.. Presence of starch grains which are circular and concentric having hilum in the centre is important observation in powder form of rhizome or corm. The other parameters observed may be useful for the future identification of the plant.
5. ACKNOWLEDGEMENT:
The authors sincerely thank Prof. P. Jayaraman (PARC) Chennai, for identifying and providing the information about plant.
Legends for the figures:
Abs-Abaxial side; AC-air chamber; Ads-Adaxial side; AB-Abaxial bundle; Ep-epidermis; Bs-Bundle sheath; La-Lamina; LB-Lateral bundle; MR-midrib; MB-Median bundle; Ph-Phloem; PM-Palisade mesophyll; VB- Vascular bundle; Abe-Abaxial epidermis; Ade-Adaxial epidermis; LM-Leaf margin; Lv-Lateral vein; SM-Spongy mesophyll; TC-Tanniniferous cell; St-Stomata; PVB-Peripheral vascular bundle; Px-Protoxylem; W-wing; X-Xylem; AbB-Abaxial bundle; SMB-Sub marginal bundle; W-wing; WB-Wing bundle; X-Xylem; GT-Ground tissue; Pe-Periderm; Hv- Horizontal vein; LMR-Lateral midrib; MLV-Median lateral vein; MR-Midrib; SC-Subsidiary cell; SLV-Secondary lateral vein; St-Stoma; Vt-Veinlet; SG-Starch grains
REFERENCES:
1. Britto JD and Mahesh R. Exploration of Kani Tribal Botanical Knowledge in Agasthiayamalai Biosphere Reserve - South India. Ethnobotanical leaflets. 1(1); 2007: 1-10.
2. Jeyaprakash K, Ayyanar M, Geetha KN and Sekar T. Traditional uses of medicinal plants among the tribal people in Theni District (Western Ghats), Southern India. Asian Pacific Journal of Tropical Biomedicine. 1(1); 2011: S20-S25.
3. Sass JE. Elements of botanical microtechnique. MC Graw Hill, New York. 1940.
4. Johansen DA. Plant Micro Technique. MC Graw Hill, New York. 1940.
5. Easu K. Plant Anatomy. John Wiley and sons, New York. 1964.
6. Sundeep Kumar HK etal. Pharmacognostical and physico-chemical studies on the root of Bambusa arundinacea. Der Pharmacia Lettre. 4 (5); 2012: 1367-1375.
Received on 16.02.2013 Modified on 01.03.2013
Accepted on 10.03.2013 © RJPT All right reserved
Research J. Pharm. and Tech. 6(4): April 2013; Page 398-402