COMPARATIVE (QUANTITATIVE AND QUALITATIVE) STUDIES OF STOMATA OF SELECTED SIX MEDICINALLY VIABLE SPECIES OF CASSIA L.

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1 Int. J. LifeSc. Bt & Pharm. Res Amal Kumar Mondal and Sayantan Tripathi, 2012 Research Paper ISSN Vol. 1, No. 3, July IJLBPR. All Rights Reserved COMPARATIVE (QUANTITATIVE AND QUALITATIVE) STUDIES OF STOMATA OF SELECTED SIX MEDICINALLY VIABLE SPECIES OF CASSIA L. Sayantan Tripathi 1 and Amal Kumar Mondal 1* *Corresponding Author: Amal Kumar Mondal, amalcaebotvu@gmail.com The Stomatal diversity (size, shapes, types and orientation) in the foliar epidermis has great value in plant systematics studies. The present paper deals with comparative study of stomatal structure of six species of Cassia which are used by rural people of South West Bengal as medicine. Leaf epidermal studies mainly stomatal studies of the 6 species of Cassia of family Caesalpiniaceae viz., Cassia alata L., Cassia fistula L., Cassia occidentals L., Cassia siamea Lamk., Cassia sophera L., Cassia tora L. were made. Leaf clearings and cuticular preparations were examined with light microscopy. The study is based on the presence and absence of stomata, types of stomata present in the epidermal surface, stomatal count/cm 2, stomatal index and epidermal cell shape. The size and shapes of stomata are also varied in the tree species which bear larger size of stomata in respect of other habits and habitats. Three types of stomata were observed viz. paracytic, anisocytic and anomocytic. Among these 3 types of stomata the paracytic type of stomata are more common and than other. Keywords: Epidermis, Medicinal use, Cassia, Paracytic stomata INTRODUCTION Various plants and its parts are used in medicinal purpose from ancient age. Now a days it is observed that the African gorillas also use various plants and its parts to cure their health. The medicinal plants have been used by Hakims and in folklore medicines as 80% of the population lives in rural areas that mostly depend on Unani system of medicines (Soomro et al., 1997). The available literature shows that leaf epidermal features are important in systematic botany similar to the use of modern techniques and chemical composition (Edeoga and Ikem, 2001; and Mbagwu and Edeoga, 2006). Epidermal 1 Plant Taxonomy, Biosystematics and Molecular Taxonomy Laboratory, Department of Botany & Forestry, Vidyasagar University. 104

2 structures and stomatal ontogeny of some Nigerian ferns have been found relevant in their recognition (Gill and Karatela, 1985). Many workers such as Edeoga (1991), Edeoga and Osawe (1996), Mbagwu and Edeoga (2006), Nwachukwu and Mbagwu (2006) stressed that epidermal and cuticular traits of plants epidermal cells, type and arrangement of stomata, size and shape of trichomes and number of vascular bundles could serve as vital tools in solving taxonomic problems in Angiosperms. Stomatogenesis has long been studied by morphologists, physiologists and taxonomist.the morphology and ontogenies of taxa are important in intrageneric systematics. Diversity in stomata types, even on the same surface of an organ, indicates the weakness in using stomata as a taxonomic character. (Pant and Kidwai, 1964). In spite of diversity, the most frequent stomata type can be used as a taxonomic character (Gopal, 1970). Caesalpiniaceae is a family of dicotyledonous plant with about 160 genera and some 2,000 species. The genus Cassia is one of the genuses of Caesalpiniaceae having about 692 species. Some of the Cassia species is used as medicine in Eastern India mainly South West Bengal. We select Cassia alata L., Cassia fistula L., Cassia occidentals L., Cassia siamea Lamk., Cassia sophera L., Cassia tora L. which are frequently used by the rural and tribal people of south West Bengal. Apart from physiognomic characters, anatomical properties of plant parts are sources for taxonomic inferences in different groups of flowering plants (Edeoga et al., 2007; Guimeraes et al., 2007; Kaplan et al., 2007; Keshavarzi and Zare, 2006). Despite the immense economic importance of the legumes and the physiological importance of the stomatal apertures, reports on the frequency and the structure of the stomata are lacking or incomplete for many species. On the basis of arrangement of epidermal all neighbouring the guard all, more than 25 main types of stomata in dicots have been recognized (Metcalfe and Chalk, 1979). Stace (1980) reported 31 different types of stomata among cotyledonous plants. But the present study is based on the paper of Metcalfe and Chalk (1950) who described the 4 types i.e. Anisocytic, Anomocytic, Diacytic and Paracytic. The aim of present study is to use stomatal characters as aid in taxonomy of these selected medicinally useful Cassia species. The study would help in the identification and authentication of these medicinal plants on the basis of stomatogenesis. MATERIAL AND METHODS Plant Material: The medicinal plant specimens Cassia alata L., Cassia fistula L., Cassia occidentals L., Cassia siamea Lamk., Cassia sophera L. and Cassia tora L. belonging the family Caesalpiniaccae under order Leguminales were taken for stomatal study. Cassia alata L. is popular in traditional treatment of asthma, leprosy and it is used for controlling of ringworm (Paria, 2005). Role of Cassia fistula L. is used as purgative and also used to treat skin disease. Its leaves are used to treat skin disease and ulcer. Powder of Cassia fistula L. root is given to cure menstrual 105

3 disorder and its pulp of seed is used in high blood pressure (Paria, 2005). Cassia ossidentales L. is popularly used as purgative. It is also used to treat hysteria, whooping cough and urinary troubles (Paria, 2005). Cassia sophera L. is an annual erect shrub of important medicinal properties. The whole plant extracts and leaves have expectorant properties, cures cough, asthma and acute bronchitis. They are specific to eliminate ring worms and also useful in the treatment of gonorrhoea and syphilis. The bark is used in the treatment of diabetes; the roots in elephantiasis, wounds. (Kirtikar and Basu,1935). The leaves of Cassia siamea Lamk. are used in the treatment of diabetes, disturbances in the bodily functions, lymph node swelling, urine stones, general deficiency conditions, beri beri, classic deficiency of avitaminose by lack of vitamin B1 (thiamine) in gastrointestinal disorders - malabsorption - meals taken with polished rice etc., antihypertensive, insomnia (sleeplessness), against dysentery and disorders of the large intestine. (Kirtikar and Basu, 1935). Cassia tora L. is popularly known for the treatment of dysentery and eye disease. Leaves are also used in boils, ring worm, leprosy, bronchitis and liver complaints. Pest of fresh pods is applied to cure lentigo. Seed powder is taken to cure intestinal problems caused by intestinal worms. Seed pest mixed with boiled rice water is applied in case of one sided headache (Paria, 2005). The species identification of the selected material was determined according to standard literature. It was done in the months from February to April. The foliar epidermal peals were taken from the middle of both surfaces of mature leaves. Isolation of Epidermis: Epidermis of leaf is isolated from both fresh and dry plant specimen. The mature leaves were fixed in FAA solution (acetic acid: alcohol: formalin: water = 2:5:1:12) for 24 hours and washed in 70% ethanol. Three circular disk samples were cut from an area adjacent to the midrib of each leaf. Disk sample was boiled in 5% aqueous solution of KOH for 5-10 minute. Epidermal peals were stripped and stained with 1% in 50% aqueous ethanol, saffranin and temporary mount in glycerine. Stomatal frequency counts made and camera lucida were drawn. The numbers of stomata were counted in each field ( cm 2 ). The stomatal frequency was based on average obtained from observations of 3 microscopic fields. Stomatal index (I) was calculated by the following formula using the no. of stomata(s) and epidermal cells was present in a unit area [I=S/(S+E)]. After preparing, slides were observed under light microscope [(40x)Leica DM 1000] and phase contrast microscope for detailed analysis and obtaining better picture as well as measuring the length and breadth of stomata including guard cells. RESULTS AND DISCUSSION The results in this investigation were summarised in tables including stomata count, stomata type, stomatal index, stomatal complex. The entire selected species share a number of common characters, i.e., shape of epidermal cell, presence of stomata, stomatal index and stomatal complex which are useful in distinguishing these species. Epidermal Cell: The shape of foliar epidermis is one of the significant taxonomic characters. 106

4 Taxonomic studies of a number of families are based on leaf epidermis anatomy (Bhatia, 1984, Jones, 1986). In this investigation it was found that the epidermal cell shape varies slightly with growth habit. The shrub species posses pentagonal to polygonal shaped epidermal cells. The epidermal cell shapes of tree species are variously shaped or undulating to irregular (Table 1). In these six species only Cassia siamea Lamk. (Figure 2E, F) bears hypostomatic leaves i.e. stomata present only at the abaxial surface of leaves. Here it was found that the shape of epidermal cells varies in their surfaces. The adaxial surface of the leaf shows tetragonal to polygonal shaped epidermal cells but the abaxial surface i.e. stomata bearing surface shows undulating to irregular shaped epidermal cells. Stomata: Three different types of stomata anomocytic, anisocytic, paracytic were observed in these selected species. It was found that abundant stomata were present on abaxial surface as compared to adaxial surface. All these selected species bear paracytic stomata except the adaxial surface of Cassia alata L. (Figure 2A). The adaxial surface of Cassia alata L. (Figure 2A) bears anisocytic stomata. Anisocytic and paracytic stomata were also found in the abaxial surface of Cassia occidentals L. (Figure 1B), Cassia sophera L. (Figure 1F) and Cassia tora L. (Figure 1D). From this investigation it was found that all the anisocytic stomata bearing species are shrub in growth habit. Anomocytic stomata are found only in the abaxial surface of Cassia siamea Lamk. (Figure 2F). All the investigated species showed paracytic stomata, a mixture of stomata in same species were also observed. Cassia ocidentales L., Cassia sophera L. and Cassia tora L. are closely related. They showed paracytic stomata in adaxial surface and in the abaxial surface they showed paracytic stomata along with anisocytic type (Table 1). Table 1: Qualitative Foliar Stomatal Characteristics of the Selected Species of Cassia Stomata Presence Stomata Type S. No. Taxa Habit Surface Leaf Epidermal Cell Shape of Anomo- Aniso- Para- Dia- Stomata cytic cytic cytic cytic 1. Cassia alata L. Shrub Ad. Pentagonal to hexagonal + + Ab. Pentagonal to hexagonal Cassia fistula L. Tree Ad. Variously shaped + + Ab. Variously shaped Cassia occidentals L. Shrub Ad. Pentagonal to polygonal + + Ab. Pentagonal to polygonal Cassia siamea Lamk. Tree Ad. Tetragonal to polygonal Ab. Undulating to irregular Cassia sophera L. Shrub Ad Pentagonal to polygonal + + Ab Undulating to irregular Cassia tora L. Shrub Ad. Pentagonal to polygonal + + Ab. Pentagonal to polygonal

5 Table 2: Quantitative Foliar Stomatal Characteristics of the Selected Species of Cassia S. No. Taxa Habit Surface Stomatal Complex. Legth Width (with guard cell) µm 2 1. Cassia alata L. Shrub Ad 23.89( ) 20.27( ) Ab 18.48( ) 12.36( ) 2. Cassia fistula L. Tree Ad 13.74( ) 11.66( ) Ab 15.22( ) 10.25( ) 3. Cassia occidentals L Shrub Ad 23.11( ) 16.49( ) Ab 22.98( ) 17.09( ) 4. Cassia siamea Lamk. Tree Ad Ab 23.49( ) 15.32( ) 5. Cassia sophera L. Shrub Ad 24.85( ) 15.24( ) Ab 22.54( ) 15.29( ) 6. Cassia tora L. Shrub Ad 22.10( ) 13.06( ) Ab 23.28( ) 15.66( ) Table 3: Stomatal Index Percentage and Stomatal Count/cm 2 of the Selected Species of Cassia S. No. Taxa Habit Surface of Leaflet Stomatal Index (SI) Percentage Stomata Count/cm 2 1. Cassia alata L. Shrub Ad Ab Cassia fistula L. Tree Ad Ab Cassia occidentals L Shrub Ad Ab Cassia siamea Lamk. Tree Ad Ab Cassia sophera L. Shrub Ad Ab Cassia tora L. Shrub Ad Ab

6 Figure 1: Leaf Epidermis Showing Stomata of A. Cassia occidentalis (adaxial surface); B. Cassia occidentalis (abaxial surface); C. Cassia tora (adaxial surface); D. Cassia tora (abaxial surface); E. Cassia sophera (adaxial surface); and F. Cassia sophera (abaxial surface) 109

7 Int. J. LifeSc. Bt & Pharm. Res Amal Kumar Mondal and Sayantan Tripathi, 2012 Figure 2: Leaf Epidermis Showing Stomata of A. Cassia alata (adaxial surface); B. Cassia alata (abaxial surface); C. Cassia fistula (adaxial surface); D. Cassia fistula (abaxial surface); E. Cassia siamea (adaxial surface); and F. Cassia siamea (abaxial surface) 110

8 Quantitative analysis of stomatal complex in the selected species shows great variation (Table 2). Here it was found that except Cassia fistula L. Cassia alata L., Cassia occidentals L., Cassia siamea Lamk., Cassia sophera L., Cassia tora L. share almost equal size of stomata which shows close relationship. In these 5 species Cassia sophera L. and Cassia occidentals L. share almost equal size of stomata and the ratio between size of stomata of adaxial and abaxial surface is almost equal. Smallest stomata were found in the adaxial surface of Cassia fistula L. ( µm 2 ). Stomatal index is one of the useful tools in order to distinguish species. It was found that stomatal index has low value on adaxial surface as compared to abaxial surface (Table 3). According to stomatal index Casia alata L. and Cassia fistula L. are closely related. Cassia occidentals L., Cassia siamea Lamk., Cassia sophera L. and Cassia tora L. showed relationship among them. Highest value of stomatal index was observed on abaxial surface of Cassia occidentals L. (28.7), where lowest value was observed on the adaxial surface of Cassia alata L. (8.3). Stomatal count is a significant character to differentiate various species. Highest stomata count per unit area was found on the abaxial surface of Cassia sophera L. (194245/cm 2 ). Lowest stomata count was found in adaxial surface of Cassia alata L. (6475/cm 2 ). All the investigated Cassia species share more or less equal number of stomata which shows they are closely related. CONCLUSION The study and investigation mainly focused on comparative study of quantitative and qualitative stomatal characters of 6 species of Cassia belonging to family Caesalpniaceae under the order Leguminales, used in medicinal purpose in the South West Bengal region. From the study it can be concluded that there is no relationship between the stomata size and growth habit. Size of stomata and epidermal cell shape prove that all the investigated species are closely related. All the investigated species showed paracytic stomata, a mixture of stomata in same species were also observed. Cassia ocidentales L., Cassia sophera L. and Cassia tora L. are closely related. They showed paracytic stomata in adaxial surface. In the abaxial surface they showed both anisocytic and paracytic type of stomata. Shape of epidermal cells, presence of stomata, stomatal type, stomatal complex, stomatal index were investigated. These parameters are helpful to differentiate the species. However, the stomatal features may prove to be a little taxonomic value unless the developments of different stomata types were studied. A greater number of information on taxa will be helpful to understand the taxonomic value of stomata type and distribution. ACKNOWLEDGMENT We are grateful to our honourable Vice Chancellor of Vidyasagar University, Prof. Ranjan Chakraborti, and we are also thankful to our friends (Santanu Dash and Dulal Chandra Bera). Without their help we cannot complete our work. REFERENCES 1. Ahmad K, Khan M A, Ahamad M, Shaheen N and Nazir A (2010), Taxonomic Diversity in Epidermal cells of some Sub-Tropical Plant Species, International journal 111

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Issued 01 November Abstract

Issued 01 November Abstract Ethnobotanical Leaflets 13:1417-25. 2009. Morphological and Anatomical Studies of the Leaf and Stem of Some Medicinal Plants: Stachytarpheta jamaicensis (L.) Vahl. and S. cayennensis (L.C.Rich) Schau.