Pharmacognostic studies of Shorea tumbuggaia roxb. A globally threatened species

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1 PHCOG J ORIGINAL ARTICLE Pharmacognostic studies of Shorea tumbuggaia roxb. A globally threatened species S. Ankanna and N. Savithramma Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh , India Submission Date: ; Review completed: ; Accepted Date: ABSTRACT Introduction: Since all parts of Shorea tumbuggaia an endemic endangered medicinal plant species using as a folklore claims has therapeutic qualities. To ensure reproducible quality of plant herbal products, proper control of starting material is utmost essential thus in recent years there has been an emphasis in standardization of medicinal plants of therapeutic potential. Methods: The present study has laid down a set of macroscopic, microscopic and fluorescence features of stem, stembark and leaf has been carried out by simple and compound microscopes; and spectro flourometer. Results: The salient anatomical features of the Shorea tumbuggaia are the stem is circular with shallow wide furrows, well defined medullary resin canals connected by phloem rays from cortical region to medulla. The stem bark revealed that major source for various secondary metabolites like phenols, flavonoids, tannins, anthocyanins, triterpenoids when compared to the stem and leaf. The leaf consisting special type of star shaped sclereid cells on abaxial surface. Paracytic stomata are distributed on abaxial side only. Fan shaped sphaerites cells consisting 4 digits are distributed towards lower side. All of the histochemical reactions showed that the presence of tannins, starch and phenols in leaf and cotyledons. The chemical potency of stem bark powder proved at excitation at 396 nm and emitted photons at 475 nm. Conclusions: With this back drop, it becomes extremely important to make an effort towards standardization of the plant material to be used as medicine and help in identification and authentification of the plant material in the drug. Keywords: Shorea tumbuggaia, medicinal tree, standardization. INTRODUCTION Standardization is the system to ensure that every packet of medicine that is sold as the correct amount and will induce therapeutic effect. According to the WHO, botanical standards should be proposed as a protocol for the diagnosis of the herbal drug quality control of crude drug and its pharmaceuticals can be attempted by different methods of evaluation depending upon the morphological and microscopical studies. [1] Pharmacognostic studies *Corresponding author. S. Ankanna Research Scholar Department of Botany, Sri Venkateswara University, Tirupati Andhra Pradesh, INDIA. Mobile: ankanna2010@gmail.com DOI: /pj can serves several purposes inducing batch to batch consistency, conformation of correct amount of dosage and positive control to indicate possible loss or degradation. [2] Standardization practices continue today because of its biomedical benefits as well as place in cultural beliefs in many parts of the world and have made a great contribution towards maintaining human health. [3] To ensure reproducible quality of plant herbal products, proper control of starting material is utmost essential thus in recent years there has been an emphasis in standardization of medicinal plants of therapeutic potential. [4] Plants are utilized extensively as raw drugs for many formulations in traditional systems of medicine. To check the genuineness of the raw drugs and to detect adulteration of these materials, an authentic pharmacognostic study is needed for each raw drug. [5] Usually the drugs are collected by traditional practitioners who have inherited Ayurvedic or other herbal practices. Their identification is mostly based on morphological features or other traditionally 52 Phcog J Dec 2012 Vol 4 Issue 34

2 known characteristics. In such cases, there is a chance of selecting incorrect raw drugs. Therefore, an extensive anatomical and phyto chemical screening is needed for each raw drug used in the formulation to avoid any ambiguity and such a study will serve also as a reference for further studies. [6] Anatomical studies are helpful in describing a particular drug with a special emphasize on quantitative microscopy such as sclerides; starch grains, crystals, stomata and trichomes; and qualitative microscopic studies such as xylem phloem and other tissues. [7] S. tumbuggaia is a tree taxon with economic and medicinal values. Stem is used in marine yards as a substitute for pitch. The tree trunk is in use as flag poles for temples. [8] The heart wood is similar to sal but much smoother and better for carpentry. The plant extracts used to cure ear-aches. Plant parts are to be used as an external stimulant. Leaf juice is used as ear drops for children. [9] The bark having anti ulcer activity. [10] The stem is a source of resin, which is used as incense. The resin used to cure duodenal ulcers and Amoebic dysentery. The oleoresin which is exuded from the stem bark of Shorea can be used to cure hydrosis and alexiteric. [11] It is also used in indigenous medicine as an external stimulant and a substitute for arbutus. [12] Hence, the present pharmacognostical study was under taken to supplement useful data in regard to its correct identity, as this plant is broadly used in indigenous system of medicine. Plant material MATERIAL AND METHODS Fresh and healthy stem, stembark and leaf of Shorea tumbuggaia were collected from Tirumala Hills of Chittoor district, Andhra Pradesh, India. Shorea tumbuggaia Roxb. or Vatica tumbuggaia. Wt. & Arn. (Dipterocarpaceae). Commonly known as Green Dammar. It is large sporadic resinous tree attains the maximum height of about m with maximum width of cm. Bark is thick, rough dark brown longitudinally furrowed, within the furrows gum is exuded. Simple leaves with ultimate reticulate venation. Inflorescence is Axillary/terminal panicles. Flowers white. Fruits with a woody pericarp with wings 2 3 times as long as capsule. Single seed with 4 unequal fleshy cotyledons. S. tumbuggaia trees bear adequate foliage during and after the rainy season. It has reduced foliage during late winter and early dry season. At the same time, gradual new leaf flushing occurs. The leaf transitional stage characterized by leaf fall and leaf formation is quite prominent during April May. Flowering occurs during April to May and fruiting May to June. Each fruit produces only one seed against the actual number of six ovules. The sepals are acrescent in that they are thickened, and three of them expand into wings and are larger than the other two sepals. The fruit wall is free from calyx, woody, with a thin inner membranous lining invaginated into the folds of cotyledons and split into two parts at the apex. Stem is long cylindrical, coated with rusty ash and attains of about meters height and 1.5 to 2.0 meters girth at Tirumala hills, Andhra Pradesh, India. Sample preparation The plant material was fixed in a mixture of solvents containing formalin, acetic acid and alcohol (70% v/v) for histological studies. After 24 hours of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol as per the schedule given by Asokan. [13] Infiltration of the specimens was carried by gradual addition of paraffin wax in tertiary butyl alcohol solution until it attains super saturation. The specimens were cast into paraffin blocks. Transverse sections (T.S) of different organs of the plant materials were taken using a rotary microtome (RMT-30) and stained with different stains. Microphotographs of the sections were made by using Nikhon labhot 2 microscopic unit. Stomatal index and frequency A thin film or a smear of quick fix was applied on the upper and lower surfaces of the leaves and it was allowed to dry for 5 minutes. The smear was gently peeled off with the help of needle and forceps. The peelings were mounted and observed under microscope on the same power in which number of stomata found in microscopic field area were noted. Stomatal index and stomatal frequency were calculated by using the following formula. Stomatal Index = Number of stomata Number of stomata + Numberofepidermal cells Stomatal Frequency Histochemical tests Number of stomata = Microscopicfield area The fresh sections of different plant parts were stained with 0.05% toluidine blue (polyphenols); 20% ferric chloride for few minutes (Tannins) Lugol s iodine (carbohydrates) and observed under microscope. [14] Fluorescence studies Plant extracts were prepared with various chemicals like Ammonia, Nitric acid, Hydrochloric acid, Ferric chloride, Phcog J Dec 2012 Vol 4 Issue 34 53

3 Alcoholic aqueous Sodium Hydroxide. A small quantity of the supernatents were subjected to Fluorolog-3-spectro fluorometer (Jobin Yvon) then the excitation and emissions were recorded, and compared with electromagnetic spectrum. RESULTS AND DISCUSSION The transverse section of the stem shows the single layered thick walled epidermis consisting of prominent lenticels (Figure 1a-1). Below the epidermis sclerenchymatous hypodermis is present followed by 2 3 layers of cortex. In between the phloem and xylem single layered cambial ring is present and divides the two tissues as secondary phloem and xylem. The phloem consists well developed prominent phloem rays towards epidermis starting from medulla. Protoxylem extends towards medullary region and metaxylem with prominent parenchymatus strands towards cambium. Central region of stem showed well developed parenchymatous medulla with special type of schizogenous cavities (Figure 1-a2) and also numerous tannin cells can be seen. Stembark is soft, thick, longitudinally fissured and corky becoming rough when wounded. The bark outer surface is grey or ash with thick membranous flaks and often covered with crustose lichen green coloured surface is shallow regular vertical fissures and exudes a gum which is reddish brown to brownish black. The anatomical studies revealed that the bark is differentiated into outer thick periderm and inner secondary phloem. Periderm can be divided into phellum and phelloderm. Phellum cells are organized in to thin tangential membranous layers and the older layers exfoliate in the form of flakes. The phelloderm zone is broad and distinct and the cells are turned into lignified sclerides. Secondary phloem differentiated into inner narrow non collapsed zone and center broad collapsed zone. Non collapsed zone consists of radial files of sieve tubes, axial parenchyma and fibres. The collapsed phloem has dilated rays, crushed obliterated sieve tube members, thick walled and lignified fibres and abundant tannin filled parenchymatous cells (Figure 2a). Phloem rays are uniseriate or multiseriate type with homocellular or hetero cellular origin. Similar results have been reported from Ficus religiosa stem bark by Babu. [5] Transverse section of the leaf showed the upper and lower epidermis, mesophyll tissue and prominent vascular bundle. The upper epidermis is formed by the arrangement of rectangular cells and is covered by thick cuticle. Below the epidermis compactly arranged two layers of palisade parenchymatous tissue can be seen followed by 3 4 layers of loosely arranged spongy parenchymatous tissue and consists sclerenchymatous patches. The vascular bundle consists of xylem tissue towards lower side and phloem tissue extends towards upper side. Hypostomatic leaf consisting stomatal index and 55% stomatal frequency. The stoma is surrounded by two prominent guard cells (Figure 3-a4) and were covered by two subsidiary cells to show the paracytic type of stomata. Same results has been reported from Senna and Coca by Kokate. [1] And also observed that 4 digitated fan shaped sphaerites cells (Figure 3-a5) on the aboxial side which are more responsible for storage of innulin polysaccharides may be used to cure diabetis. Star shaped sclereid cells are scattered (Figure 3-a3) on abaxial side. Same results were recorded from Innula helenium, Taraxacum officinale, Saussurea lappa, Chichorium intybus by Kokate. [1] Figure 1. Anatomy of Shorea tumbuggaia stem (a) TS of stem, (a1) lenticels, (a2) Resin canals, (a3) Phloem rays, (a4) Xylem, (a5) Xylem rays, (a6) Parenchyma strand, (b) Starch Locations, (c) Tannin Locations, (d) Phenols Locations. Histolocalization The Histolocalization studies revealed that the secondary phloem and phloem rays of stem bark contains more poly 54 Phcog J Dec 2012 Vol 4 Issue 34

phenols; tannin cells have been observed at the medullary region of the stem and also secondary phloem; and the leaves contains more number of starch grains both in upper and lower epidermis.

4 S. Ankanna, et al.: Pharmacognostic studies of Shorea tumbuggaia roxb. A globally threatened species Figure 2. Anatomy of Shorea tumbuggaia stembark (a) TS of stembark, (b) Starch Locations, (c) Tannin Locations, (d) Phenols Locations. phenols; tannin cells have been observed at the medullary region of the stem and also secondary phloem; and the leaves contains more number of starch grains both in upper and lower epidermis. But there is no evidence for crystals in the all parts of Shorea tumbuggaia (Figure 1(b d); Figure 2 (b e), Figure 3 (b d)). Figure 3. Anatomy of Shorea tumbuggaia leaf (a) TS of leaf, (a1) adaxial side, (a2) abaxial side, (a3) Scleride cells, (a4) Paracytic stomata, (a5) Sphaerites, (b) Starch Locations, (c) Tannin Locations, (d) Phenols Locations. responsible for highest excitation wavelength (420 nm) but hydrochloric acid proved at 330 nm. The highest rate of the colour emission is recorded in stem bark combination with alcoholic sodium hydroxide followed by aqueous sodium hydroxide and ammonia. Flourescence studies Under day light conditions stem bark powder as such emitted light brown colour whereas in U.V. light emitted violet colour where it has excited at 396 nm and emitted photons at 475 nm (Graph-1). And also most of the combinations emitted black to brown colour (Table 1). Maximum emitted wavelength (486 nm) has been observed in stem bark combination with nitric acid. Whereas, minimum emittance wave length obtained at 394 nm by the stem bark addition with glacial acetic acid. Nitric acid alone and also combination with ammonia is Phcog J Dec 2012 Vol 4 Issue 34 Graph 1. Flourescences studies of Shorea tumbuggaia stem bark power as such. 55

5 Table 1. Fluorescence studies of Shorea tumbuggaia stem bark. S. No Experiment Day Light Excitation Emission UV Light 1 Stem bark powder as such Light brown 396 nm 475 nm Blue 2 Powder + 1N NaOH (aqueous) Dark brown 363 nm 458 nm Blue 3 Powder + 1N NaOH (alcoholic) Yellow 367 nm 466 nm Blue 4 Powder + 1N HCl Orange 330 nm 400 nm Violet 5 Powder + H 2 SO 4 Black 373 nm 448 nm Blue 6 Powder + Acetic acid Orange 348 nm 394 nm Violet 7 Powder + Ferric Chloride Black 350 nm 397 nm Violet 8 Powder + HNO 3 Brick red 420 nm 486 nm Blue 9 Powder + NH 3 Dark brown 343 nm 434 nm Violet 10 Powder + HNO 3 + NH 3 Red 420 nm 488 nm Blue CONCLUSION Because of having highly medicinal importance of Shorea tumbuggaia, pharmacognistic studies are inevitable, the present study helps in understanding their identification, taxonomical determination, medicinal importance and to differentiate from the closely related other species of Shorea. REFERENCES 1. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 39 th edition published by Nirali Prakashan, Aug 1, pages. 2. Chaudhury RR. Herbal medicine for human health. New Delhi, World health organization. 1992, Nasreen S, Radha R. Assessment of Quality of Withania somnifera Dunal (Solanaceae) Pharmacognostical and phytophysiochemical profile. Int J Pharmacy and Pharmaceutical Sciences. 2011; 3(2): Faiyaz Ahmed, Asna Urooj. Pharmacognostical studies on Ficus racemosa stem bark. Pharmacognosy Journal 2011; 3(19): Babu K, Gokal Shankar S, Rai Sadananda. Comparative pharmacognostic studies on the bark of four Ficus species. Turk J Bot 2010; 34: Vaibhav S, Kamlesh D. Pharmacognosy; the changing: Scenario, Pharmacog Rev 2007; 1(1): Brinda P, Saraswathy A, Jayaraman P. Micro morphological identification of the medicinal bark of Ventilago madraspatana, J Med Arom Plant Sci 2000; 23: Savithramma N. Threat to indigenous species of Tirumala Hills of Eastern Ghats. EPTRI ENVIS News letter 2003; 9(2). 9. Madhavachetty K, Sivaji K, Tulasi Rao K. Flowering plants of Chittoor district Andhra Pradesh, India. Published by Students Offset Printers, Tirupati, 2008; Patil KS, Chaturvedi SC. Anti ulcer activity of stem bark of Shorea tumbuggaia. J Natural Remedies. 2004; 4(1): Sumy O, Ved DK, Krishnan R. Tropical Indian Medicinal Plants propagation methods; Foundation for Revitalization of Local Health Traditions. Bangalore, India. 2000; Savithramma N. Shorea tumbuggaia Roxb. An endemic endangered and medicinal tree taxon under threat. Non-wood News, 2011; 22: Asokan J. Botanical microtechnique principles and practice. Sky Graphics, Chennai, Treare GE, Evans WC. Pharmacognosy 17 th edn, Bahive Tinal, London. 1985; Phcog J Dec 2012 Vol 4 Issue 34

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