MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY ASPECTS OF NATURALIZED EXOTIC SOLANUM DIPHYLLUM L.

8/3/2019 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.MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY ASPECTS OF

NATURALIZED EXOTICSOLANUM DIPHYLLUML.

FATMA A. HAMADA, ARAFA I. HAMED*, MOHAMED G. SHEDED ANDABDEL 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 andgrows 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), andCapsicum 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. al176Boulos (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).

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) (Cheng-

yi and Raven, 1994) and amatillo (FDT, 1995).

**

A B

*


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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 177

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 currentresearch 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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FATMA A. HAMADA, et. al178A. 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-JXA-

840A electron probe micro-analyzer, Japan.

Analysis of certain metabolites of the Solanum diphyllum L. leaf, stem and root:

1. Total tannins were estimated using the gravimetric method (Copper acetate

method) according to Ali et al. (1991).

2. Total carbohydrates were determined spectrophotometrically (Cherry,

1973).

3. Total flavonoids were determined spectrophotometrically (Karawya and

Aboutable, 1982).

4. Total alkaloids were estimated using the gravimetric method according toBalbaa (1986).

5. Total saponins were determined colorimetrically (Honerlogen and Tretter,

1979).

In vitro cytotoxisity essay experimental:

Plant material: Different plant parts ofSolanum 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.


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Extraction and isolation:

A. Shoot extract:

The dried and powdered aerial parts of stem and leaves ofSolanum 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 ofSolanum

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 withvarious 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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FATMA A. HAMADA, et. al180The 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 LSDtest at level0.05 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-reproductiveshoots 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 baseattenuate; 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 0.75-

1.7cm 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 0.15-

0.4 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 flattened-

reniform 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 ofSolanum 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 ofSolanum diphyllum L. are pentamerous, actinomorphic

A B


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FATMA A. HAMADA, et. al182and 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).

.

Figure (3): A, B Solanum diphyllum L. roots; showing main root and root laterals.

(Bar = 1 cm.)

It is well known thatthe primary role of roots in addition to providing stability isto 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 (2003a) 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

ShootAnother shoot

A B

Underground lateralconnection (root lateral)

Shoot

Undergroundlateral connection

Vertical main root


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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 andthe 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.).

AB


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FATMA A. HAMADA, et. al184Thick 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 3-6layers 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 bi-

collateral 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) Eglandularmulticellular 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

B

C

D

E

F

A


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FATMA A. HAMADA, et. al186(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 shootleaves 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 ofS. 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).

A B


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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 ofSolanum diphyllum L. leaf under light

microscope showing anisocytic stomata (Bar = 0.05 mm). B) Abaxial

epidermal surface ofSolanum 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 tothe 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.

A B


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FATMA A. HAMADA, et. al188In 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 cellscontaining 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 likestructure. 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. Crystalcontaining 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, asthere 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 inleaf epidermis (Bar = 0.05 mm.) D) Mucilage excertion cracksleaf

epidermis (Bar = 0.05 mm).

BA

C D


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FATMA A. HAMADA, et. al190Epicuticular 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 ofEucalyptus 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 ofSolanum 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; thatconcerned 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).

Figure (10): Analysis of certain metabolites in Solanum diphyllum L. different parts.

0

2

4

6

8

10

12

14

16

Leaf Stem Root

plant part

percentage%

(gm/100gmd

ry.w

t.)

Total tannins

Total carbohydrates

Total flavonoids

Total saponins

Total alkaloids


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FATMA A. HAMADA, et. al192

According to Morgan (1984) and Munns (1988) one of the main adaptivemechanisms 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 indrier 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 wasobserved 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 carrierprotein 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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FATMA A. HAMADA, et. al194Table (1): Growth inhibitory activity percentage of the three crude extracts ofSolanum

diphyllum L. against HCT116, MFC7, HEPG2 as human cancer cell lines, andHFB4 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 resultsshowed 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 (IC 50) = 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 ofDioscorea

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


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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 IC 50

(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 El-

Sayed et al. (2009) from the methanolic extract ofSolanum 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


24/32

FATMA A. HAMADA, et. al198antineoplastic 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-(-D-

Glucopyranosyl) Etioline was separated by El-Sayed et al. (2009) from the

methanolic extract ofSolanum 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 mannerand 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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