Science, Technology and Development ISSN : 0950-0707

Pharmacognostic Evaluation of paniculatum Linn. Leaves Jeenu Joseph 1*, Akhila S 2, Presannakumaran P.N 3, Santhosh M. Mathews 4 1Associate Professor, Department of Pharmacognosy, Pushpagiri College of Pharmacy, Thiruvalla, Kerala 2 Professor, Department of Pharmacognosy, KVM College of Pharmacy, Cherthala, Kerala 3Professor, Department of Pharmaceutical Chemistry, Pushpagiri College of Pharmacy, Thiruvalla, Kerala 4Principal, Professor, Department of Pharmaceutics, Pushpagiri College of Pharmacy, Thiruvalla, Kerala

Abstract - Clerodendrum paniculatum Linn. is traditionally used in , and Japan for the treatment of various afflictions like inflammation, neuralgia, rheumatism, ulcer and wounds. An effort has been made to draw attention to this folklore herbal medicine that aid in identification of fresh and dried crude leaves samples morphologically and pharmacognostically. The present study also discuss about the morphological and microscopic standardization parameters of the leaves that will furnish referential information for exact identification of the crude drug and enable to prevent adulteration of genuine samples utilized in the formulation of herbal medicines. Keywords : Microscopic, pharmacognostic, standardization, Clerodendrum paniculatum Linn.

INTRODUCTION India is one of the world’s ponderous biodiversity centers that consist of more than 44,000 profuse . This helped the traditional ethnobotanical healers and herbal health practitioners a great resource of natural pharmacy to select the natural herbal ingredients to produce remedies for the treatment, control and management of diverse human diseases[1]. Majority of world’s population hang on herbal medicine to meet the daily demand of health requirements as it has less side effects, nontoxic, easily available and comparatively cheap. As per WHO all around world 80% population of developing countries depend on herbal medicines for primary healthcare [2]. The Clerodendrum of the Family consist of exceedingly 550 species. In various indigenous medicines and folk medicines, many species of ethnomedical significance of the genus are pointed out. In Korean Indian, Chinese, Thai and Japanese systems of medicines the genus is popularly used for the therapy of several ailments such as cancer, malaria, syphilis, thyroid, inflammation, diseases of skin, lung and blood [3]. Clerodendrum paniculatum Linn. of the Family Lamiaceae is an annual or biennial semi woody shrub of 1 to 2 m height. It is called Pagoda plant orange red to scarlet terminal panicle resembles a Japanese Pagoda. It is native of India, Malasia and that is popularly cultivated in tropical gardens of the world [4,5]. The plant is traditionally used in India, China and Japan for the treatment of various afflictions like inflammation, neuralgia, rheumatism, ulcer and wounds. Traditionally it is utilized in Thai medicine as anti-pyretic and anti- inflammmatory[6]. In Andaman and Nicobar islands plant is used traditionally for treating snake bites, wound, jaundice, body ache and giddiness [7]. Adulteration or substitution is an intentional practice done in authentic plant by replacement with another plant material or another foreign material to decrease the cost and/or to increase the weight and potency. Therapeutic efficacy of the crude drugs rely on the quality and quantity of the active chemical constituents. Exploitation of herbal medicines or natural products begins with wrong identification[8]. These problems can be fully resolved by performing the pharmacognostic study of medicinal . It is very important and indispensible to formulate pharmacognostic specifications of crude drugs from natural origin. Pharmacognostic study incorporate parameters which help in detecting adulteration in powdered drug as once the plant is dried and made into crude powder it is deprived of its morphological identity and easily subjected to adulteration. Thus pharmacognostic study assures authentication of the plant and assure reproducible quality, safety and efficacy of herbal products [9]. MATERIALS AND METHODS Collection and authentication of Plant materials The fresh leaves of Clerodendrum paniculatum Linn . were collected from Mallappally, Pathanamthitta District on may 2018. The plant’s identification and authentication was confirmed by the botanist Dr. Jacob Thomas, Herbarium

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Curator, PG and Research Department of Botany, Mar Thoma College, Tiruvalla. Voucher specimens was deposited in the Herbarium of PG and Research Department of Botany (No: MTCHT 1051) on 07/06/2018. Organoleptic and Macroscopic Studies Macroscopic characters of fresh leaves were analyzed according to Brain and Turner[10].Quantitative leaf microscopic studies included determination of surface constants like stomatal index, stomatal number, vein-islet, vein termination number, palisade ratio using fresh leaves with the aid of camera lucida[11].( Table 1) Various diagnostic characters present in the leaves were studied with or without staining. Microscopic Studies Microscopic studies were conducted by taking free hand transverse section of leaves, and staining with safranin which was then mounted in glycerine. Cellular and anatomical studies were performed by using camera lucida. Dried leaves powder was utilized for investigating powder microscopic characters after staining with safranin.[12- 14]. Microphotographs were taken with Trinocular ‘Leica’ microscope attached with ‘Leica DFC 295’ digital camera [15] Microscopical study of leaf using Scanning Electron Microscope A square piece of the sample is placed in the centre of the carbon coated conductive double sided tape fixed on the specimen stub of the equipment Hitachi TM 3000.The specimen height was adjusted and placed inside analysis chamber of the instrument. The chamber was evacuated and sample was observed at different resolutions and photographs were taken using software of the equipment[16]. Physicochemical Analysis Loss on drying, extractive values, ash value, swelling Index, foaming index and leaf constants were determined.[17,18]

RESULTS

Organoleptic and macroscopic evaluation Clerodendrum paniculatum Linn is a semi woody erect shrub that attain a height of 1 to 2 m. Leaves are large, simple and oppositely arranged. Leaves have characteristic odour and characteristic taste. Leaves are cordate in shape consisting of 3 to 7 lobes. Length of the leaf is up to 55cm that has a large lamina up to 30 cm long and a long petiole up to 25 cm. Upper surface is dark green, leathery and glossy. Lower surface is light green and consist of glandular hairs. Leaf margin is sinuate or dentate. Venation is alternate and have 5 to 6 veins on each side of midrib that anastomose near the margin. Apex is acuminate and base symmetric. (figure 1) Leaf Microscopy Transverse section of leaf revealed outermost single layer of compactly arranged barrel shaped epidermis. Outer cell wall was thickened and coated with heavy cuticle. Stomata and trichomes were present in both upper and lower epidermis. The mesophyll tissue was differentiated into single layered compactly arranged radially elongated palisade cells containg chloroplast and spongy parenchyma. Spongy parenchyma contain loosely arranged 6-8 layered parenchymatous cells with intercellular spaces. Midrib region is slightly raised on adaxial side and broadly semicirculay in abaxial side. Midrib region consist of epidermis, collenchymas, spongy parenchyma. Vascular bundle contain lignified xylem vessels and non lignified phloem fibres which are collateral, conjoint and closed. It consist of 12 to 14 vascular bundles arranged as a ring in the midrib covered by endodermis (figure 2) Leaf surface showed mainly anomocytic stomata. It also contain anisocytic and rarely diacytic stomata with bumbbell shaped guard cells. Epidermal cells are wavy. Lower surface consist of more stomata and trichomes than upper epidermis. Epidermis contain three types of trichomes which include non lignified glandular trichomes, non lignified uniseriate multicellular covering trichomes and non lignified wooly branched trichomes. Leaf surface showed numerous veins, vein terminations and vein islets. Vein islets were distict square or rectangular in shape.(figure 3) Petiole Petiole was reniform in cross section with two protuberances at the two sides. Arrangement was similar to that of midrib region. Transverse section revealed outermost single layer of compactly arranged barrel shaped epidermis. Outer cell wall was thickened and coated with heavy cuticle. Anomocytic stomata and the three different trichomes present in the TS of midrib region were present in both upper and lower epidermis. Below the epidermis petiole contain 10-12 layers of collenchymas, loosely arranged spongy parenchyma and vascular bundles arranged as a ring in the centre. Vascular bundles are collateral, conjoint and closed covered by endodermis. Other than the central ring of vascular bundles, one each was present in the two protuberances at the two sides of the petiole. (figure 4)

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Apex Tr Col Margin

Base VB

50 cm Petiole

Col

Figure 1 Morphology of C.paniculatum Linn. leaves Figure 2 T.S of C.paniculatum Linn. leaves through midrib Col-collenchyma,VB-vascular bundle, Tr-trichomes

Ade Tr Pal

Sp Vb

Abe

Figure 3 T.S of C.paniculatum Linn. leaves through lamina Figure 4 T.S of C.paniculatum Linn. leaves through petiole Ade-Adaxial epidermis, Pal-palisade tissue, Tr-trichomes, Vb-vascular bundle Sp-spongy Parenchyma, Abe-Abaxial epidermis

Powder Microscopy The powder microscopic characters were epidermis containg anomocytic stomata. Three types of trichomes were present which include a) wooly branched unicellular trichomes with bulbous base b) lignified uniseriate multicellular dagger shaped covering trichomes (one to eight celled) and c) nonlignified glandular trichomes with large multicellular head and a short single stalk It also consist of loosely arranged parenchyma containing large oil cells, annular xylem tracheids and bordered pitted vessels. Calcium oxalate crystals were present as rhombohedral prisms, and acicular raphides. When stained with potassium iodide, starch grains with star shaped hilum were also seen. (figure 4)

Figure 4 a) branched trichome b)multicellular trichome c)glandular trichome d)stomata and epidermal cells

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e) long fibres f) vessels and tracheids g)epidermal cells of lamina h)starch grains i)calcium oxalate crystals

Microscopical study of leaf using Scanning Electron Microscope Anomocytic stomata, multicellular covering trichomes and epidermal cells with wavy walls were clearly seen (figure 5)

Figure 5 SEM of C.paniculatum Linn. leaves a)upper epidermis with trichome, stomata, epidermal cells b)lower epidermis with trichome ,stomata, epidermal cells

Physicochemical analysis Loss on drying or moisture content was found to be 6.9±0.09%w/w, petroleum ether soluble extractive value was 1.5%w/w, ethanol soluble extractive value was 1.6%w/w, water soluble extractive value was 12%w/w, foreign organic matter was 0.06%w/w, total ash 7.86%w/w, acid insoluble ash 2.98%w/w, water soluble ash 6.45%w/w, swelling index 1.71, foaming index less than 100.

Table 1 Quantitative microscopy of Clerodendrum paniculatum Linn

Variables Adaxial Surface Value Abaxial Surface Value (in 1 mm 2 area) (in 1 mm 2 area) Stomatal number 9 24 Stomatal index 18.3 19.2 Epidermal cells 40 101 Vein islet number 5±3 Vein termination number 16±3 Palisade ratio 9 to 11

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DISCUSSION Though modern sophisticated research tools are available for the evaluation of the plant materials, one of the simplest and cheapest method to establish the correct identity of the source material is the macroscopic and microscopic study[15]. According to WHO the macroscopic and microscopic study of the plant material is the initial step to establish the correct identity and purity of the herbal material and these studies should be performed prior any other tests [16]. Macroscopic qualitative study rely on study of morphologic and organoleptic profile of the drug set out as diagnostic parameters. Organoleptic studies revealed the colour, characteristic odour and taste of the plant. Other morphological characters were also revealed. The microscopic study revealed the presence of anomocytic and anisocytic stomata and three different type of trichomes on the adaxial and abaxial leaf surface. CONCLUSION For Clerodendrum paniculatum Linn, no detailed standardized research work has been reported in the literature. Macoscopic, microscopic and preliminary pharmacognostic evaluation provide relevant information that may be helpful in authentication of plant material and serve as a referential standard for the powdered crude drug. This study helps to provide relevant information to prevent adulteration which is helpful for quality control of crude drugs. It will also aid in differentiating the leaves of Clerodendrum paniculatum Linn. from closely related species of the genus or family. Thus this study will be absolutely useful for the authentication, standardization and development of formulation of crude drug for treatment of various illness.

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