International Journal of Pharma and Bio Sciences V1(2)2010. PHARMACOGNOSTIC AND PHYTOCHEMICAL INVESTIGATION OF STEM BARK OF Tectona grandis Linn.

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1 D.V. GOSWAMI* 1, M.J. PATIL 2, ANUJ MODI 3 AND R. TIWARI 4 1 Department of Pharmacognosy, Vidyawati College of Pharmacy, Jhansi, U.P. India 2 Department of Pharmacognosy, M.M College of Pharmacy, Pune, M.S. India 3 Department of Pharmacognosy, Adina Institute of Pharmaceutical Sciences, Sagar, M.P. India 4 Department of Pharmaanalysis, B.R.Nahata College of Pharmacy, Mandsor, M.P. India * Corresponding Author dgoswamipharma@gmail.com ABSTRACT Bark of Tectona grandis Linn. commonly known as Sagwan belongs to the Verbenaceae family and, traditionally used in diabetes, bronchitis, constipation and in various skin ailments. Present work is related to standardization of Tectona grandis by using pharmacognostic (macroscopy, microscopy and physical constants) and phytochemical investigation on stem bark of Tectona grandis. All the parameters were studied according to the WHO and pharmacopoeial guidelines to standardize the Tectona grandis. KEYWORDS Tectona grandis, Pharmacognostic evaluation, Physicochemical Analysis. INTRODUCTION Standardization of natural products is a complex task due to their heterogeneous composition, which is in the form of whole plant, plant parts or extracts obtained thereof. To ensure reproducible quality of herbal products, proper control of starting material is utmost essential. The first step towards ensuring quality of starting material is authentication. Thus, in recent years there has been a rapid increase in the standardization of selected medicinal plants of potential therapeutic significance 1,2. Despite the modern techniques, identification of plant drugs by pharmacognostic studies is more reliable. According to the World Health Organization, the macroscopic and microscopic description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such materials and should be carried out before any tests are undertaken 3. Tectona grandis Linn. (Verbenaceae) is a large deciduous tree. Branchlets are quadrangular, channeled and stellately 1 tomentose. The tree is growing in higher situations, native to central India, Konkan, Western Deccan peninsula, South India and Burma 4. It is commonly known as sagwan (Hindi), saka (Sanskrit) and teak tree (English) 5, 6. Teak is a hardwood species of worldwide reputation 7. Leaves are by cm, elliptic or obvate, acute or acuminate. Upper surface of leaf is rough but usually glabrous and the lower clothed with dense stellate grey or tawny tomentum. Flowers are shortly pedicellate with lanceolate bracts at the forks. Fruits are 1-3 cm in diameter, subflobose; pericarp is soft with dense felted stellate hairs 4. Root contains lapachol, tectol, tectoquinone, β-sitosterol and a diterpene, tectograndinol 8. Roots are used in the treatment of anurea and urine retention 9. The flowers are acrid, bitter and useful in the treatment of bronchitis, biliousness and urinary discharges. Bark is astringent, acrid, sweet and useful in the treatment of bronchitis. The wood is acrid, sedative, anthelmintic, expectorant and useful in the

2 treatment of gravid uterus, piles, leucoderma, dysentery, headache and burning pain over liver region. The ashes of wood applied to swollen eyelids and are said to strengthen the sight. The oil of nuts promotes the growth of hair and removes itchiness of skin. The flowers and the seeds are diuretics 4. MATERIALS AND METHODS 1. Plant Material Collection Bark of T. grandis were collected from Ahmednagar district of Maharashtra in September 2008 and authenticated by P.G. Diwakar, Botanical Survey of India, Pune, where a sample specimen (Voucher number: DGTEG1) has been deposited. The fresh barks were separated and used for the study of macroscopic and microscopical characters, whereas dried bark powder material was used for the determination of physical constant and phytochemical constituents. 2. Chemical and Reagents Used All the reagents used were of analytical grade obtained from Sigma Chemical Co., St. Louis, USA and Fine Chemicals Ltd., Mumbai, India. 3. Macroscopic Evaluation Different macroscopic parameters were studied for evaluation of Tectona grandis bark like color, odour, taste and size as per WHO guideline Microscopic Evaluation The crude bark was subjected for microscopical evaluation. The paraffin embedded specimens were sectioned with the help of rotary microtome. The thickness of the section was µm. Dewaxing of the section was done by customary procedure 10. The sections were stained with toluidine blue as per the method of O Brien 11. Since toluidine blue is a polychromatic stain, the staining results were remarkably good and some cytochemical reactions were also obtained. The dye rendered pink color to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Wherever necessary sections were also stained with safranin, fast-green and Iodine (for starch). 5. Determination of Physical Constants (i) Ash Values Different Ash values like total ash, water soluble ash and acid insoluble ash were evaluated 12. (ii) Extractive Values Different extractive values like water-soluble and alcohol-soluble extractive values were evaluated 12. (iii) Foreign Organic Matter and Moisture Content Foreign organic matter was determined from the weight of the drug taken. Moisture content is determined by Loss on drying method in terms of percent w/w 12. (iv) Extraction The coarse powder of bark was used for extraction. Continuous hot extraction was carried out by using different solvents of increasing polarity [petroleum ether ( C), ethyl acetate and ethanol] in Soxhlet extractor. Finally the marc was allowed for maceration about 24 hours with distilled water to obtain aqueous extract 13, 14. (v) Qualitative Phytochemical Screening Freshly prepared bark extracts were tested for the presence of phytochemical constituents by using reported methods 15, 16, 17. 2

RESULTS AND DISCUSSION 1. Macroscopic Evaluation Parameters Color Odor Taste Size Shape Table 1.

Microscopy of Stem Bark The bark consists of the following zones (i) Periderm The periderm is superficial, wide and undulates with wavy outline. The fissures are shallow and irregular.

Phelloderm is not evident. Inner of the periderm is a parenchymatous zone resembling cortex.

of Bark -Periderm Figure 1 (Pe - Periderm, DPhR- Dilated Phloem Ray, CPh-Collapsed Phloem, PhSc- Phloem Sclereids (ii) Collapsed Secondary Phloem Collapsed phloem constitute the major portion

3 RESULTS AND DISCUSSION 1. Macroscopic Evaluation Parameters Color Odor Taste Size Shape Table 1. Macroscopy of Bark Features Outer surface brown, inner surface whitish brown to buff Characteristic Acrid 5 12 mm thick Quadrangular, fluted 2. Microscopy of Stem Bark The bark consists of the following zones (i) Periderm The periderm is superficial, wide and undulates with wavy outline. The fissures are shallow and irregular. It is consists of homogeneous phellem, which is 300µm wide. The phellem cells are homogenous comprising of regular radial files of thin walled, suberised tabular cells. Phelloderm is not evident. Inner of the periderm is a parenchymatous zone resembling cortex. This zone is formed by dilated phloem rays which extent up to the inner border of this periderm, so this zone is called Pseudo-cortex (Figure 1). T. S. of Bark -Periderm Figure 1 (Pe - Periderm, DPhR- Dilated Phloem Ray, CPh-Collapsed Phloem, PhSc- Phloem Sclereids (ii) Collapsed Secondary Phloem Collapsed phloem constitute the major portion and occupies almost 3/4 th of the width of the bark. It consists of highly dilated rays and long, narrow radial bands of outer phloem element. The phloem rays are narrow in the inner parts and become progressively wider toward the periphery. They become undulated in the outer region. The cells of this dilated rays consists of horizontal rows of small, rectangular thin compact cells. The cells are parenchymatous and thick walled. Alternating with the dilated rays are the radial bands of crushed 3

phloem elements and tangential segments of fileses.

The crushed phloem element can be seen in the form of tangential or radially oblique dark lines. A part from dark lines there are also wide parenchymatous cells which remain intact.

The short blocks of fibers are seen in the form of continuous cylinders which are radially intercepted by phloem rays (Figure 2). T. S.

4 phloem elements and tangential segments of fileses. The radial bands of crushed phloem and filuse segments are wider towards the inner part and become gradually narrow and tapering in the outer portion of the bark. The crushed phloem element can be seen in the form of tangential or radially oblique dark lines. A part from dark lines there are also wide parenchymatous cells which remain intact. Alternating with the crushed lines and parenchymatous cells, there are thick tangential segments of phloem fibers towards the interior portion of bark. The short blocks of fibers are seen in the form of continuous cylinders which are radially intercepted by phloem rays (Figure 2). T. S. of the Bark- Collapsed Secondary Phloem Figure 2 (DPhR- Dilated Phloem Ray, CPh- Collapsed Phloem, PhSc- Phloem Sclereids) (iii) Non-Collapsed Phloem The non collapsed phloem is seen in the inner most portion of the bark and the outer part of the secondary xylem. This portion consists of intact well preserved sieve elements, companion cells and narrow straight phloem rays. The sieve elements are polygonal in outline, random in distribution and have the companion cells at the corners. The phloem parenchyma cells are similar to the sieve elements but lack the companion cells. Starch grains are seen stored in the parenchyma cells of the phloem (Figure 3). (iv) Crystals Calcium oxalate crystals are seen associated with the phloem fibers bands. The crystals are prismatic type. They occur along the outer and inner boundaries of the fiber bands (Figure 4). 4 (v) TLS view of the Secondary Phloems In TLS section of secondary phloem in the bark, the phloem rays and the sieve tubes exhibit certain characters. The rays are non-storied and uniseriate to multiseriate. The rays are heterocellular consisting procumbent body cells and marginal upright cells. The procumbent cells are horizontally oblong forming a wide panel; the upright cells are vertically oblong. The rays are (400)µm in height, 20-70µm wide. Ray frequency is 7 per mm (Figure 5).

5 International Journal of Pharma and Bio Sciences V1(2)2010 T. S. of the Bark (a) Non Collapsed Phloem (b) Periderm and Pseudo-Cortex Figure 3 T. S. of the Bark - Crystal in Phloem Fibers Figure 4 T.L.S. of the Bark-Secondary Phloem (a) Phloem under Low Magnification (b) Phloem under Enlarged Magnification Figure 5 (CPh- Collapsed Phloem, PhSc- Phloem Sclereids, NCPh- Non Collapsed Phloem, Pe - Periderm, PCo Pseudo Cortex, Cr - Crystals,, PhF - Phloem Fibres, PhR- Phloem Ray, SC- Scalereids, ST- Sieve Tube, SP- Sieve Plate) 5

(vi) Sieve Tube Members The sieve tube members are short, narrow and straight. They are 170µm long, 20-25µm wide.

In the phloem parenchyma cells are seen large, circular bodies which are starch grains. These bodies are adhering the lateral walls or free in the cell lumen (Figure 6). T.L.S.

Determination of Physical Constants (i) Ash Values Table 2. Ash Values Evaluation parameter Yield (% W / W) Total ash 7.25 Water- soluble ash 1.5 Acid insoluble ash 3.

6 (vi) Sieve Tube Members The sieve tube members are short, narrow and straight. They are 170µm long, 20-25µm wide. The sieve plate is simple, slightly oblique and callose is deposited on the sieve plate which is thick mass masking the sieve plate. In the phloem parenchyma cells are seen large, circular bodies which are starch grains. These bodies are adhering the lateral walls or free in the cell lumen (Figure 6). T.L.S. of the Phloem - Sieve Tube, Phloem rays, Axial Parenchyma and Callose Figure 6 (UC - Upright cell, AP - Axial parenchyma, Ca- Callose, PC - Procumbent cell, R- Rays, ST- Sieve tube) 3. Determination of Physical Constants (i) Ash Values Table 2. Ash Values Evaluation parameter Yield (% W / W) Total ash 7.25 Water- soluble ash 1.5 Acid insoluble ash 3.5 (ii) Foreign Organic Matter and Moisture Content Table 3. Foreign Organic Matter and Moisture Content Values Particulars Results (%W / W) Foreign organic matter 0.04 Moisture content 14.6 (iii)extraction All the extracts were vaccum dried to produce petroleum ether extract (2.81%w/w), ethyl acetate extract (6.48% w/w), ethanol extract (5.19% w/w) and aqueous extract (8.86% w/w) respectively. 6

7 Table 4. Percentage Yield of Extract in Different Solvent Extract Color Yield (%w/w) Petroleum ether extract Yellowish-brown 2.81 Ethyl acetate extract Brown 6.48 Ethanol extract Reddish-brown 5.19 Aqueous extract Dark Brown to black 8.86 (iv) Qualitative Phytochemical Screening The qualitative chemical tests of the petroleum ether, chloroform, ethyl acetate, ethanol and aqueous extracts revealed the presence of alkaloids, tannins, saponin, flavonoid, amino acid, flavonoids, steroids, glycosides and carbohydrates. Table 5. Qualitative Phytochemical Screening of Tectona grandis Bark Extracts Constituents Pet. Ether Ethyl acetate Ethanol Aqueous Carbohydrate Proteins Amino acids Steroids Glycosides Saponin Flavonoids Alkaloids Tannins CONCLUSION In conclusion, the present study on pharmacognostical characters of Tectona grandis Linn. bark will be providing useful information in regard to its correct identity and help to differentiate from the closely related other species of Tectona. The other parameters observed may be useful for the future identification of the plant. REFERENCES 1. Wallis TE, Ed. Textbook of Pharmacognosy. 5 th Edn, CBS Publications: , (1985). 2. Trease GE and Evans WC, Ed. Pharmacognosy, 12 th Edn., Bailliere Tindal, East Bourne: , (1986) World Health Organization: Quality control methods for medicinal plant materials, WHO Library: , (1998). 4. Adcock IM and Mattew JC, New drugs for asthma. Drug Discovery Today. 3: , (1998). 5. Aguinaldo AM, Osler PM, Ocampo D, Bowden BF, Grey AI and Peter G. Tectograndone, an anthraquinonenaphthoquinone pigment from the leaves of Tectona grandis, Phytochemistry; 33(4): , (1993). 6. Aletta DK, Hanneke PM, Mirjam K, Anneke HH, Andrys CD, Frank AM and Redegeld

8 Frans PN. Key role for mast cells in nonatopic asthma. J Immunol, 169: , (2002). 7. Ansari SH., Ed. Essentials of Pharmacognosy, 1 st Edn. Birla publications Pvt. Ltd.: 2-4, (2004). 8. Atkinson RW, Anderson HR, Sunyer J, Ayres J, Baccini M and Vonk JM, Acute effects of particulate air pollution on respiratory admissions: results from APHEA 2 project. Am J Respir Crit Care Med, 164(10): , (2001). 9. Barar FSK. Ed. Essential of Pharmacotherapeutics, 3 rd Edn., S. Chand and Company Ltd: , (2003). 10. Johansen DA., Ed. Plant Microtechnique. Mc Graw Hill Book Co.: , (1940). 11. O Brien TP, Feder N and Mc Cull ME. Ed. Polychromatic staining of plant cell walls by toluidine blue-o Protoplasma; 59: , (1940). 12. Indian Pharmacopoeia, Government of India, Ministry of Health, Controller of Publications, Vol. II, 3rd Edn., 74, (1985). 13. Harborne JB,.Ed. Phytochemical Methods. 3 rd Edn. Springer International: 7, (1998). 14. Mukherjee PK., Ed. Quality Control of Herbal Drugs, 1 st Edn, Business Horizone, Pharmaceutical Publushers: , (2002). 15. Khandelwal KR, Ed. Practical Pharmacognosy Technique and Experiments, 23 rd Edn: 15-29, , (2005). 16. Kokate CK. Ed. Practical Pharmacognosy, 4 th Edn., Vallabh Prakashan: , (1994). 17. Farnsworth NR., Biological and phytochemical screening of plants. J. Pharm. Sci. 55: , (1966). 8

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