Comparative anatomical features of cineraria (L.) Druce and (Sw.)

r ticle DC (Mimosaceae) A Stellaa Robertson, N. Narayanan1, N. Deattu1, N. R. Ravi Nargis2 Department of Pharmacognosy, Maharaji College of Pharmacy, Besant Nagar, 1Department of Pharmaceutical Sciences, College of Pharmacy, Madras Medical College, Chennai; 2Department of Ayurvedic Medicine, Sri Sai Ram College of Ayurvedic Medicine, Tamil Nadu, India

A comparative study of the leaflets of two domestic species ofProsopis is reported. Both the species, Prosopis cineraria and Prosopis juliflora, have been reported to possess antibacterial, antifungal, anti-inflammatory and the most concerning anticancer properties.

r igin a l In view of its medicinal importance and taxonomic confusion, the individual morphological and histological characteristics of these two species have been described through certain parameters such as structural profile of the leaflets, stomatal morphology, venation

O pattern, petiolule and vascular system of the rachis. Evaluation of the fresh and anatomical sections of the leaves of both species was carried out to determine its macro- and microscopical (histological) characters. The studies indicated the presence of bipinnately compound leaf, an entire margin, apiculate apex, obtuse base, reticulate venation, thick and straight anticlinal walled epidermal cells, prismatic type of calcium oxalate crystals in the mesophyll tissue, dense deposition of tannin content and paracytic type stomata in the P. cineraria, whereas P. juliflora has a bipinnately compound leaf with an entire margin, blunt apex, round base, reticulate venation, thick and straight walled epidermal cells, large mucilage cavities in the mesophyll tissue and paracytic type stomata. The above findings provided referential information for identification of the species P. cineraria and P. juliflora.

Key words: Prosopis cineraria, Prosopis juliflora, sacred tree, Vanni

INTRODUCTION P. juliflora is wildly distributed and is a perennial deciduous thorny shrub or small tree [Figure 2]. It is Both the species Prosopis cineraria and Prosopis juliflora an exotic species and found in most of arid and semi- [6] belong to the family Leguminosae and subfamily arid parts of India. The products obtained from P. Mimosaceae. P. cineraria Linn. (syn. Prosopis spicigera juliflora have been used for human consumption in Linn. and cineraria Linn.)[1] is a large tree, up to 10 bread, biscuits, sweets, syrup and liquors. Extracts m in height, with branches prickly, prickles curved and of P. juliflora seeds and leaves have several in vitro compressed [Figure 1]. The species is found throughout pharmacological effects such as antibacterial, antifungal and anti-inflammatory properties. These properties India extending to Persia. It is known as Vanni or jambu have been attributed to piperidine alkaloids. A number in Tamil, Jand or Khejra in Hindi and Shami in Sanskrit.[2] of compounds have also been reported from this , It is called Kalpa Plant in Ayurveda and Siddha literature. the most common of these being steroids, tannins, The whole plant is used in the Indigenous System of leucoanthocyanidin and ellagic acid glycosides. A new Medicine as a folk remedy for various ailments like monocyclic diketone, prosopidione, and two alkaloids, leprosy, dysentery, bronchitis, asthma, leucoderma, namely, juliprosinene and juliflorinine, have been piles, muscular tremor and wandering of the mind. It isolated from the leaves.[7-9] is also known to possess anthelmintic, antibacterial, antifungal, antiviral and anticancer activities. Leaf paste The bipinnate compound leaves of P. cineraria exhibit of P. cineraria is applied on boils and blisters, including close resemblance with the leaves of P. juliflora. So, there mouth ulcers in livestock and leaf infusion on open sores is always a possibility of confusion and adulteration of on the skin.[3-5] The smoke of the leaves is considered to the leaves of both the species. Therefore, the present be a good remedy for ailments of the eye. The chemical study aims at developing microscopical standards to constituents isolated include spicigerine from the whole distinguish the leaves of P. cineraria and P. juliflora. plant; and steroids, namely, campesterol, cholesterol, MATERIALS AND METHODS sitosterol, stigmasterol; alcohols, namely, octacosanol and triacontan-1-ol; and alkane, hentriacontane, all Plant Material

www.greenpharmacy.info isolated from the leaves. The plant specimens of P. cineraria were collected

Address for correspondence: Mrs. Stellaa Robertson, Department of Pharmacognosy, Maharaji College of Pharmacy, Besant Nagar, Chennai – 600 090, Tamil Nadu, India. E-mail: [email protected] Received: 29-05-2010; Accepted: 27-08-2010; DOI: 10.4103/0973-8258.74138

275 International Journal of Green Pharmacy | October-December 2010 | Robertson, et al.: Comparative anatomical features of P. cineraria and P. juliflora from the forest of Thiruvallur district, Tamil Nadu, India, measuring 50–70 μm in height, and spongy mesophyll tissue whereas those of P. juliflora were collected locally from the has six or seven layers of loosely arranged parenchyma roadside vegetation. The specimens were collected from the cells [Figure 4a]. upper portion of the tree facing east side during morning time between 7.00 a.m. and 11.00 a.m., then identified and In P. juliflora, the leaflet is dorsiventral and 2.7 mm wide. authenticated by Plant Anatomy Research Centre, West The lamina of the leaflet is 210 μm thick along the midrib Tambaram, Chennai. Voucher specimen (No: A-43/PARC and 170 μm thick along the lamina. The mesophyll has a for P. cineraria and No: A-44/PARC for P. juliflora) has been palisade cell layer measuring 70 μm in height and spongy deposited in the same Institution. mesophyll tissue has three or four layers of loosely arranged parenchyma cells [Figure 4b]. First, the morphological characteristics of leaves of P. cineraria and P. juliflora were studied. Then, the required T. S. of midrib samples of leaves were cut and removed from the plant In P. cineraria, the midrib of the leaflet is biconvex in and fixed in FAA (Formalin, 5ml + Acetic acid, 5ml+ 70% transectional view and 300 μm thick in vertical plane. Ethanol, 90ml) for 24 hours. After fixing, the specimens The vascular bundle is collateral with a conical mass of were dehydrated and the paraffin-embedded specimens thick-walled, angular xylem elements and a thin arc of were sectioned using rotary microtome (10 µm thick);[10] the phloem elements; bundle size is 130 × 160 μm. A thick arc sections were stained with Toluidine blue O. of gelatinous sclerenchymatous cells occurs on both upper and lower sides of the bundle [Figure 5a]. The histological profile, namely, T.S. of lamina, T.S. of midrib and cellular content, of both the P. cineraria In P. juliflora, the midrib of the leaflet is flat and not and P. juliflora were studied. Then, T.S. of petiolule, T.S. projecting above the leaf surface. The vascular system is of secondary rachis and T.S. of primary rachis were also single-stranded and collateral. The xylem is thick and broad. studied. Photomicrographs were prepared with Nikon-lab The phloem is thin and less prominent. It has very thick phot 2. sclerenchyma bundle sheath [Figure 5b].

RESULTS Cellular contents In P. cineraria, calcium oxalate crystals of prismatic type are Morphological Profile of P. cineraria and P. juliflora common in the mesophyll tissue and in the bundle sheath P. cineraria sclerenchyma [Figure 6a]. It has bipinnately compound leaves, alternate in arrangement. The leaflets are 15–18 pairs, and shaped In P. juliflora, large mucilage cavities are very common in oblong with an entire margin, apiculate apex, obtuse base, the mesophyll tissue [Figure 6b]. glabrous surface, reticulate venation, petiolate, and the petiole is 0.5–4 cm long. The average leaf size is 2.5 cm Stomatal morphology (length) and 1 cm (breadth). Fresh leaves are green in colour, In P. cineraria, the stomata are predominantly paracytic and are odourless with a bitter taste [Figure 3a]. type with two subsidiary cells. The epidermal cells are polyhedral with thick, straight anticlinal walls [Figure 7a]. P. juliflora It has bipinnately compound leaves, alternate in arrangement. In P. juliflora, the epidermal cells are small and polyhedral. The leaflets are 15–18 pairs, and shape is oblong with an The epidermal walls are thick and straight. The stomata entire margin, blunt apex, round base, glabrous surface, are paracytic type with two unequal parallel subsidiary reticulate venation, petiolate, and the petiole is 2.5–3 cm cells [Figure 7b]. long. The average leaf size is 1.8 cm (length) and 0.3 cm (breadth). Fresh leaves are green in colour, and are odourless Venation pattern with a less palatable taste [Figure 3b]. P. cineraria has reticulate venation system with distinct vein islets and short, thick vein terminations [Figure 8a]. Histological Profile of P. cineraria and P. juliflora T.S. of lamina P. juliflora has reticulate venation system with distinct vein In P. cineraria, the lamina of the leaflet is 150μ m thick, and islets and short, thick vein terminations [Figure 8b]. dorsiventral. The adaxial and abaxial epidermal layers are 15–20 μm thick with semicircular or dome-shaped cells. T. S. of petiolule The cuticle is prominant. Both the epidermal layers are In P. cineraria, the petiolule is planoconvex in transectional stomatiferous. The mesophyll has a palisade cell layer view with flat adaxial side and short lateral wings. The

| October-December 2010 | International Journal of Green Pharmacy 276 Robertson, et al.: Comparative anatomical features of P. cineraria and P. juliflora epidermal layers are large and thick with compact, T.S. of primary rachis tanniniferous ground tissue. The vascular strand is In P. cineraria, the primary rachis is circular in cross-sectional horizontally flat with broken masses of xylem and phloem, view with shallow adaxial depression. Its vertical height ensheathed by thin sclerenchyma layers [Figure 9a]. is 800 μm and horizontal width is 650 μm. The epidermal layer is distinct with papillate cells and prominant cuticle. In P. juliflora, the petiolule is shield-shaped in transectional The ground tissue is rectangular and squarish with densely view with a prominent adaxial cone and thick, short lateral tannin filled regular mass of parenchyma cells. The vascular wings. The vascular system consists of adaxial and abaxial system consists of a wide, circular hollow cylinder of main main bundles and small circular adaxial cone bundles which bundle measuring about 600 μm in diameter. The xylem get surrounded by thick sclerenchyma sheath [Figure 9b]. elements of the main cylinder are wide, circular, fairly thick-walled, circular, angular or elliptical and embedded T. S. of secondary rachis in the sclerenchymatous ground tissues. The phloem occurs In P. cineraria, the secondary rachis is vertically oblong with in thick, continuous sheath around xylem.. The vascular short, less prominant ridges. It is 730 μm in vertical plane bundle gets ensheathed by sclerenchymatous cylinder. Two and 550 μm in horizontal plane. The epidermal layers are circular, prominent adaxial accessory strands are placed prominant and undulate with papillate epidermal cells. within the adaxial ridges. The accessory strands with a The ground tissue is four or five layered, narrow and central mass of xylem get surrounded by a thin layer of densely tanniniferous. The vascular system consists of a phloem and thick sclerenchyma sheath [Figure 11a]. wide, bowl-shaped abaxial plate, and a short flat adaxial plate and three circular adaxial strands placed in the upper In P. juliflora, the primary rachis is circular in cross-sectional portion [Figure 10a]. view with bowl-shaped main bundle and three adaxial bundles which form a circle [Figure 11b]. In P. juliflora, the secondary rachis is adaxially flat with two prominent adaxial bundles and one closed wide cylinder of DISCUSSION main bundle. The sclerenchyma sheath is thick and found all around the vascular bundles [Figure 10b]. As per World Health Organization (WHO) norms, an

Figure 1: Plant of P. cineraria Figure 2: Plant of P. juliflora

Figure 3a: Leaflet ofP. cineraria Figure 3b: Leaflet ofP. juliflora

277 International Journal of Green Pharmacy | October-December 2010 | Robertson, et al.: Comparative anatomical features of P. cineraria and P. juliflora

Figure 4a: T.S. of lamina of P. cineraria Figure 4b: T.S. of lamina of P. juliflora

Figure 5a: T.S. of midrib of P. cineraria Figure 5b: T.S. of midrib of P. juliflora

Figure 6a: Cellular contents of P. cineraria Figure 6b: Cellular contents of P. juliflora

Figure 7a: Stomatal morphology of P. cineraria Figure 7b: Stomatal morphology of P. juliflora

| October-December 2010 | International Journal of Green Pharmacy 278 Robertson, et al.: Comparative anatomical features of P. cineraria and P. juliflora

Figure 8a: Venation pattern of P. cineraria Figure 8b: Venation pattern of P. juliflora

Figure 9a: T.S. of petiolule of P. cineraria Figure 9b: T.S. of petiolule of P. juliflora

Figure 10a: T.S. of secondary rachis of P. cineraria Figure 10b: T.S. of secondary rachis of P. juliflora

Figure 11a: T.S. of primary rachis of P. cineraria Figure 11b: T.S. of primary rachis of P. juliflora

279 International Journal of Green Pharmacy | October-December 2010 | Robertson, et al.: Comparative anatomical features of P. cineraria and P. juliflora examination to determine the sensory, macroscopic CONCLUSION and microscopic characteristics is the first step towards establishing the identity and the degree of purity of Since the leaves of the two taxa, P. cineraria and P. juliflora, medicinal plant materials and should be carried out have close resemblances in external profile and there is no before any further tests are undertaken.[11] Organoleptic anatomical work on records, there are always possibilities evaluations can be done to establish the identity and purity of adulteration of the leaves of these two plants. The present and thereby ensure the quality of a particular sample. work is taken up with a view to lay down the macroscopic A number of different bases are used for morphological and microscopic standards, which could be used in deciding studies and a natural variation in these characteristics the genuineness of the above-described drugs, irrespective plays an important role for preliminary evaluation of crude of their collection from different sources. Macroscopic and drugs. The basis of analysis by evaluation of microscopic microscopic descriptions are provided from a diagnostic characters is that there are always sufficient differences in point of view. In conclusion, the comprehensive analysis the same type or different types of plants as far as the cell of the anatomical features of leaflet of P. cineraria and characteristics are concerned. P. juliflora helps to evaluate the usefulness of the characters establishing the botanical identity of the taxon and to P. cineraria and P. juliflora, both belonging to the same family differentiate these two taxa, which are often mistaken for Mimosaceae, share several common features, especially identification. with reference to foliar morphology. In both the species, the leaves are bipinnate, pinnae one or two pairs and leaflets are REFERENCES 15–18 pairs. These two species can be easily confused and to distinguish one from the other poses little difficulty. This 1. Anonymous. The Wealth of India, Raw materials. Vol. 8. New renders easy adulteration of P. cineraria by P. juliflora.Therefore, Delhi: CSIR; 1969. p. 247-9. some diagnostic features have been evolved to identify and 2. Yoganarasimhan SN. Medicinal Plants of India. Vol. 2 Tamil Nadu, to differentiate the two taxa. The leaflets ofP. cineraria and P. Bangalore: Vedams Books (P) Ltd; 2000. p. 443. 3. Nadkarni KM. Indian Materia Medica. Vol. 1. Bombay: Popular juliflora are bipinnately compound, petiolate and dorsiventral. Prakashan; 1976. p. 2032, 1011. There are many microscopic common features between these 4. Malik A, Kalidhar SB. Phytochemical examination of Prosopis two taxa. However, structure of the petiolule, venation pattern cineraria L (Druce) Leaves. Indian J Pharm Sci 2007;69:576-8. of the rachis and structure, organisation of the midrib tissue 5. Stellaa R, Narayanan N. Pharmacognostical and antimicrobial are different in these two species. studies of the stem barks of Prosopis cineraria (L.) Druce. Res J Pharmacogn Phytochem 2009;1:227-31. 6. GoelU, Saxena DB, Kumar B. Comparative study of allelopathy Some microscopic differences between these two species as exhibited by Prosopis juliflora Swartz and Prosopis cineraria (L) include Druce. J Chem Ecology 1989;15:591-4. 1. When the waxy sections of leaf tissues were stained 7. Van Den Eynden V, Cueva E, Cabrera O. Wild foods from Southern with Toluidine blue O, P. cineraria stained blue-green Ecuador. Econ Bot 2003;57:576-603. which showed that P. cineraria has a dense deposition of 8. Ahmad A, Ali Khan K, Ahmad VU, Qazi S. Antibacterial activity tannin content, whereas P. juliflora stained red and has of juliflorine isolated from Prosopis juliflora. Planta Med 1986; no significant content of tannin. 1:285-8. 9. Ahmad A, Khursheedm AK, Sabiha Q, Viqaruddin A. Antifungal 2. P. cineraria has no mucilage secretion but dilated activity of some hydrosoluble Prosopis juliflora alkaloids. mucilage secreting cells, which are rarely common in Fitoterapia 1989;60:86-9. the mesophyll of the P. juliflora. 10. Johansen DA. Plant Microtechnique. New York: McGraw-Hill; 3. The cross-sectional outline of petiolule is shield-shaped 1940. p. 126-54. with adaxial cone in P. juliflora, whereas it is roughly 11. WHO Geneva. Quality Control Methods for Medicinal Plant elliptical in P. cineraria. Material. New Delhi: AITBS Publishers; 1998. p. 10. 4. The size and shape of the rachis and their vascular Source of Support: Nil, Conflict of Interest: None declared. bundles also show differences between these two taxa.

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