MACROSCOPICAL, MICROSCOPICAL AND PHYSICO-CHEMICAL STUDIES ON LEAVES OF DALBERGIA SISSOO LINN. (FABACEAE)

MACROSCOPICAL, MICROSCOPICAL AND PHYSICO-CHEMICAL STUDIES ON LEAVES OF DALBERGIA SISSOO LINN. (FABACEAE)

Rohit Kumar Bijauliya ^*1, S. K. Jain¹, Shashi Alok¹, V.K. Dixit¹ and Vijay Kumar Singh¹ and Man Singh²

Institute of Pharmacy ¹, Bundelkhand University, Jhansi - 284128, Uttar Pradesh, India.

Department of Pharmacy ², Motilal Nehru Medical College, Allahabad – 211002, Uttar Pradesh, India.

ABSTRACT: Objective: To rationalize the macroscopial, anatomical and physico-chemical studies on leaves of plant Dalbergia sissoo Linn. (Fabaceae). Methods: The crude ethanolic extract of leaves of Dalbergia sissoo Linn. (Fabaceae) was using physico-chemical parameters, fluorescence analysis, and preliminary phytochemical investigation (TLC, HPTLC, column chromatography). Results: An attempt has been made to highlight this folk herbal medicine through present study which will assist in the identification of fresh as well as dried crude samples of leaves anatomically and physiochemically. TLC fingerprint profiling and fluorescence analysis of powdered leaves were also carried out and the salient qualitative and quantitative parameters are reported. Conclusions: The present study will provide referential information for correct identification and help in checking adulteration in market samples used in the preparation of various herbal medicines. The present observation will also be helpful in macroscopical, microscopical and physiochemical studies on leaves of Dalbergia sissoo Linn.(Fabaceae).

Dalbergia sissoo Linn., Ethanolic extract, Leaves

INTRODUCTION: Dalbergia sissoo, commonly known as Indian Rosewood, is a deciduous tree, also known as sisu, sheesham, tahli and Tali. Dalbergia sissoo is the state tree of Punjab state (India) and the provincial tree of Punjab province (Pakistan). It is found growing along river banks below 900 metres (3,000 ft) elevation, but can range naturally up to 1,300 m (4,300 ft). It can withstand average annual rainfall up to 2,000 millimetres (79 in) and droughts of 3–4 months. It prefers soils from pure sand and gravel to rich alluvium of river banks.

Shisham can grow in slightly saline soils. Seedlings are intolerant of shade. ¹ Dalbergia sissoo is a medium to large tree of about 25 meters high with grey yellow trunk, 2-3 meters in diameter.

Leaves: are leathery, pinnately compound, alternate leaflets, petiolated leaf stalk, measures about 15 cm long, each leaflet widest at the base, to 6 cm long with a fine pointed tip.

Flowers: are whitish to pink, fragrant, nearly sessile, and in dense clusters.²

Pods: are oblong, flat, thin, strap-like 4–8 cm long, 1 cm wide and light brown. They contain 1–5 flat bean-shaped seeds 8–10 mm long. It has a long taproot and numerous surface roots which produce suckers. Young shoots are downy and drooping, stems have light brown to dark grey bark up to 2.5 cm (0.98 in) thick, shed in narrow strips, large upper branches support a spreading crown.³

Seeds: are 6-8 x 4-5 mm, kidney shaped, thin and flat, light brown. The fruit is dry and hard. The sapwood is white to pale brown in colour and the heartwood is golden to dark brown in colour. It develops a long taproot from an early age, and numerous lateral ramifying roots. ^4-5

Different parts such as roots, bark, wood, leaves and seeds are being used as remedy in many diseases including skin diseases, blood diseases, syphilis, stomach problems, dysentery, nausea, eye and nose disorders, aphrodisiac, expectorant. Leaf extract has been used to treat sore throats, heart problems, dysentery, syphilis, and gonorrhea. In India and Nepal rural people use Dalbergia sissoo leaves to treat animals suffering from non-specific diarrhoea. ⁶

FIG. 1: LEAVES OF DALBERGIA SISSO

Pharmacognostic studies and phytochemical action on leaves of Dalbergia sissoo L. have promoted us to undertake the present study. (Table 1)

TABLE 1: ETHNOMEDICAL INFORMATION OF DALBERGIA SISSOO LINN.

MATERIALS AND METHODS:

Plant Material: The plant material was collected from the local region of Bundelkhand Jhansi in the month of December 2015. The plant was identified by local people of that Bundelkhand region and authenticated by Dr. Gaurav Nigam (Asst. Professor) Department of Botany, Bundelkhand University, Jhansi (U.P.) India. A herbarium specimen of the plant (BU/Bot./Spe./Pha./01-2016/01) was preserved in the Department of Pharmacognosy of our Institute for further reference. The fresh leaves were used for the study of macroscopic and microscopical characters. Whereas collected plants leaves were separated and dried under shade, pulverized by mechanical grinder, passed through 40 mesh sieve and stored in a closed vessel for further use. This coarse powder was used for the determination of ash values, extractive values and preliminary phytochemical investigation as standard methods.

Chemicals: The various chemical are used such as ethanol, chloroform, benzene, n-hexane, ethyl acetate, toluene, formic acid, petroleum ether, sodium & pot. hydroxide, sulphuric acid, hydrochloric acid, nitric acid, glacial acetic acid, iodine silica gel 60-80 mesh and Silica gel 60 F254 precoated Aluminium plates 0.2 mm.

Macroscopy: The observed macroscopical characters of leaves of Dalbergia sissoo Linn.    were as follows:

• Leaf arrangement- Alternate
• Leaf type- Odd pinnately compound
• Leaf margin- Entire
• Leaflet venetion- Pinnate
• Leaflet shape- Elliptic (oval) orbiculate
• Leaf size-2.57-2.80cm length & 2.19-2.40 mm breadth
• Leaf colour- Green (dark)
• Odour- Characteristic
• Taste - Bitter followed by astringent

Microscopic:

T.S of leaf:

Midrib: Transverse section of midrib shows a flat surface on the adaxial side and convexity on the abaxial side (Fig. 2 A, B). The epidermis is made up of single layer of rectangular transversely elongated cells (Fig. 2 A, B). Abaxial epidermis is papillose and inner walls are gelatinized. The hypodermal region of adaxial and abaxial epidermis is composed of 2 to 4 rows of collenchymas cells.

A large arc shaped collateral vascular bundle is situated in the centre. Sclerenchyma fibres are present on the adaxial and abaxial side of the vascular bundles.

Leaf is dorsiventral in structure. Adaxial epidermal cells are larger than the abaxial epidermal cells. Hypodermis on the upper side is made up of large rectangular parenchyma cells (Fig. 2. C).The palisade tissue is made up of 2 rows of columnar closely packed cells. The spongy tissue is composed of 5 to 7 rows of loosely arranged round parenchyma cells. A small crystalline grains or prisms or rod shaped crystals are seen in the mesophyll tissue. The stomatal index for abaxial epidermis is 17 to 21 (Fig. 2.D); palisade ratio 3 to 4; vein islet number 18 to 22 (Fig. 2.E). The smaller veins of the leaf are vertically transcurrent.

Petiole: Transverse section of petiole is circular in outline (Fig 2.F). The outer most epidermis is made up of single layer of cells. Most of the cells elongate to form uniseriate trichome. Epidermal cells are papillose. The cortex is broad and composed of round, closely arranged parenchyma cells (Fig 2.G and Fig 2.H). In the centre ‘U’ shaped with strongly incurved ends and approximately circular, leaving a small gap on the adaxial side, large, collateral vascular bundle is seen. Vascular bundle is surrounded by sclerenchyma fibres(Fig 2.I).

Epidermis in surface view: The adaxial foliar epidermis is made up of polygonal parenchyma cells with straight wall and devoid of stomata. Uniseriate trichomes are noticed (Fig. 2.J).

Trichome: Trichomes are numerous, simple, uniseriate with a short basal cell accompanied by an elongated terminal cells with blunt tip (Fig. 2.J, K). The abaxial foliar epidermal cells are also polygonal in shape with straight walls but smaller in size. It is perforated by rubiaceous stomata or stomata surrounded by a rosette of cells (Fig. 2. L).

Fig. 2. A: T.S. of leaf

Fig. 2. B: T.S. lamina

Fig. 2. C: Adaxial foliar epidermis                                         

Fig. 2. D: Stomata

Fig. 2. E: Vein islets

Fig. 2. F: T.S. of petiole                           

Fig. 2. G: T.S. of petiole – A portion enlarged

Fig. 2. H: T.S. of petiole showing cortical cells

Fig. 2. I: T.S. of petiole Vascular

Fig. 2. J: Trichome – Enlarged                                                

Fig. 2. K: Trichome

Fig 2. L: Stomata enlarged

FIG. 2: MICROSCOPIC OF DALBERGIA SISSOO LINN. LEAVES

Extraction of plant materials: A total of 250 g coarse powder of air dried leaves of Dalbergia sissoo were packed in muslin cloth and subjected to soxhlet extractor for continuous hot extraction with petroleum ether and ethanol for 8 h separately. Then the each extracts were filtered and filtrate was evaporated to dryness. The percentage yield of the petroleum ether and ethanol extracts is given below in Table 2 respectively.

TABLE 2: EXTRACTION OF PLANT MATERIALS

Physico-chemical parameters: Leaves of Dalbergia sissoo such as total alcohol soluble extractive, water soluble extractive, ash value, acid insoluble ash, water-soluble ash, loss on drying, are presented in Table 3.

TABLE 3: PHYSICAL PARAMETER OF LEAVES OF DALBERGIA SISSOO

Fluorescence analysis: The fluorescence analysis of the powdered drug of Dalbergia sissoo in various solvents and chemical reagents was performed under normal and 254 nm and 366 nm UV light. The fluorescence analysis of the powdered drug of Dalbergia sissoo Observed the different colour under UV light is given in Table 4.

TABLE 4: FLUORESCENCE ANALYSIS OF POWDERED LEAVES OF DALBERGIA SISSOO

Qualitative and Quantittive evaluation parameters: The calculated Quantitative values like stomatal number, stomatal index, vein-islet number, vein termination numer and Palishade ratio, of the leaves of Dalbergia sissoo are showed in Table 5.

TABLE 5: RESULT OF QUANTITATIVE MICROSCOPY OF DALBERGIA SISSOO LEAVES

Preliminary photochemical investigation: Photochemical tests were done in plant extracts for the detection of presence of different chemical constituents such as; alkaloids, glycosides, flavonoids, essential oils, carbohydrates, proteins, tannins and other substances which are responsible for the biological activity. So the chemical tests are performed in the ethanolic extract of Dalbergia sissoo. For the detection of different chemical constituents are observed in the Table 6 given below respectively.

TABLE 6: DATA FOR THE PHYTOCHEMICALS SCREENING OF POWDERED LEAVES OF PET. ETHER AND ETHANOLIC EXTRACT OF DALBERGIA SISSOO LINN.

Thin layer chromatography: “Their relative polarities which related to the type and number of functional    groups present on a molecule capable of hydrogen bonding”

Where R_f =Retention factor

The ethanolic extract of leaves of Dalbergia sissoo Linn was subjected to thin layer chromatography studies, to find the presence of number of compounds which support by the chemical test. R_f value and colour of TLC spots, in solvent system of Toluene: Ethyl acetate: Formic Acid (7:3: few drops). These TLC spots with R_f value and colour are in Table 7, and TLC plate in Fig. 3 is given below.

FIG. 3: TLC FINGER PRINTING OF ETHANOLIC EXTRACT ON LEAVES OF DALBERGIA SISSOO

TABLE 7:TLC FINGER PRINTING OF ETHANOLIC EXTRACT OF LEAF OF DALBERGIA SISSOO SPOTS

HPTLC finger printing: Ethanolic extract was developed on chromatographic plates with many ratios of different solvents and the best eluent mixture was used further for HPTLC profile to minimize errors in TLC pattern. The preliminary HPTLC studies revealed that the solvent system toluene: ethyl acetate and few drops of formic acid (7:3:few drops) was ideal and gave well resolved sample peaks. Fig. 4, 5, 6 and 7 HPTLC finger printing of ethanolic extract on leaves of Dalbergia sissoo given the spots of the chromatogram were visualized at 254 nm and 366 nm.

Preparation of sample: Dissolved liquid extract in 10 ml ethanol. Filtered using (0.45μm) (milipore) filter paper and applied 8μl of the solution for Chromatography.

Procedure: High Performance Thin Layer Chromatography (HPTLC) was carried out on Silica gel 60 F254 precoated Aluminium plates 0.2 mm thickness, Merck India Limited Mumbai. An Applicator from Camag Linomat-5 (Camag Switzerland: 140443) was used for band application and photo documentation unit (Camag Reprostar-3: 140604) was used for documentation of chromatographic fingerprints. The mobile phase used Toluene: Ethyl acetate (7:3) solution. The plate ware developed over a distance of 9 cm in a saturated development chamber (Twin through chamber 10X10 cm with SS lid and visualized under as given in each separate sample report. After spraying with 5% methanolic sulphuric acid reagent followed by heating at 105 °C for 5-10 minutes.

TABLE 8: SHOWING R_f VALUES OF HPTLC FINGERPRINTS PROFILE IN TEST SOLUTION OF DALBERGIA SISSOO L. LEAVES

TABLE 9: SHOWING R_f VALUES OF HPTLC FINGERPRINTS PROFILE IN TEST SOLUTION OF DALBERGIA SISSOO L. LEAVES

HPTLC Finger prints of test solution of Dalbergia sissoo L. Leave

T1                 T2

FIG. 4: AT 254 nm (BEFORE DERIVATIZATION 

T1                  T2

FIG. 5: AT 366 nm (BEFORE DERIVATIZATION)

T1               T2 

FIG. 6: UNDER UV LIGHT (AFTER DERIVATIZATION)

T1               T2

FIG. 7: AT 366nm (AFTER DERIVATIZATION)

Where Track T1 and Track T2 (in duplicate)

Column chromatography: The basic principle lying in the column chromatography is adsorption of component at solid-liquid interface. For good separation, the component of mixture should have different degree of affinity for the solid support. The component having strong adsorption for column material is held up while that component having less affinity moves down the column at faster rate as the elute passes through the column.

FIG. 8: COLUMN OF THE ETHANOLIC EXTRACT OF DALBERGIA SISSOO LEAVES

Column chromatography is separated into two categories depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity or percolation, it is called gravity column chromatography. If the solvent id forced down the column by the air pressure, it is called flash chromatography. Data of column chromatography ethanolic extract of Dalbergia sissoo Linn. leaves is given below respectivly in Table 10 and colum chromatography in Fig. 8 is given below.

TABLE 10: DATA OF COLUMN CHROMATOGRAPHY ETHANOLIC EXTRACT OF  DALBERGIA SISSOO LEAVES

RESULTS: As a part of standardization study, the macroscopically examination of drug was studied. The results showed greater extractive values in hot extraction, indicating the effect of elevated temperature on extraction. Percentages of the extractive values were calculated with reference to air-dried drug. The percent extractives in different solvents indicated the quantity and nature of constituents in the extracts. The extractive values are also helpful in estimation of specific constituents soluble in particular solvent.

ACKNOWLEDGEMENTS: We are grateful to Dr. S.K. Jain (Associate professor) and Dr. Shashi Alok (Assistant Professor), Faculty of Pharmacy, Bundelkhand University, Jhansi for his assistance and encouragement. We extend our sincere thanks to Mr. Vighnesh Dixit (Assistant Professor) and Mr. Vijay Kumar Singh (Assistant Professor) Faculty of Pharmacy, Bundelkhand University, Jhansi, for critically reading the Manuscript and providing the valuable suggestions.

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    How to cite this article:

    Bijauliya RK, Jain SK, Alok S, Dixit VK, Singh VK and Singh M: Macroscopical, microscopical and physico-chemical studies on leaves of Dalbergia sissoo Linn. (Fabaceae). Int J Pharm Sci Res 2017; 8(4): 1865-73.doi: 10.13040/IJPSR.0975-8232.8(4).1865-73.

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