1 Department of Botany, K.M. Centre for Post Graduate Studies, Puducherry, India
2 Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C. Mahe, Puducherry, India
Due to non-availability or short supply of original medicinal plants the crude drugs from the plants are adulterated by similarly resembling plants. Micromorphological studies of leaves could help in the identification and authentication of the original medicinal plant from the crude drug materials.
To investigate the qualitative and quantitative micromorphological parameters of the leaves of Cardiospermum halicacabum.
The foliar micromorphological parameters such as orientation, stomatal types and morphology; density, distribution of trichomes and stomata; stomatal index and trichome types, paradermal sections were obtained manually by the standard method. The leaves were excised and fixed primarily in formalin, acetic acid and ethyl alcohol solution; cleared in 70% ethanol, bleached with 5% NaOH and rinsed in distilled water. The cleared leaves were used for the study of venation pattern, vein-islets, veinlet terminations and arrangement of crystals.
The microscopic examinations revealed the presence of anomocytic and anisocytic types of stomata with the abaxial epidermis of leaves. The stomatal density was 72 and stomatal index reported as 33.1. Glandular trichomes were few with uniseriate stalk, capitate multiseriate and multicellular head. Non-glandular trichomes frequent which were unicellular, uniseriate and bristle in nature. The density of vein islets and veinlet termination was observed as 18 and 13 respectively.
The foliar micromorphological (stomata, vein clearing etc.) findings could be used in the proper identification and authentication of C. halicacabum from the crude plant materials which is essential in quality control measures.
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* Address correspondence to this authors at the Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C. Mahe, Puducherry, India; Tel: +918943157187; E-mail: firstname.lastname@example.org
Cardiospermum halicacabum L. belongs to the family Sapindaceae and is universally recognized as the Balloon vine, Heart pea and Love in a puff. It is a dioecious, woody perennial climber, native to the tropical America, distributed in the tropical and subtropical Africa, Asia and throughout India. C. corumdum, C. glabrum and C. inflatum are the synonyms of Balloon vine [1Murti K, Panchal MA, Lambole V, Gajera V. Pharmacologicl properties of Cardiospermum halicacabum – a review. Pharmacologyonline 2010; 2: 1005-9.]. The plant grows up to 2 to 4 m in length; leaves are deltoid with two highly lobed leaflets. Each leaflet possesses 2-ternate, lanceolate segment with inciso-serrate margins. Stem is long and wiry. Flowers are white, small and hermaphrodite, arranged in axillary, umbellate cymes. Two circinate tendrils situated at the top on a slender peduncles. The fruits are inflated, green and thin-shelled with triangular capsule. The seed coat is black, opaque, smooth and thick with a white finely porous, chordate spot at the micropyle [2Ferrucci MS. Cytotaxonomy of Sapindaceae with special reference to the tribe Paullinieae. Genet Mol Biol 2000; 23(4): 941-6. [http://dx.doi.org/10.1590/S1415-47572000000400039] ].
The chemical profile of C. halicacabum reveals the presence of saponins, alkaloids, glycosides, apigenin, quebrachitol, luteolin, chrysoeriol, flavonoids, proanthocyanidin, stigmasterol, tannins, fatty acids and vital minerals [3Broadley MR, Bowen HC, Cotterill HL, et al. Phylogenetic variation in the shoot mineral concentration of angiosperms. J Exp Bot 2004; 55(396): 321-36. [http://dx.doi.org/10.1093/jxb/erh002] [PMID: 14739259] , 4Deepan V, Alekhya P. Saravanakumar, Dhanaraju MD. Phytochemical and anti-microbial studies on the leaves extracts of Cardiospermum halicacabum Linn. Adv Biol Res 2012; 6(1): 14-8.]. This plant has been exploited enormously for the commercial production of Allergy Relief LiquidTM and Bioforce Pollinosan® Tablets (marketed by Bioforce, USA) in treating hay fever, allergies, sneezing and watery eyes. Florasone Cardiospermum Cream (Boericke and Tafel, USA) is used for skin ailments such as swelling, scaling, blisters/vesicles, burning and pain. It is an active ingredient in lotions for dermatitis, eczema, and psoriasis [5Subramnyam R, Newmaster SG, Paliyath G, Newmaster CB. Ethnobotany 2007; 19: 1-6.]. Traditionally, the plant is reported to be useful in treating stiffness of limbs, chronic bronchitis, snakebites, urinary and stomach disorders [6Wang J, Wang N, Yao X, Kitanaka RI. Inhibitory activity of Chinese herbal medicines toward histamine release from mast cells and nitric oxide production by macrophage-like cell line. J Nat Med 2006; 60: 73-7. [http://dx.doi.org/10.1007/s11418-005-0010-6] ]. The leaves are used to cure malaria, diarrhea, dysentery and haemorrhoids, asthma, pertusis, oedema, nephritis and oliguria, earaches, ophthalmias and muscular pains [7Khare CP. Indian Medicinal Plants: An Illustrated Dictionary. New York: Springer 2007; p. 121.]. The whole plant possesses various biological activities including anthelmintic, anti-blenorrhagic, antidiarrhoeal, antifertility, anti-inflammatory [8Huang MH, Huang SS, Wang BS, et al. Antioxidant and anti-inflammatory properties of Cardiospermum halicacabum and its reference compounds ex vivo and in vivo. J Ethnopharmacol 2011; 133(2): 743-50. [http://dx.doi.org/10.1016/j.jep.2010.11.005] [PMID: 21073940] ], antipyretic, antiparasitic, analgesic, antibacterial, antioxidant, antiulcer, larvicidal, ovicidal [9Govindarajan M. Mosquito larvicidal and ovicidal activity of Cardiospermum halicacabum Linn. (family: Sapindaceae) leaf extract against Culex quinquefasciatus (Say.) and Aedes aegypti (Linn.) (Diptera: Culicidae). J Ethnopharmacol 2009; 124(1): 39-44. [PMID: 19393729] ], antihyperglycemic and nephroprotective properties [10Parameshappa B, Ali Basha MS, Sen S, et al. Acetaminophen-induced nephrotoxicity in rats: protective role of Cardiospermum halicacabum. Pharm Biol 2012; 50(2): 247-53. [http://dx.doi.org/10.3109/13880209.2011.596843] [PMID: 22103691] ].
The natural population of this plant is depleting day-by-day due to the uncontrolled harvest of the plants from the wild by the traditional drug manufactures and pharmaceutical companies. The plant materials are adulterated by the cheaper and similar type of plants due to non-availability of the original plant in the forest areas.
Light microscopy has long been used to study the internal and exomorphic features of vegetative and reproductive characteristics of plants [11Gandhi D, Albert S, Pandya N. Morphological and micromorphological characterization of some legume seeds from Gujarat, India. Environ Exp Biol 2011; 9: 105-13.]. Micromorphological characterization provides information for the investigation of taxonomic/systematic relationship between varieties of species, resolving problems in evolutionary relationships [12Segarra JG, Mateu I. Seed morphology of Linaria species from Eastern Spain: identification of species and taxonomic implications. Bot J Linn Soc 2002; 135: 375-89. [http://dx.doi.org/10.1111/j.1095-8339.2001.tb00788.x] ], and to understand the environmental parameters, which support the survival of species under particular climate etc. Developments in herbal medicine researches require good quality of raw material, standard methodology of drugs formulations and quality control parameters [13Bhagwat GG, Chaulang GM, Ghodke DS, Patil KS, Yeole PG, Ganjiwale RO. Pharmacognostic study of plant Euphorbia hirta L. J. Pharm Res 2008; 1: 39-43.]. Microscopic examination of plant species or powdered materials is easy and cost effective method of identification and authentication [14Chanda S. Importance of pharmacognostic study of medicinal plants: an overview. J Pharmacogn Phytochem 2014; 2: 69-73.].
Therefore, the objective of the present investigation is to establish foliar micromorphological and architectural parameters of traditional medicinal vine C. halicacabum, which could be useful in plant identification and authentication from the crude drugs material, monograph preparation and to understand the adaptation mechanism of plants developed under various environments.
MATERIALS AND METHODS
Collection and Identification of Plant Materials
Healthy vines of C. halicacabum were collected during the months of January to August, 2016 from the coastal areas of Puducherry (India) and identified with the help of Gamble flora [15Gamble JS. Flora of the Presidency of Madras, London (Repr Ed. 1995. Bishen Singh, Mahendra pal singh, dehradun), Vol.1, 1921.]. Plants were randomly selected and the required leaf samples were collected at third to seventh leaves (4-5 leaves per plant) from the base with sterilized scissor. Fresh leaves were used for qualitative as well as quantitative micromorphological and leaf architectural studies. Ten sites were marked and ten plants per site were collected.
Foliar Micromorphological Studies
Experiments were conducted to study the micromorphological features of C. halicacabum leaves under normal environment. The foliar micromorphological parameters such as orientation, stomatal types and morphology; density, distribution of trichomes and stomata, stomatal index and trichomes types, the paradermal sections were obtained manually by standard method [16Johansen DA, Ed. 1, Plant Microtechnique. New York, London: McGraw Hill Book Co. 1940; pp. 182-97.].
Venation Pattern and Evaluation of Vein Density
The leaves were excised and fixed primarily in formalin, acetic acid and ethyl alcohol (FAA in the ratio of 1:1:3, v/v) solution. The fixed leaves were cleared in 70% ethanol (v/v) until chlorophyll was completely removed (12-24 h), bleached with 5% (w/v) NaOH for 24-48 h, rinsed three times in distilled water and allowed to remain in saturated Chloral hydrate solution for 24-48 h [17Sass JE. Elements of Botanical Microtechnique. New York, London: McFraw-Hill Book Co. 1940; p. 222.]. The cleared leaves were used for the study of venation pattern, vein-islets, veinlet terminations and arrangement and morphology crystals.
The materials were stained with 1% (v/v) safranine (Loba chemie, India) aqueous solution for 3-5 min, mounted in water, examined under photomicroscope (Labomed iVu 3100, USA) and analyzed using software Pixelpro. These micrographs with different magnifications were used for identification of micromorphological and leaf architectural parameters. Magnifications of the figures were indicated by the scale-bars. The data for various parameters like stomatal density, frequency and stomatal index were calculated by the methods suggested by Salisbury [18Salisbury EJ. The interrelations of soil climate and organisms and the use of stomatal frequency as an integrating index of relation of the plant. Bech Bot Zbl 1932; 99: 402-20.]. Types of stomata have been described by following the classification and terminology as suggested latest by Prabhakar [19Prabhakar M. Structure, delimitation, nomenclature and classification of stomata. Acta Bot Sin 2004; 46: 242-52.]. The terminology adopted for venation pattern was of Hickey and Wolfe [20Hickey LJ, Wolfe JA. The bases of angiosperm phylogeny. Vegetative morphology. Ann Misso Bot Gard 1975; 62: 538-89. [http://dx.doi.org/10.2307/2395267] ].
The statistical analyses were performed by ANOVA using SPSS version 16 (SPSS Inc., Chicago, USA). The significance of differences among mean values was calculated by Duncan’s multiple range tests at P < 0.05 and results were presented as mean ± standard deviation (SD) of three experiments.
RESULTS AND DISCUSSION
The study of micromorphological and architectural parameters of C. halicacabum leaves using light microscopy revealed a number of important features, which were intrinsic to its habitat. The leaves were ovate, biternate with dentate margins. The leaves have prominent, thick crescent midrib towards abaxial epidermis, which was less prominent and conical on the adaxial epidermis.
The epidermal cells were compactly arranged and interrupted by stomatal apparatus. Almost all the cells were amoeboid in shape and visibly larger than the stomatal guard cells. The paradermal sections of the abaxial epidermis illustrate the presence of large, distinct and undulated regular anticlinal walls. In addition to these, glandular and non-glandular trichomes were also observed.
The guard cells of the stomata were equal and interpreted with some intercellular spaces. Stomata positioned at the same level of epidermal layers. Subsidiaries were not distinguished except in developing stomata. Stomata were observed on both the epidermal surfaces (amphistomatic leaves), but abaxial epidermis presented higher stomatal density than the adaxial epidermis. Predominant anomocytic stomata with indistinct subsidiaries and anisocytic stomata were observed on the abaxial surface (Fig. 1A). Stomata were distributed randomly, facing all directions over the epidermis on the intercoastals regions and lying close to each other (Fig. 1B). Stomatal density and index of the abaxial epidermis were 72 and 33.1 respectively (Table 1).
Paradermal section of abaxial epidermis showing epidermal cells and stomatal density (CC – Costal Cells).
Magnified view of stomatal apparatus on abaxial epidermis.
Table 1 Stomatal density and stomatal index of abaxial surface of leaves of C. halicacabum.
Abaxial surface of the midrib is triangular in shape and made up of thick walled, angular cells with a prominent vascular strand. The values of stomatal frequency and index are directly proportional to the rate of transpiration [21Xu Z, Zhou G. Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. J Exp Bot 2008; 59(12): 3317-25. [http://dx.doi.org/10.1093/jxb/ern185] [PMID: 18648104] ]. Orientation of stomata on epidermis and their spacing are the indicative parameters of the nature of physiological plasticity of the guard cells. The leaves are liable to change towards climatic changes with great variations within the species and same individuals to some extent. Foliar micromorphological studies could be used as a constructive key in taxonomy between the families [22Zahra NB, Ahmad M, Shinwari ZK, Zafar M, Sultana S. Systematic significance of anatomical characterization in some Euphorbiaceous species. Pak J Bot 2014; 46: 1653-61.]. Stem surface possesses flattened epidermal cells with anomocytic stomata, and glandular and non glandular covering trichomes in this plant species as reported by Patil [23Patil AG, Joshi KA, Patil DA, Chandra N. Pharmacognostical standardization and HPTLC fingerprint of Cardiospermum halicacabum L. stem. Res J Pharm Biol Chem Sci 2011; 2: 343-52.].
Two types of foliar trichomes were observed in C. halicacabum, glandular capitate trichome and bristle like non-glandular hairy trichomes. Glandular trichomes possessed multicellular head (4 cells arranged in two rows and two columns), one celled stalk (subsessile), uniseriate and covered by a thick wall (Fig. 1C). The non glandular hairy trichomes were unicellular with fine curved tip and broad base (Figs. 1E and 1F). The density of non glandular trichomes was more than capitate glandular trichomes (data not shown). Zalke et al. [24Zalke AS, Duraiswamy B, Gandagule UB, Singh N. Pharmacognostical evaluation of Cardiospermum halicacabum Linn. leaf and stem. Anc Sci Life 2013; 33(1): 15-21. [http://dx.doi.org/10.4103/0257-7941.134561] [PMID: 25161325] ] measured the size of the trichomes (glandular trichomes 50 × 80 μm with dense cytoplasm, non-glandular trichomes 15 × 50 μm). Mucilagenous cells were also observed with both the epidermal cells (Fig. 1D). Distribution of trichomes and their types are considered as the structural defense of plants and these are prejudiced by various external factors like, to reduce herbivore attack, tolerance to wind and frost, and to check water loss due to transpiration [25Hanley ME, Lamont BB, Fairbanks MM, Rafferty CM. Plant structural traits and their role in anti-herbivore defence. Perspect Plant Ecol Evol Syst 2007; 8: 157-78. [http://dx.doi.org/10.1016/j.ppees.2007.01.001] ]. Glandular trichomes are known to secrete certain exudates such as resin and mucilage and prone to reduce the attack of herbivores [26Konno K, Nakamura M, Tateishi K, et al. Various ingredients in plant latex: Their crucial roles in plant defence against herbivorous insects. Plant Cell Physiol 2006; 47(Suppl. S): S48.].
Glandular capitate trichome (GTC) on abaxial epidermis.
Mucilaginous cell (MC) on the abaxial surface.
Non-glandular trichome (NGT) on abaxial epidermis.
Glandular, Non-glandular trichomes and crystals on major vein (GTC– Glandular Capitate Trichome, NGT – Non-glandular Trichome, CR – Crystals).
Venation Pattern and Vein Density
Vein clearing study showed the presence of thick primary veins and the noticeable uniform lateral veins arose from the major vein. The major and minor veins were straight and exhibited clear reticulate venation pattern in C. halicacabum. The lateral veins ran towards the margins, joined with one another and encircled some discrete space. Vein-islets were distinct, mostly polygonal and rectangular in shape (Fig. 2A). Each vein islet presented with simple, straight veinlet terminations (vein endings), mostly unbranched but rarely branched dichotomously. The density of vein islets and veinlet termination observed as 18 and 13 respectively (Table 2). The developments of vein density in a species are indicative of proper transport of minerals and mechanical reinforcement of leaves which results in the formation of characteristic venation pattern [27Evans LT. Transport and distribution in plants Transport and transfer processes in plants. New York: Academic Press 1976; pp. 1-13. [http://dx.doi.org/10.1016/B978-0-12-734850-6.50006-X] ]. Growth and increased vein system of species also implies the fitness of the plants towards environmental factors [28Hastings HM, Sugihara G. Fractals, A user's guide for the natural sciences. Oxford Univ. Press 1993.]
Venation pattern and vein density through cleared leaf and distribution of crystals over the veins (VI - Vein Islet, VT - branched Veinlet Termination).
Table 2 Leaf architectural parameters in C. halicacabum.
Crystals in C. Halicacabum Leaves
Oxalates of calcium (sand crystals) were observed all along the primary and secondary veins, arranged solitary as well as clustered on mesophylls (Fig. 2B). Presence of crystals and their type in a species is determined genetically. The quantitative parameters of crystals/crystal density are influenced by several environmental factors such as intensity of light, existence of herbivory and concentration of calcium in the soil [29Kuo-Huang LL, Ku MS, Franceschi VR. Correlations between calcium oxalate crystals and photosynthetic activities in palisade cells of shade-adapted Peperomia glabella. Bot Stud (Taipei, Taiwan) 2007; 48(2): 155-64.].
Arrangement of calcium oxlate sand crystals over major and minor veins (CR – Crystals).
The findings of the present study revealed the peculiar foliar micromorphological features of C. halicacabum collected from ten sites of Coromandel coastal areas of Puducherry, India. It could be useful for systematics as well as diagnostic studies of this plant in future. Besides authentication and identification, micromorphological evaluation of leaves could serve as a source of information to ascertain the environmental influence and plant’s physiological performances. The present study may open new platform to analyze the response of leaf epidermal micro-parameters under various climatic conditions.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.
Authors are grateful to the University Grants Commission, New Delhi, Government of India and Department of Science, Technology and Environment, Government of Puducherry for providing financial support to our laboratory as Major Research Project and Grant-In-Aid Scheme respectively.
Murti K, Panchal MA, Lambole V, Gajera V. Pharmacologicl properties of Cardiospermum halicacabum – a review. Pharmacologyonline 2010; 2: 1005-9.
Wang J, Wang N, Yao X, Kitanaka RI. Inhibitory activity of Chinese herbal medicines toward histamine release from mast cells and nitric oxide production by macrophage-like cell line. J Nat Med 2006; 60: 73-7. [http://dx.doi.org/10.1007/s11418-005-0010-6]
Khare CP. Indian Medicinal Plants: An Illustrated Dictionary. New York: Springer 2007; p. 121.
Huang MH, Huang SS, Wang BS, et al. Antioxidant and anti-inflammatory properties of Cardiospermum halicacabum and its reference compounds ex vivo and in vivo. J Ethnopharmacol 2011; 133(2): 743-50. [http://dx.doi.org/10.1016/j.jep.2010.11.005] [PMID: 21073940]
Govindarajan M. Mosquito larvicidal and ovicidal activity of Cardiospermum halicacabum Linn. (family: Sapindaceae) leaf extract against Culex quinquefasciatus (Say.) and Aedes aegypti (Linn.) (Diptera: Culicidae). J Ethnopharmacol 2009; 124(1): 39-44. [PMID: 19393729]