Macro, Micro-Morphological and Bioactivity Aspects of Naturalized Exotic Solanum Diphyllum L.

Al-Azhar Bull. Sci. (ISCAZ 2010). (March, 2010): pp. 175-206.
MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY ASPECTS OF NATURALIZED EXOTIC SOLANUM DIPHYLLUM L. FATMA A. HAMADA, ARAFA I. HAMED*, MOHAMED G. SHEDED AND ABDEL SAMAI M. SHAHEEN South Valley University, Faculty of Science, Botany Department, Egypt. * To whom correspondence should be addressed. Abstract
Solanum diphyllum L. is a native plant to Mexico and Central America, and distributed in many places around the world. Currently escaped from cultivation as an ornamental plant and grows wildly as a naturalized exotic plant in some places in the Egyptian Nile Valley region. Solanum diphyllum L. morphology and anatomy showed similarities with its close affinities in its genus. The presence of lenticel-like structures may be a response to the plant defence needs and/or may be a result of an adaptation to its surrounding environment; in which the plant discard excess storage materials to decrease the effect of stored osmolytes, and the presence of epidermal wax and tannins may help the plant to acclimate to the surrounding light intensities. Solanum diphyllum L. showed a promising cytotoxisity against colon (HCT 116) and breast (MCF7) carcinoma cell lines, and could be considered as a potent source of anticancer compounds. The plant might represent a good model in terms of morphological, anatomical and physiological adaptation to its surrounding environment; revealing a hidden symphony. Key words: Solanum diphyllum L.; anatomy; tannin sacs, lenticels; cytotoxicity.
Introduction The Solanaceae contains between 3,000 and 4,000 species in about 90 genera (Knapp et al. 2004). The members of the Solanaceae have a very important relationship with human being as some are noxious weeds, while others are of a great economic importance as a source of food; e.g. Solanum tuberosum L. (potato), Lycopersicum esculentum Mill. (tomato), Nicotiana tabacum L. (tobacco), and Capsicum frutescens L. (chilies), ornamentals (Cestrum spp., Petunia hybridia and Solanum spp.), and as an extremely important drug sources in medicinal pharmacology and drug therapy (e.g. Hyoscyamus, Datura and Belladona). The Genus Solanum L., has a world-wide distribution, and is one of the largest genera of the flowering plants; is considered as the largest and the most diverse genus in the Solanaceae, with 1500-1700 species, and is one of the ten most species-rich genera of flowering plants (Mabberly, 1997; Frodin, 2004).
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FATMA A. HAMADA, et. al
Boulos (2009) reported that the Solanaceae is represented in Egypt by eight genera and 30 species; nine of them are related to genus Solanum. This poor representation of wild and naturalized species is compensated by relatively large number of introduced and cultivated 42 species (Hepper, 1998). Solanum diphyllum L. is an attractive shrub, has tiny white flowers and bright orange fruits. It has been widely cultivated in tropical and subtropical areas where it escaped in many of these regions, e.g.; Florida, Java, the Antilles, Southern France, Italy, Philippines and China. This plant is usually widespread in drier habitats (D'Arcy, 1974; Knapp, 2002). In Egypt the plant is spread in Aswan and Giza governorates (Figure, 1).
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Figure (1): (A) Solanum diphyllum L. distribution in the world. (B) Its distribution in Egypt.
Solanum diphyllum L. belongs to genus Solanum section Geminata (G. Don) Walp. (Knapp, 2002). Solanum diphyllum L. has been recorded as a new record for the Egyptian flora by Shaheen et al. (2004). It has the following common and local names; twinleaf nightshade, two-leaf nightshade, tomatillo (Mexico) (Knapp, 2002), huang guo long kui (China) (Chengyi and Raven, 1994) and amatillo (FDT, 1995).
MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY .
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The relationship between the plant structure and its environment is one of the most attractive subjects; it is of great interest to plant anatomists and taxonomists at the morphological level, or even at the physio-morphological level. No one can deny that nature has provided many effective anticancer agents which are currently in use, some derived from microorganisms such as dactinomycin and doxorubicin, or from plants in the case of vinblastine, irinotecan, topotecan, vincristine, taxanes, etc. (Ruffa et al., 2002). Despite growing research on flora, only ten percent of the approximately 250 000 species of the higher plants is estimated to have been chemically and pharmacologically investigated. The potential of many plants remains virtually untapped and could be studied (Cragg and Newman, 1999). Continuing efforts of scientists around the world are not stop to seek bioactive components and new cytotoxic agents from natural and traditional herbal sources. As interest in herbal medicine and herbal anticancer therapy in particular has increased, one of the main goals of anticancer therapy and prevention is the discovery of compounds that are relatively potent and selective to tumor cells and less toxic for the normal cells. To our knowledge, little is known about the morphological and anatomical characteristics of Solanum diphyllum L.. So, one of the objectives of the current research is to reveal the most important morphological and anatomical features of the Solanum diphyllum L. Moreover, the present work investigates the relationship between its morphological and anatomical characters, its chemistry and its surrounding environment. Also, the work aims to evaluate the anticancer bioactivity via testing the in vitro cytotoxicity of the methanolic extracts of different parts of Solanum diphyllum L. against three different cell lines of common malignant tumors in Egypt and in many developing countries. Experimental The Identification and world distribution of the collected Solanum diphyllum L. specimens were insured through the Missouri botanical garden web site, New York botanical garden web site and Aluka digital herbarium web site. The plant specimens were compared with a loaned herbarium sheet from Kew botanical garden. The specimens were collected from Giza and Aswan governorates confirming the presence of Solanum diphyllum L. as a naturalized exotic species in the Egyptian Nile Valley area. A detailed list of specimens including herbarium voucher references can be obtained from the authors.
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A. Morphological investigation: Specimens' surface patterns were first investigated by the 7-14 magnification using an Olympus stereomicroscope. Measurements for length, width and thickness were done for 15 replicates using a ruler, a microscopic micro-ocular lens, and a digital caliper. B. Anatomical investigation: Cross-sections were performed on fresh samples of stems, leaves, petioles and roots; replicates of four. Plant specimens were fixed, preserved, and stained according to Alsahar and Nassar (1998). The slides were examined and photographed using Bresser Biolux Al 20x-1280x light microscope Germany, LCD Micro Bresser 40x-1600x light microscope Germany, and Leica DMLB microscope Germany; coupled to JVC TK 1380 Japan, still digital camera. C. Scanning microscope investigation: Leaf and stem specimens were fixed in 10% formalin, and dehydrated in cold ethanol series (National Research Centre technician, person. comm.). All tissues were sputter-coated with 15 nm of gold-palladium and viewed with a JEOL-JXA840A electron probe micro-analyzer, Japan. Analysis of certain metabolites of the Solanum diphyllum L. leaf, stem and root: 1. 2. 3. 4. 5. Total tannins were estimated using the gravimetric method (Copper acetate method) according to Ali et al. (1991). Total carbohydrates were determined spectrophotometrically (Cherry, 1973). Total flavonoids were determined spectrophotometrically (Karawya and Aboutable, 1982). Total alkaloids were estimated using the gravimetric method according to Balbaa (1986). Total saponins were determined colorimetrically (Honerlogen and Tretter, 1979).
In vitro cytotoxisity essay experimental: Plant material: Different plant parts of Solanum diphyllum L. were collected from Aswan, Egypt, in February 2005. A voucher specimen (No. 10.520) was retained in the Botany Department Herbarium, Faculty of Science at Aswan, Egypt.
MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY . Extraction and isolation: A. Shoot extract:
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The dried and powdered aerial parts of stem and leaves of Solanum diphyllum L. (321 gm) were exhaustively extracted with MeOH: H2O (8: 2) using Soxhlet apparatus. The alcoholic extract was condensed to syrupy consistency (54 gm). B. Root extract: The dried and powdered roots (76 gm) were exhaustively extracted with MeOH: H2O (80%) using Soxhlet apparatus. The alcoholic fraction was condensed to syrupy consistency (8 gm). C. Fruit extract: The dried and powdered mature yellow-green fruits (54 gm) were exhaustively extracted with MeOH: H2O (80%) using Soxhlet apparatus. The alcoholic extract was transferred to a separating funnel and was shaken with hexane till exhaustion. The defatted alcoholic fraction was condensed to syrupy consistency (12 gm). In vitro cytotoxic acitvity assay: (cell survival test; growth assay and viability). The cytotoxic activities of the methanolic extracts of different parts of Solanum diphyllum L. were studied against three human tumor cell lines; HCT116 (colon carcinoma), MFC7 (breast carcinoma), HEPG2 (liver carcinoma), and normal skin melanocytes cell line (HFB4). The cell lines were obtained from (ATCC, Mo, USA) culture collection, they were maintained by serial sub-culturing. The cytotoxicity of the tested crude extracts was carried out by a colorimetric assay using sulforhodamine- B (SRB) dye (Skehan et al., 1990) at Pharmacology unit, Cancer biology department, National Cancer Institute, Cairo University. Briefly, the cells were seeded into a 96-well microtiter plate at a cell density of (5 x 103 cells/well). After 24 hrs incubation, the monolayer cells were treated with various concentrations (0, 5, 12.5, 25, 50 g/ml) of different crude extracts, in which different crude extracts (1mg/ml) were diluted to the required concentration using 1% DMSO, and each concentration was triplicate and the experiment was repeated twice. After 48 hrs of incubation at 37C in 5% CO2 incubator, cells were fixed, washed and stained with sulforhodamine-B (SRB) stain, excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. The optical density (O.D) of each well was measured spectophotometrically at 570 nm using ELISA microplate reader (Sunrise Tecan, Tokyo, Japan).
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The cell survival was calculated as follows: survival fraction = O.D (treated cells)/ O.D (control cells). The relation between surviving fraction and each crude extract concentration was plotted to get the survival curve of each tumor cell line and the IC50 (inhibitory concentration 50%) of each extract was calculated using statistical computer program PRISM 5 (Sato and Kameya, 2001). Statistical analysis: Investigated parameters were subjected to analysis of variance (ANOVA) performed using SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). Values with p < 0.05 were considered significant. The results were expressed as mean S.E. The significant differences among means were compared by LSD test at level 0.05 and 95% confidence intervals. Charts were performed using Microsoft Excel software. Results and discussion Morphological description: Small perennial shrubs 0.5-1.5 m tall occasionally reach 2m (Figure, 2, A), root is a normal woody tap root, 10-30 cm length, 0.5-2 cm diameter, gives underground reproductive structures forming underground root laterals (Figure, 3). Upright woody stem green to brown green terete with 2-3 ridges when young, terete lenticellated pale brown when old; minutely puberulent with minute uniseriate eglandular trichomes 0.01-0.05 mm long, glandular hairs may present on young branches. Internodes 1-3 cm, branches and main axis thickness 0.2-2.5 cm, difoliate sympodial units with geminate leaves; leaf pair is markedly anisophyllous (unequal paired). Leaves, oblanceolate, 10-16 x 2.5-4 cm on young non-reproductive shoots and lower branches, leaf margins entire; glandular and eglandular trichomes sparsely present on the margins and on the main vein beneath, the apex acute, the base attenuate; decurrent on the petiole and stem, petiole 2.5-10 mm long. leaves on the reproductive shoots; major leaves elliptic to oblong, geminate, widest at the middle; 5-9 x 2.5-3 cm, the apex rounded, short acuminate or acute, the base acute to attenuate, decurrent on the stem and on the petiole, petioles 1-8 mm long; leaf margins and the main vein beneath have sparse trichomes, minor leaves ovate to obovate, 1-5 x 0.8-2.5 cm, the apex rounded, the base attenuate, decurrent on the petiole; petioles 1-4 mm long (Figure 2; B). Venation is eucamptodromous in which the secondaries veins are not terminated at the margin but turned upward, gradually
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apically diminished in size, and connected to superadjacent secondaries by a series of cross veins. Inflorescences opposite the leaves (leaf-opposed), short racemose, often subumbellate, peduncle 0.7 -1.1 cm; have minutely spare trichomes, pedicel 0.751.7cm long; glaborous, flowers 5-30, pentamerous; sepals 1.9-3 x 0.8-1.5 mm deeply deltoid lobed, papillose nectaries on internal tips of the sepals.
Figure (2): A) Solanum diphyllum L. (whole plant). B) Solanum diphyllum L. (a branch). Bar = (A = 7 cm, B = 2 cm)
Corolla 3-4 x1-2 mm, deeply lobed, white the outer tinged with lavendar in sunny places and become obovoid directly before anthesis, papillose nectaries on internal tips of the petals. Stamens stout, equal, anthers 1.3-1.6 mm x 0.5-1 mm, the filaments glaborous, 0.3-0.6 x 1-7 mm. Ovary glabrous 0.8-1.1 x 0.7-1.1 mm; style glabrous 3-7 x 0.150.4 mm; straight, stigma minutely papillose capitate. Fruit a bright fleshy berry, yellow or orange globose when ripe; 0.5-1.2 cm in diameter. Seeds flattenedreniform yellow to pale brown 2-3 x 1.5-2.5 x 0.4-0.6 mm (Length x Width x Thickness), the surfaces minutely pitted, a single fruit contains 20-65 seeds. The above ground part description was consistent with the description given by Knapp, et al. (2002) with little differences that may result due to differences in the geographical and environmental conditions. The growth of Solanum diphyllum L. is sympodial type; the sympodial type of growth has long been reported for Solanaceae (Sachs, 1882; Wettstein, 1891; Solereder, 1908). As in most other Solanums in section Geminata, flowers of Solanum diphyllum L. are pentamerous, actinomorphic
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and gamopetalous, and well-developed ovaries are globose and glabrous, seeds are flattened-reniform typical of those found in family Solanaceae as mentioned by Knapp (2002).
Another shoot Shoot
Shoot
Underground lateral connection (root lateral)
Underground lateral connection Vertical main root
Figure (3): A, B Solanum diphyllum L. roots; showing main root and root laterals. (Bar = 1 cm.)
It is well known that the primary role of roots in addition to providing stability is to obtain water and nutrients and in some cases work as organs for re-growth and reproduction (Jackson, 1900). Solanum diphyllum L. has underground reproductive root laterals. The function of these laterals is similar to that of rhizomes as they are vegetative reproduction underground organs and carbohydrates store. This type of vegetative propagation; to have growth from adventitious buds on roots was recorded in other families as Podostemataceae or genera including Euphorbia (Euphorbiaceae) and Rorippa (Brassicaceae) (Klimesova and Martinkova, 2004). Moreover, it was well documented by Cuda et al. (2002) and by the NAPPO (2003 a) for Solanum elaeagnifolium and Solanum carolinense. Moreover, Boyd and Murray (1982) stated that Solanum elaeagnifolium can spread by root fragments Several species in the genus Solanum showed underground reproductive structures, which give the ability to propagate clonally from these underground laterals (DAWM, 2006; NAPPO, 2003 b). This phenomenon was observed in arid Solanum spp. as those plants, which have been mentioned previously. Solanum diphyllum L. has been reported to grow in arid habitats by Knapp, 2002 and D'Arcy
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(1974), so it seems that plants that have the same morphological characters, and grow in the same environment are taxonomically related (Dennett, 2006). Anatomical description: A) The Root Secondary thickening was present, as transverse section of the root showed periderm, secondary cortex, secondary vascular bundle, and pith. Periderm composed of multi-layered thin walled cork cells arranged in regular radial rows. Within and underneath the periderm, tannin sacs were detected. Followed by the secondary cortex; starch granules were detected within these cells. The pericycle and the endodermis were not detected. Scattered patches of lightly lignified fibers, and mucilage idioblasts were scattered within the cortex. Complete vascular bundle was detected, secondary phloem externally followed by 2-3 strips of fascicular cambium. Xylem forms a continuous cylinder, traversed by narrow rays. Vessels were very variable in size, protoxylem strands were 2 or 4 at the center of the section arranged in loose radial oblique rows. Parenchyma usually was scanty few limited cells; rays were exclusively uniseriate occasionally biseriate (Figure, 4). In general our observations are in accordance with those of Metcalfe and Chalk (1950) detected for family Solanaceae and genus Solanum.
Figure (4): A) Solanum diphyllum L. middle-aged root B) old root (Bar = 0.05 mm.).
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Thick laterals and vertical roots were found to have similar root anatomy with an obvious secondary growth. According to Bell et al. (1996), Klimesova and Martinkova (2004); Guerrero-Campo et al. (2006) there is a distinction between root-sprouters and rhizomatous plants. Dicotyledonous stems whether they are above or below the soil surface, possess dicotyledonous stem anatomy with secondary thickening and a clearly defined vascular region, and pith in the centre of the stem. The underground lateral connections were found to possess root rather than stem anatomy, therefore the suggestion that the underground connections are rhizomatous is rejected. B) The Stem The stem cross section outline is terete with 2-3 ridges in young stem, terete in older one. The epidermal cells are single layered of radially elongated cells covered by thin layer of cuticle. The epidermis has sparse of three types of trichomes; unicellular and multicellular (2-3 cells) uniseriate non-glandular and multicellular glandular trichomes particularly in young branches. The epidermis replaced by the periderm in the secondary growth. Tannins deposited in the epidermis or the periderm outer most cells as tannin sacs. Cortex consisted of three cell types, 1-3 layers of chlorenchymatous cells followed by 3-6 layers of tangentially expanded angular-lamellar collenchymatous cells, and wide part of paranchymatous cells, some of which filled with dark material; idioblast mucilage cells containing druses crystals. Mucilaginous idioblasts were observed; varied from being very sparsely scattered to very dense grouped in the cells. The vascular bundle represented by a complete cylinder of vascular tissue of bicollateral vascular bundle. Secondary thickening is present as secondary external phloem and secondary xylem, which can be easily distinguished, two to three rows of fascicular cambium in between the secondary phloem and the secondary xylem were present (Figure, 5). The vascular bundle showed to some extent well developed internal phloem if compared with the external one. The outer phloem consisted of considerable number of sieve cells that mixed with numerous parenchyma cells, with lignified scattered fibers which may represent the pericycle. The inner phloem was noticeably wider. Xylem showed inconsistent size of vessels, as they varied in diameter in a radial arrangement, the small narrow primary xylem vessels were towards the inner side, while the wide secondary vessels were towards the outside, and xylem rays were uniseriate.
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Figure (5): Solanum diphyllum L. stem. A) Lenticels like structures have brown external excretion on the bark (Bar = 0.3cm). B) T.S in young stem (Bar = 0.05 mm). C) T.S in mature stem shows tannin sacs (Bar = 0.05 mm). D) T.S in the mature stem has lenticel- like structure (Bar = 0.05 mm). E) Eglandular multicellular uniseriate trichome, using SEM (Bar =100 m). F) Idioblast mucilage cell with druses crystal inside (Bar = 0.05 mm).
The stem characterized by wide pith especially in young branches with relatively thin walled polygonal parenchyma cells which increase in size towards the centre and have crystal sands. Between the innermost xylem elements and the pith lay the internal phloem. Some of the pith cells were sclerosed between pith and the internal phloem. This character was found in some species of Solanaceae e.g. Salpichroa and Sessa
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(Metcalfe and Chalk, 1950). There were other characters, which are common in Solanaceae such as the presence of bicollateral vascular bundles (Solereder, 1908; Fahn, 1982) and the presence of crystal sands (Metcalfe and Chalk, 1950). In addition to lenticels that were formed as a result of the secondary growth, there were other lenticel-like structures which result from the pressure of the tannins and mucilage on the epidermis or the periderm, which cause rupture of the epidermis (Figure, 5 [A,D]). C) The Leaf Generally, there were no anatomical differences among the reproductive shoot leaves and the non-reproductive shoot leaves, the differences were mainly morphological.
Figure (6): A) Papillae trichome on Solanum diphyllum L. leaf . B) Multicellular glandular trichome on Solanum diphyllum L. leaf margin (Bar = 0.05 mm.).
The leaf blade: The lamina of S. diphyllum L. is dorsiventral, the leaf epidermis has few sparse trichomes of four types; unicellular papillae hairs which were rare, simple unicellular and multicellular uniseriate non-glandular and multicellular glandular hairs which consisted of a short multicellular stalk and unicellular secretory head at the top round to oval in shape. Glandular trichomes were more numerous in young leaves. Trichomes mainly were present along the margin of the leaves and the main vein on the abaxial laminaside (Figure, 6).
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Stomata were anisocytic (unequalcelled); as stoma are surrounded by three cells, one of them is distinctly smaller than the other two (Van Cotthem, 1970). Stomata were very rare in the adaxial surface and more frequent on the abaxial epidermis (Figure, 7 [A]).
Figure (7): A) Abaxial epidermal surface of Solanum diphyllum L. leaf under light microscope showing anisocytic stomata (Bar = 0.05 mm). B) Abaxial epidermal surface of Solanum diphyllum L. leaf under scanning microscope showing the crystalloid wax and the stomata ( Bar = 70 m).
Our result agrees with Metcalfe and Chalk (1950), Ahmad (1964), and Dwelle et al. (1983), Murthy and Inamdar (1980) who worked on other Solanum species and recorded the presence of cruciferous types i.e. three subsidiary cells per stoma. In other words, on the dorsiventral leaf, anisocytic stomata were confined to one surface (Watson and Dallwitz, 1992) and simple uniseriate or multicellular eglandular or glandular hairs (Edmonds, 1972) were the characters that have been detected and shared with other members of family Solanaceae and genus Solanum. The stomatal openings were ovate-rounded shaped. The electron microscope scanning for the leaf showed that the epidermis is sculptured by islands of rounded crystalloids epicuticular wax. The stomata were not present on the same level. As a result of wax elevation, stomata were raised, slightly depressed or depressed due to the presence of the rims, which give the shape of depressed stomata (Figure 7 [B]). The mesophyll was distinguished into palisade and spongy tissues. Palisade tissue cells were large, elongated more or less cylindrical and arranged in a single layer. The spongy tissue cells were rounded or lack regularity and arranged loosely in 3-5 layers, large intercellular spaces were occurred among these cells. The palisade and spongy tissues were equally thick, with the ratio of approximately 1:1. There were raphides crystals in the spongy tissue. In the wings of the leaf blade there were undifferentiated small or subsidiary vascular bundles along the two wings.
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In the midrib region, the main vein is prominent, especially on the abaxial side. On the adaxial side underneath the epidermis were present the chlorenchymatous cells; 3-5 layers, followed by lacunar collenchyma; 3-7 layers, and parenchyma cells of different sizes, rounded in shape, with numerous small intercellular spaces among them; 3-5 layers. Druses crystals were detected in the parenchymatous cells. At the middle of the midrib region there was a large bicolateral vascular bundle, with patches of external and internal phloem (Figure, 8 [B]). Sclerenchymatous fibers were present around the vascular bundle especially in the middle-aged and old leaves which may be considered as bundle sheath. Some cells of the parnchymatous and spongy tissues were mucilage cells containing dark mucilage and druses crystals. Some other epidermal cells has tannin sacs (Figure, 8 [C]). Tannins are polyphenolic substances that have a scattered distribution through various plant families (e.g. Myrtaceae). The function of tannins to some extent is still little understood, they may act as an ultraviolet light shield in some plants where they are present in epidermal cells as very strong sunlight can damage chloroplast so such a screen could be beneficial. In addition it can act as herbivore deterrent as its stringent taste may protect leaves from being eaten (Cutler, 1978). With the pressure of the tannins the periderm cracked forming lenticels like structure. Usually the tannin cells often form connected systems and may be associated with vascular bundles (Esau, 1977), these observation emphasizes our suggestion that the dark components observed in the epidermal cells of Solanum diphyllum L. may be due to tannin sacs and mucilage idioblasts (Figure, 8 [C, D]). According to Franceschi and Homer (1980) idioblasts mucilage cells containing crystals occur widely in the plant kingdom and have been observed in most plant organs. These crystals may be raphides, styloids, druses, prismatics, or crystal sands (Wang et al., 1994) in our case mucilage cells included druses crystal. Crystal containing cells may not differ from other parenchymatous cells, but they are more or less specialized in form and content (Esau, 1977). Generally Lenticels are limited interruptions in the cork and phelloderm through which gaseous exchange with the surrounding takes place. Leaves differ from most stems and roots in that they are almost all primary organs, although some secondary growth can occur, as for example in the vascular supply of some gymnosperm leaves and in the leaf bases of some monocotyledons with secondary stem growth. Large changes do not occur in the shape or thickness of dicotyledonous leaves after
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primary growth has ceased (Cutler, 1978). Thus the presence of lenticels on Solanum diphylllum L. leaves happened in response to something! It was detected that most of the secreted material was discarded to the outside only after repture of the cuticle (Schnepf, 1969), e.g. as seen in Inula. Generally, the inner secretory tissues are characteristic of certain plants; their development may or may not depend on external factors such as injuries, pathogens or physiological stresses (Fahn, 1987). This secreted material is either exuded through minute pores in the cuticle, or accumulates below the cuticle and discarded outside after cuticle rupture (Fahn, 1988). This was not the first time to detect lenticels associated with tannin presence, as there were several rows of radially arranged tannin cells observed in lenticels of Ficuss microcarpa, and the reason of its presence was not known (Kuo-Huaung and Hung, 1995).
Figure (8): Solanum diphyllum L. leaf. A) Leaf has excretions of dark spots (Bar = 2cm). B) T.S in the leaf shows lenticel-like structure (Bar = 0.1 mm). C) Tannin sacs in leaf epidermis (Bar = 0.05 mm.) D) Mucilage excertion cracks leaf epidermis (Bar = 0.05 mm).
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Epicuticular wax crystals were detected in many plant species on the surface of the cuticle (Baker, 1982). These aggregates of wax serve a variety of ecological and physiological functions. As for example it led to enlarging the exposed hydrophobic surface, render the leaf highly unwettable (Holloway, 1970), thereby protecting the plant from dirt (Barthlott et al. 1998) and pathogenic microorganisms (Deising et al., 1992). Wax crystals can also help to maintain stomatal gas exchange (by keeping water off the pores) (Brewer and Smith, 1997). Also, it serves as a protecting and selecting barrier by reducing insect mobility (Knoll, 1914). The presence of wax may lead to give the shape of slightly sunken stomata, the manner of distribution of the wax particles sometimes is considered as a beneficial character. Thomas and Barber (1974) and Cameron (1970) studied the effect of cuticular waxes on light absorption in leaves of Eucalyptus spp. they found that differences in characteristics caused by the amount and orientation of waxes on the leaf cuticle caused variation in the ability of the leaves to absorb light in the 400-700nm waveband, so the presence of wax and tannins help the plant to acclimate to the surrounding light intensities. Generally it was observed in the field that Solanum diphyllum L. grows widely in shade places under trees. The distribution of stomata on the abaxial surface probably is an adaptation to water loss or light strength. This is in agreement with Metacalf and Chalk (1950). The leaf petiole The cross section outline of the petiole is more or less half a circle with straight to shallow raised adaxial surface. Epidermal cells are radially elongated covered with thin layer of cuticle, followed by 2-3 layers of chlorencyma, lacunar collenchyma 5-7 layers and then isodiametric parenchyma. Two small lateral ridges or subsidiary bundles were present near the adaxial surfaces which were responsible to feed the fimbrial veins (Figure, 9).
Figure (9): T.S in leaf petiole of Solanum diphyllum L. (Bar = 0.05 mm).
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The rest of the vascular tissue of the petiole, which extends into the lamina as the mid-vein, appears as a median arc-shaped (horse-shoe shaped). Bicollateral vascular bundle, in which internal phloem is well developed than the external one, is surrounded by scattered lignified fibers. Crystal sands were present. Tannin sacs, mucilage cells and lenticel-like structures were present as the case in the leaf and stem. The petiole nearly has the same tissues as the leaf and in a similar arrangement. Generally the collected specimens from the two different localities (Giza and Aswan governorates) were morphologically and anatomically similar. Analysis of certain metabolites: The analyses of certain metabolites in the leaves, stems, and roots of Solanum diphyllum L. showed that the total tannins and carbohydrate were found in relatively large amounts in root and stem in comparison with other metabolites (data not shown). However, tannins, flavonoids and saponins were found in large amounts in leaf tissues (Figure, 10). Beside the functions that previously recorded for the tannin sacs, in general, polyphenols, which were proved to be high in Solanum diphyllum L. leaves extract (Hossain et al., 2009), have a wide range of possible functions in plants; that concerned with basic processes of growth and development, or involved in protection and varied according to the environmental and ecological pressures, which they might help to minimize their hazards (Smith, 1975). Moreover, accumulation of total phenols was detected in most xerophytic plants and could be considered as an adaptive physiological response and safeguards against drought stress (Abd Alla et al., 1999; Hegazi, 2000).
16 percentage % (gm/100gm dry.wt.) 14 12 10 8 6 4 2 0 Leaf Stem plant part Root Total tannins Total carbohydrates Total flavonoids Total saponins Total alkaloids
Figure (10): Analysis of certain metabolites in Solanum diphyllum L. different parts.
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According to Morgan (1984) and Munns (1988) one of the main adaptive mechanisms contributing to drought resistance in plants is the ability to actively accumulate solutes, in order to decrease the internal osmotic potential and thus prevent or reduce an important loss of water from the stressed tissues. It was observed by (Soliz-Guerrero et al., 2002; Moges et al., 2001; Watkinson et al., 2003, 2006; Leatherwood, 2005; Misra and Gupta, 2006; Abdul Jaleel et al., 2007) that the ability of the plant to tolerate drought is due to the accumulation of compatible solutes such as polyphenols, carbohydrates, alkaloids and saponins, etc. Knapp (2002) observed and stated that Solanum diphyllum L. is widespread in drier habitats, and has a woody tap roots, which may persist through dry or otherwise unfavorable periods, also it was observed by Sheded et al. (2010) that Solanum diphyllum L seeds have the ability to germinate under low osmotic potential induced by PEG 6000, in other words it has the ability to resist drought. All these emphasis that the tannin sacs and mucilage cells may play a role in this process (drought resistance), and the discard of these substances via the plant stem and the leaves epidermis may be a regulatory process; as the plant under favourable conditions has no need for the extra stored osmolytes, this hypothesis is in need to further investigation. In addition, there are other morphological character such as root systems of plants adapted to arid regions, which possess a range of unique features to survive and reproduce in such dry conditions and poor soils. Solanum carolinense is a good example as mentioned previously; has an extensive root system with lateral root branches, and recorded by Bradbury and Aldrich (1957) as a drought tolerant plant. Solanum diphyllum L. has to some extent the same root morphological character of Solanum carolinense, and its seeds showed the ability to resist drought, moreover, from literatures it was detected to grow in dry habitats, and it is taxonomically related to Solanum carolinense so it could have the same ability of drought resistance. The relationship between plant structure and the environment in which the plant grows was a fascinating subject to early plant anatomists, and still to be of great interest, as the adaptations in the anatomy of plants might be an ecological benefit. Moreover we should realize that not all adaptations are evident at the morphological level, some are physiological (Cutler, 1978) or might be at both levels; in which the
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plant uses both of them in order to persist in the unfavorable conditions and insure its existence. Cytotoxicity assay: The growth inhibition percentage was found to be increased with increasing concentration of different crude extracts. Results are tabulated in tables (1, 2) and graphically represented in figure (11). The methanolic extracts were found to exhibit activity against the human colon, breast and hepatoma cancer cell lines. The cytotoxic activity of various Solanum diphyllum L crude extracts was clearly paralleled to the added concentration; it was observed that at concentration 25 g/ml the inhibition percentages of root crude extract against HCT116, MFC7, HPEG2 carcinoma cell lines were (88%, 86.9% and 73.5%), shoot crude extract (85.5%, 83.8% and 73.3%) and fruit crude extract (87.2%, 85.3% and 68.3%) respectively, and were less toxic for the normal human melanocytes cell line (HFB4) in comparison with these cancer cell lines. It was observed that the inhibition percentage begin to decline with conc. 50 g/ml and this may refer to the absence of response in the survival fraction with increasing concentration of the crude, which may be due to saturation of the carrier protein which transports the drug intracellularly with the drug. In addition, with increasing time the drug is inactivated by metabolizing enzymes. Moreover, the portions of cells that were not affected or killed by the drug proliferate normally due to absence of the drug; as there was no longer effect of the drug in the cells (Prof. Shouman, person. comm.). The HCT116, MFC7, HEPG2 cells which were treated with different concentrations of crude extracts showed antiproliferative effects under BRS assay. The IC50 (inhibitory concentration 50%) values for root crude extract were (4.04, 5.26 and 11.5 g/ml), shoot crude extract (4.5, 9.80 and 10.6 g/ml) and fruit crude extract (5.11, 9.07 and 10.4 g/ml) respectively, indicating that the susceptibility of HCT116 and MFC7 cells is higher than that of HEPG2 cells. In other words, colon and breast cancers were the most sensitive, while liver cancer is the least sensitive, and the crude extracts were less toxic for normal cells (IC50=22 g/ml).
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Table (1): Growth inhibitory activity percentage of the three crude extracts of Solanum diphyllum L. against HCT116, MFC7, HEPG2 as human cancer cell lines, and HFB4 melanocytes as a normal cell line.
In terms of safety, the potent plant extracts should be less cytotoxic for the normal human skin melanocytes cell line, than for the cancer cell lines. The results showed that Solanum diphyllum L. root crude is the most potent inhibitor of HCT116 (human colon carcinoma cells) by growth inhibition, followed by shoot crude extract and seed crude extract respectively. Root crude extract was a potent inhibitor against breast cancer MCF7 cells demonstrated maximum cytotoxicity (IC
50=
5.26 g/ml), and according to SRB assay results, plant crude extracts were less
toxic for normal human skin melanocytes with an IC50 of 22 g/ml. The root crude extract was more potent than shoot and fruit crude extracts, and the cytotoxicity value of HCT116 under the effect of root crude extract (IC50) = 4.04
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g/ml was significantly lower than that for doxorubicin (a drug reference), which its IC50 were (4.5 and 5 g/ml) respectively in case of HCT116 and MCF7 cancer cell lines. Shoot crude extract (IC50 = 4.50 g/ml) was more potent in its effect than fruit extract (IC50 = 5.11 g/ml) against HCT116 cell line. The crude extracts were proved to have cytotoxic activity against cancer cell lines in vitro, and were less toxic for normal human melanocytes than for the three cancer cell lines. However the 25 g/ml concentration induced the higher inhibition, actually it was not the best concentration, because the inhibition was not low enough in case of the normal cell line.
Table (2): IC50 for the crude extracts and for doxorubicin as a drug reference.
Statistical analysis showed obviously that the best concentration of Solanum diphyllum L. different crude extracts affected the carcinoma cells, and in the same time has low toxicity on the normal cells (HFB4) was 12.5 g ml-1 (Figure 11). Our results suggested that medium-low doses of root extract may cause apoptosis or suppression for HCT116 and MCF7 carcinoma cells potentially, besides shoot and seed extracts affected HCT116 and are safe as they did not affect the normal cells severely. According to Lacaille-Dubois (2005) several pharmacological in vitro and in vivo researchers found various pharmacologically active compounds in many plants e.g. Dioscorea species having cytotoxic activity, even water extracts of Dioscorea membranacea were found to be specifically active on breast cancer cells (IC50 7.7 g/ml) with little activity on normal cells (IC50 78.4 g/ml).
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Figure (11): Total mean surviving fraction of different crude extracts at conc. 5, 12.5 and 25 g/ml. (*) Shows no significance between different cell lines at the same concentration at p<0.05.
Continuing efforts not stop to seek bioactive components from the traditional sources. The traditional herbal remedy was used for the treatment of many carcinomas for centuries, several compounds with anticancer activities have been isolated from different parts of plants e.g. Dioscorea collettii var. hypoglauca (Dioscoreaceae) (Hu et al., 1996; Hu et al., 1999). Many species contain specific active compounds that were reported to possess cytotoxic, antitumor activity (Lacaille-Dubois and Wagner, 2000; Lacaille-Dubois, 2002). Various plant extracts have been evaluated for its cytotoxic or cytostatic effects against variety of cancer cell lines. Solanum lyratum extract was tested against human colon adenocarcinoma cell line (Colo 205) (Hsu, et al., 2008), Annona glabra against human leukemia cell lines (CEM/VLB) (Cochrane et al., 2008), Blumea balsamifera (Norikura et al., 2008) against rat and human hepatocellular carcinoma cells (McA-RH7777 and HEPG2) and showed that Blumea balsamifera MeOH extract (BME) induced growth inhibitory activity in rat and human
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hepatocellular carcinoma cells (McA-RH7777 and HEPG2). Also, It was detected that Sukun wood crude (Artocarpus altilis) against human breast cancer (T47D) cells has a cytotoxic effect in a concentration dependent manner, with an extract IC50 (6.19 g/ml) (Arung et al. 2009). Tribulus terrestris in addition to its aphrodisiac properties, it was proved to contain compounds that efficiently affect tumors and have been used extensively in Indian and Chinese traditional medicine for the treatment of various urinary, cardiovascular and gastrointestinal disorders (Wang et al., 1990; CHEMEXCIL, 1992; Anand et al., 1994). Moreover, there were compounds isolated from fenugreek (Trigonella foenum graecum L.) induced cell death and morphological change indicative of apoptosis in leukemia carcinoma cell line H-60 (Hibasami et al., 2003). Solanum aculeastrum Dunal has long been used to treat various cancers in the Eastern Cape Province of South Africa, and compounds that has inhibitory effect on Hela carcinoma cells were separated from it (Koduru et al., 2007). Moreover a steroidal alkaloid compound, 3-O-(-D-Glucopyranosyl) Etioline; that exhibit a cytotoxic effect against the Hela cells (cervical cancer cell line) was separated by ElSayed et al. (2009) from the methanolic extract of Solanum diphyllum L. roots. And It was reported that Solanum lyratum extract (SLE) inhibited cell proliferation of gastric carcinoma SGC- 7901 cells, human hepatoma BEL-7402 cells, and A375-S2 cells in vitro and in vivo (Sun et al., 2006; Ren et al., 2006). Doxorubicin is an anthracycline antibiotic of wide spectrum of action has been used in healing cancer tumors since the late 1960s. It considered as a powerful drug against cancer. The tumors are most commonly responding to doxorubicin when it is given as a single agent or in combination with other antitumor agents. Other cancers that are less responsive to doxorubicin are still treated with the drug because of its overall benefits (Singal and Liskovic, 1998). Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of solid and hematopoietic tumors (Xin, et al., 2007). Doxorubicin is used to treat neoplastic diseases such as acute lymphoblastic leukemia, Wilms tumor, soft tissue and osteogenic sarcomas, Hodgkins disease, non-Hodgkins lymphomas, Ewings sarcoma, and bronchogenic, genitourinary, breast, and thyroid carcinoma (IARC, 1976). Doxorubicin is a highly effective
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antineoplastic drug, but its clinical use is limited by its adverse side effects on the heart as it induced cardiotoxicity and congestive heart failure, and affected the mature and the growing skeletons (Li et al., 2006; Van Leeuwen, et al., 2000). Thus having a new drug that avoids the adverse side effects of Doxorubicin is considered as a promising good merit. Our results showed that the IC50 using the crude extracts were more potent than that of Doxorubicin, especially that of root extract against HCT116. Moreover, a steroidal alkaloid compound, 3-O-(-DGlucopyranosyl) Etioline was separated by El-Sayed et al. (2009) from the methanolic extract of Solanum diphyllum L. root, which exhibited a cytotoxic effects against the Hela cells (cervical cancer cell line). All these suggest the possibility of using Solanum diphyllum L. as a potent source for anticancer compounds that could be utilized pharmaceutically after further investigations.
Conclusion In conclusion, the plant showed a morphological and anatomical affinity to its family, section and genus. Root, shoot and fruit methanolic extracts of Solanum diphyllum L exhibited cell viability decreasing in a concentration-dependent manner and potent activity against variety of carcinoma cell Lines (breast, colon and hepatoma cancer cells) with relatively mild effect on normal cell line, therefore it could has a potential anti-cancer effect. The isolation and characterization of the compound(s) responsible for the observed activities of these extracts are currently being conducted. The plant needs further studies in order to explore its drug therapy possibilities.
Acknowledgement Thanks due to Kew Botanical Garden Herbarium, U.K. for providing the loaned specimen herbarium sheet of Solanum diphylllum L. used in confirming the identification of the plant species.
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Macro, Micro-Morphological and Bioactivity Aspects of Naturalized Exotic Solanum Diphyllum L.

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Macro, Micro-Morphological and Bioactivity Aspects of Naturalized Exotic Solanum Diphyllum L.

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Al-Azhar Bull. Sci. (ISCAZ 2010). (March, 2010): pp. 175-206.

FATMA A. HAMADA, et. al