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UK Journal of Pharmaceutical and Biosciences Vol. 4(4), 80-84, 2016 RESEARCH ARTICLE UK Journal of Pharmaceutical and Biosciences Available at www.ukjpb.

Soni * Department of Biosciences, Veer Narmad South Gujarat University, Udhna Magdalla Road, Surat, 395007, Gujarat, India Article Information Received 16 December 2015 Received in revised form 30

1 UK Journal of Pharmaceutical and Biosciences Vol. 4(4), 80-84, 2016 RESEARCH ARTICLE UK Journal of Pharmaceutical and Biosciences Available at ISSN: A Contribution on the Microcharacters of Barleria prionitis L. Useful in Pharmcognosy Milvee K. Vyas, Kailash Patel, Kunjal V. Soni * Department of Biosciences, Veer Narmad South Gujarat University, Udhna Magdalla Road, Surat, , Gujarat, India Article Information Received 16 December 2015 Received in revised form 30 Aug 2016 Accepted 31 August 2016 Keywords: Pharmacognosy, Barleria prionitis; Anatomy, Stomata, Trichomes Corresponding Author: milveevyas4u@gmail.com Mob.: , Abstract Barleria prionitis (Acanthaceae) is an important medicinal plant distributed throughout the various regions of India and useful in urinary and paralytic affection, stomach disorders, catarrh, cough and anasaraca and lacerated sole. In the present investigation an attempt was made to study the anatomy of root, stem, lamina, midrib, petiole, stomata and trichomes as well as histochemical localization of various substances viz., calcium oxalate, lignin, suberin, lipids in the organs of Barleria prionitis. The present study revealed that the plant distinguishing microcharacters viz., bicollateral vascular bundles, internal phloem, uni - biseriate xylem rays, inverted omega shape vascular bundle, diacytic stomata and eglandular as well as glandular trichomes which are useful in solving taxonomic problems and pharmacognosy. They have significance in identification of crude drugs from this taxon. 1 Introduction Barleria prionitis is an erect much branched prickly shrub of family Acanthaceae growing in various regions of India. It is a medicinal plant useful in urinary and paralytic affection, stomach disorders and applied to lacerated sole 1. Various plant parts are useful in catarrh, cough and in anasaraca 2. A paste of the roots applied to boils and glandular swellings 3. This plant has alternative, diuretic, tonic and febrifuge action 4-6. The anatomical characters are useful in identification and detection of adulterants in crude drug 7. From the survey of literature it has been revealed that the study about the internal structure of various parts of Barleria prionitis is scanty. The present work was carried out on the anatomy of various organs viz., root, stem, lamina, midrib and petiole. In addition to this, stomata, trichomes and histochemical localization of calcium oxalate crystals, lignin, suberin and lipids were also investigated. The study was aimed to investigate whether or not microcharacters can provide some distinguishing features helpful to plant taxonomy and pharmacognosy. 2 Materials and Methods Different organs of Barleria prionitis were collected from the field and small pieces were fixed in FAA (Formaldehyde Alcohol Acetic acid) and preserved in 70% ethnol. The small species of various organs were dehydrated by TBA (Tertiary butyl alcohol), infiltrated and embedded in paraffin wax. 8 to 10 micron thin sections were taken and stained with safranin O and FCF fast green 8. For the stomatal studies, epidermal peels were taken with the help of forceps and stained with Delafield's hematoxylin and mounted in glycerin jelly For the histochemical studies hand sections of fresh materials were used. The histochemical substances localized and their tests are presented in table 1. The microphotographs were taken on Axion scope A1 photomicroscope of Carl Zeiss. 3 Results 3.1 Stem Epidermis is unilayered, with a thin and smooth cuticle. Hypodermis is made up of collenchyma and cortex is of parenchyma cells. The cells of outer two layers of cortex contain chloroplasts (Fig. 1A, B). Stem has eglandular multicellular uniseriate and filiform trichomes as well as clavate or club shaped glandular trichomes (Fig. 1B). The primary vascular system consisted of two large

bicollateral vascular bundles. The pith was parenchymatous. Needle shape crystals of calcium oxalate were observed in certain pith parenchyma (Fig. 1C, D).

2 bicollateral vascular bundles. The pith was parenchymatous. Needle shape crystals of calcium oxalate were observed in certain pith parenchyma (Fig. 1C, D). Table 1: Histochemical reagents to localize substance Histochemical Substance reagent localized Reference Phloroglucinol and HCL Lignin Jensen 11 Sudan black B Oily contents (Lipids) Bancroft 12 Sudan IV Suberin Johansen 13 Hydrogen Calcium oxalate peroxide and Pizzolato 14 crystals Silver nitrate In old stem a ring of vascular cambium is produced by the union of inter and intra fascicular cambium which forms secondary vascular tissues (Fig. 1A). The secondary xylem consisted of solitary, spherical or oval, vessel elements, polygonal xylem fibers and uniseriate xylem rays. The walls of xylem fibers have higher deposition of lignin. Ray cells were short (Fig. 1A, F). The secondary phloem was made up of sieve tube elements, companion cells and phloem parenchyma. Lipid globules were observed in cortical cells of old stem (Fig. 1F). A complete ring of internal phloem was present on the inner side of xylem cylinder (Fig. 1E). 3.2 Root Root has uniseriate epidermis of thin walled, closely arranged tubular cells. A cortex of 6 to 8 layers of parenchyma cells was present beneath the epidermis. Endodermis and pericycle are unilayered. The endodermal cells were tubular or barrel shaped whereas the cells of pericycle are much smaller and more or less rectangular in the shape. Root has tetrarch xylem alternating with four small groups of phloem. Protoxylem is exarch. Asmall parenchymatous pith was observed in the center (Fig. 2A). A ring of vascular cambium of 3-4 layers of rectangular cells was formed in old root which produces more secondary xylem then secondary phloem. Secondary xylem has spherical or oval vessel elements, polygonal xylem fibers with thick lignified cell wall and uni - biseriate xylem rays. Vessel elements are solitary or found in groups of two (Fig. 2B, C). The cellular components of secondary phloem are similar to the stem phloem. Periderm was composed of phellogen of 2 to 4 layers of thin walled rectangular cells, phellum of radially arranged cells with thickening of suberin on the walls and phelloderm of tangentially elongated parenchyma cells (Fig. 2C). 3.3 Leaf Lamina The epidermal cells of lamina were squarish or rectangular and larger on adaxial side (Fig. 2D). The wall of epidermal cells was sinuous on both, adaxial and abaxial surfaces. Lamina was hypostomatic with diacytic stomata (Fig. 2E). The abnormal stoma without guard cells was occasionally observed (Fig. 2F). Lamina was dorsiventral with elongated palisade cells in a single row (Fig. 2D). Lipid globules were observed in palisade and spongy tissues (Fig. 2D). The larger mesophyll vascular bundles were bicollateral with scanty phloem on adaxial side and smaller were collateral. Fig 1: A - F, Transactions of stem. A - C, X 480; D - F, X 800.; co - collenchyma; cr crystals; gt glandular trichome; li lipid globules; pi pith; sp secondary phloem; sx secondary xylem; vb vascular bundle; ux- uniseriate xylem rays; xf xylem fibres Midrib The ground tissue of midrib was differentiated into adaxial and abaxial hypodermis of collenchyma and large cortex of parenchyma. The palisade tissues of lamina were extended in UK J Pharm & Biosci, 2016: 4(4); 81

Arumugam and Natesan 23 described the anatomical characters of stem and leaves of Barleria noctiflora.

3 the midrib up to certain distance on adaxial side. The midrib has a single central inverted omega shape bicollateral strand (Fig. 3A). Numerous research has been conducted on the epidermal structures in Acanthaceae However, the study on the anatomical structures of various organs of Barlaria is less Arumugam and Natesan 23 described the anatomical characters of stem and leaves of Barleria noctiflora. Present study shows that Barleria prionitis can be distinguished from it by the presence of multicellular trichomes, single layer of palisade cells and heterogeneous ground tissues of midrib. Crystals of calcium oxalate were found by Bhogaonkar and Lande 20 in the leaves of Barleria prionitis. Inamdar 18 reported, rod like and square crystals of oxalate in the members of Acanthaceae. In the investigated species the needle shape crystals are observed in the stem and petiole. Fig 2: A F; A D transactions; A C root; D lamina; E, F surface view of abaxial epidermis of lamina. A - D, X 480; E, F, X 800.; bx - biseriate xylem rays; ds diacytic stomata; li lipid globules; pd phelloderm; pe phellum; sp secondary phloem; sw stoma without guard cells; ux- uniseriate xylem rays; xf xylem fibres Petiole Petiole has adaxial flattened surface with median groove and convex abaxial side in transactional view. Hypodermis was composed of 4-8 layers of collenchyma. Cortex was large and made up of parenchymatous cells (Fig 3C). Needle shape oxalate crystals were observed in cortical cells (Fig. 3B). The peripheral cortical cells contain chloroplasts. The vascular system of petiole was formed by one large central crescent shape bicollateral strand and two small adaxial collateral bundles (Fig. 3D). Latter were placed in the adaxial corners on the inner side of collenchyma of hypodermis (Fig. 3C). 4 Discussions Barleria prionitis commonly known as "Vajradanti" and "Porcupine flower" is widely used in traditional medicine in India. Fig 3: A D transactions; A - midrib; B - D petiole. A, C, D, X 480; B, X800. ad adaxial vascular bundle; co - collenchyma; cr crystals Daicytic (Caryophyllaceous) stomata are common in Acanthaceae 7. The ontogeny of diacytic stomata in Acanthaceae was first traced by Pant and Mehra 24 and Paliwal 25. Later, Inamdar 18 studied the ontogeny of stomata of 31 species of Acanthaceae. He had described diacytic stomata with two to four subsidiary cells. In Barleria prionitis diacytic stomata with two subsidiary cells are observed similar to that reported by Vasoya 26. UK J Pharm & Biosci, 2016: 4(4); 82

4 The family Acanthaceae is rich in different types of trichomes which are used as aid for identification 27. Structure and distribution of folier epidermal trichomes of 109 species of Acanthaceae have been studied by Ahmad 16 and observed both glandular and eglandular trichomes in different species. Arumugam and Natesan 23 reported only eglandular unicellular trichomes in Barleria noctifolra. Barleria prionitis can be distinguished from this as it has both multicellular eglandular as well as glandular trichomes similar to the observations of Bhogaonkar and Lande 18 According to 20 in Barleria prionitis stem the internal phloem is present in the form of small patches. Present study shows that it is present in the form of a complete ring on the innerside of the cylinder of secondary xylem. 5 Conclusions From the present study it was revealed that several microscopic features characterized to Berlaria prionitis viz., two bicollateral vascular bundles in young stem ring of internal phloem on the inner side of secondary xylem cylinder of stem, uniseriate xylem rays in stem and uni to biseriate rays in root, hypostomatic lamina with daicytic stomata, presence of a single layer of palisade cells in lamina, inverted omega shape bicollateral vascular bundle in midrib and crescent shape in petiole. This peculiar microcharacter can be useful in solving taxonomic problems and pharmacognosy as this plant has significance in Ayurved. The histochemical study shows the presence of crystals of calcium oxalate, higher thickening of lignin on walls of xylem fibers, deposition of suberin on walls of phellum cells and lipid globules in cells of stem and lamina. This information has significance in pharmacognosy as it is suggestive of medicinal utility of plant. 6 Conflict of interests Authors have no conflict of interest. 7 Author's contributions KS has collected literature and helped in preparation of slides for microscopic studied. KPP has done microphotography and written conclusion. MV carried out entire practical work, written manuscript and approved it. 8 References 1. Joshi SG. Medicinal Plants. Oxford and IBH Pub. Co. Pvt. Ltd. New Delhi, India; Singh MP, Panda H. Medicinal herbs with their formulations. Daya Pub. House, Delhi, India; Ambasta SP (ed.). The useful plants of India. National institute of science communication and information resources. CSIR, New Delhi, India; Chopra RN, Chopra IC, Handa KL, Kapur LD. Indigenous drugs of India. Academic Pub., Calcutta, India; Nadkarni AK. Dr. KM Nadkarni's. Indian Meteria Medica, Revised ed., Popular Book Depot, Bombay, India; Gujral ML, Saxena PN, Misra SS. An experimental study of the comparative activity of indigenous diuretics. J. Indian Med. Asso. 1955; 25 : Metcalfe CR, Chalk L. Anatomy of the dicotyledons. Vol. I. and II. Clarendon press, Oxford; Berlyn GP, Miksche JP. Botanical microtechnique and cytochemistry. IOWA State Univ. Press, Ames, IOWA; Pal VC, Singh OV, Singh B, Ahmad A. Pharmacognostical Studies of Amaranthus Spinosus Linn. UK Journal of Pharmaceutical and Biosciences. 2013; 1(1): Pal VC, Singh B, Ahmad A. Pharmacognostic Evaluation of Leaves and Stem of Murraya koenigii. UK Journal of Pharmaceutical and Biosciences. 2015; 3(1): Jensen W. Botonical histochemistry. W. H. Freeman and Company, San Francisco, California Bancroft JD. Histochemical techniques. Butterworths, London Johansen DA. Plant microtechnique. McGraw - Hill Book Company, New York, Pizzolato P. Histochemical recognition of calcium oxalate. J. Histochem. Cytochem. 1964; 12(5): Dipa C, Daniel M. Foliar trichomes of some members of the family Acanthaceae and their taxonomic utility. Int. J. Pharm. Biosci. 2011; 2(3): Ahmad KJ. Epidermal hairs of Acanthaceae. Blumea. 1978; 24(1): Inamdar JA, Bhatt DC, Chaudhari GS. Structure and development of stomata in some Acanthaceae. Proc. Indian Acad. Sci. (Plant Sci.) 1983; 92(3): Inamdar JA. Epidermal structure and ontogeny of caryophyllaceous stomata in some Acanthaceae. Bot. Gaz. 1970; 131(4): Jani S, Harisha CR, Patel BR. Variations of trichomes in family Acanthaceae W.S.R. to Kalmegh - a micromorphological and micrometric study. Int. J. Universal Pharm. Biosci. 2013; 2(6) : UK J Pharm & Biosci, 2016: 4(4); 83

5 20. Bhogaonkar PY, Lande SK. Anatomical characterization of Barleria prionitis Linn. : A well-known medicinal herb. Biological forum - an Int. J. 2012; 4(1) : Chiwapreecha B, Janprasert K, Kongpakdee C. Comparative anatomy of three medicinal plants in Acanthaceae. Int. Symposium Med. Aromatic Plants. ISHS Acta Horticulture. 2014; 33: Nag S, Sinha K, Dutta R, Pal KK. Phytochemical and anatomical studies of the medicinal plant Barleria lupulina Lindl. Asian Resonance. 2013; 2 (4): Arumugam S, Natesan SK. Pharmacognostical studies and phytochemical investigation of Barleria noctiflora Linn (Acantheceae). Int. J. Pharmcog. Phytochem. Res. 2015; 7(3): Pant DD, Mehra B. Development of caryophyllaceous stomata in Asteracantha longifolia Nees. 1963; 27(4): Paliwal GS. Structure and ontogeny of stomata in some Acanthaceae. Phytomorphology. 1966; 16(4): Vasoya U. Pharmacognostical and phytochemical studies on the leaves of Barleria prionitis (L.). Int. J. Pharmaceu. Sci. Res. 2012; 3(7): Hayat MQ, Asraf M, Khan MA, Yasmin G, Shaheen N, Jabeen S. Diversity of foliar trichomes and their systematic implications in the genus Artemisia (Asteraceae). Int. J. Agric. Biol. 2009; 11 (5): UK J Pharm & Biosci, 2016: 4(4); 84

ANATOMY OF PLANTS Introduction: The study of gross internal structure of plant organs by the technique of section cutting is called plant anatomy.

ANATOMY OF PLANTS Introduction: The study of gross internal structure of plant organs by the technique of section cutting is called plant anatomy. (Pandey, 2002). Various plant organ viz. root, stem, leaves,