Anatomical and Phytochemical Studies on Benincasa hispida ( Thunb . ) Cogn . ( Cucurbitaceae )

The morphological and anatomical studies of Benincasa hispida (Thunb.) Cogn. from Nigeria was carried out with the view to reporting morphological and anatomical characteristics for the first time. Physical and microscopic (microtomy) observations were used. B. hispida is a monoecious climbing or trailing herb, stem hairy, 5-angled, with suborbicular stipuliform bract at the petiole-base; leaves simple, very hairy on both surface, alternate, blade palmately or ovate in young plant, base cordate. Flowering occurs between April and May. Female flowers solitary, male flowers solitary or in a slenderpedunculate racemes, petals-5, cream, yellow or pale yellow, ovary ellipsoid, ovules many, stigma 3-lobed and stamen 3. Fruits are large, weighs 8.5 9.0 kg, succulent, densely hairy when young, with a thick waxy deposit when mature, cylindrical to oblong with hairy stalk. Seeds are ovate-obovate, cream. Leaves and petals of male flower are amphistomatic with anomocytic, tetracytic and anisocytic stomata while petals of the female flower are hypostomatic with anomocytic stomata only on the abaxial surface. It has glandular and non-glandular trichomes with uniseriate stalk, clavate and multicellular gland heads. The midrib, petiole, stem, tendril, male and female flower stalks and tendril have hollow pith with 3, 9, 6-7, 16, 14 and 10 bicollateral vascular bundles respectively. The percentage crude protein, ash, carbohydrate, lipid, crude fibre, alkaloid, flavonoid, tannin and phytate could account for the numerous medicinal properties.

Benincasa (Cucurbitaceae) is a monotypic genus with a single species.It is indigenous to both arid and temperate regions of the earth and requires long periods of warm, dry weather for their optimal growth (Whitaker and Davis, 1962).It is under cultivation in large areas in Indian states (Cheran et al., 2010).B. hispida has a chromosome number of 2n = 24 (Mini-Raj et al., 1993;Robinson and Decker-Walters, 1993) and four cultivars based on the size, shape, fuzziness, waxiness, and presence or absence of a dusty or ashy layer (Rubatsky and Yamaguchi, 1997).The fruits of this species could be medium or large and weigh from 7.5 to 45 kg depending on genotype and the production system (Rubatzky and Yamaguchi, 1997;Stephens, 1988) with varying shapes (oblong to cylindrical or elongated) (Stephens, 1988;Rubatzky and Yamaguchi, 1997).

Proximate and phytochemical studies
The leaves of these plant species were detached washed with distilled water, air-dried, ground into powder using and electronic blender, sieved with 80 micron mesh and 200g of each sample stored in glass bottles.The samples were analysed for crude protein, ash and crude fibre according to the AOAC Official Method (AOAC, 2000), 920.152, 930.05, 948.22 and 935.53 respectively.The method of determination of phytochemicals were oxalates (Sanchez-Alonso and Lachica, 1987), alkaloids and flavonoid (Harborne, 1973), saponins (Obadoni andOchuko, 2001), lipid (Yaniv et al., 1999) and tannin (AOAC, 2006).

Epidermal characteristics
Three stomata types namely anomocytic, tetracytic and anisocytic occurred in this species (Fig. 3 and Table 1).

Leaves
The leaf is amphistomatic.The abaxial (upper) and adaxial (lower) epidermises have anomocytic, tetracytic and anisocytic stomata with the anomocytic and tetracytic stomata predominating.The shape of the adaxial epidermal cells are irregular (Fig. 3A) while the abaxial epidermal cells are polygonal (Fig. 3B).The adaxial anticlinal cell walls are slightly straight or curved while the abaxial ones are undulating.The stomatal indexes are 80.86 ± 2.59 and 32.39 ± 5.11 on the lower and upper epidermises respectively (Table 1).

Source of plant material
The plant material studied in this work was collected from the University of Port Harcourt Biodiversity Center (a forest over 50 year old), South-South, Nigeria.The plant was identified, processed and deposited in the University of Port Harcourt Herbarium (UPH/V/1264).The morphological attributes of the species were described and recorded.

Epidermal studies
Foliar materials for epidermal studies were collected fresh from plants growing in the wild.The adaxial and abaxial epidermal surfaces were peeled, stained with 1% safranin or alcian blue, rinsed with distilled water to remove excess stain, mounted in a drop of pure glycerine on clean glass slides; coverslips placed over the peels and sealed with nail varnish to prevent dehydration (Ndukwu and Okoli, 1992).The slides were observed using a microscope and the epidermal features described by Metcalfe and Chalk (1979) and stomatal types described according to Malvey (2004).

Anatomical studies
Cut sections of petiole, midrib from matured leaves and young stems were fixed in FAA (formaldehyde:glacial acetic acid:ethanol in the ratio of 1:1:18 parts of 70% ethanol v/v) for at least 48 hours.These materials were washed in several changes of distilled water, dehydrated through alcohol series (30%, 50%, 70% and 100%) solution 2 hours in each and embedded in wax.Sections were cut on a Leitz 1512 rotary microtome at thickness between 15 -20 µm.The thin sections selected were de-waxed and stained with 1% Safranin O and counterstained with Alcian blue, mounted on slides and photomicrographs of the anatomical sections taken with Leitz Diaplan photomicroscope fitted with Leica WILD MPS 52 camera.

Male flower
The petals of the male flower are amphistomatic having anomocytic and tetracytic stomata.Anomocytic and tetracytic stomata were recorded on the abaxial surface while adaxial surface had only anomcytic stomata (Figs.3C  and 3D).The adaxial anticlinal cell walls are slightly straight or curved while the abaxial ones are undulating.The stomatal indexes are 7.45 ± 0.24 and 0.77 ± 0.51 on the abaxial and adaxial surfaces respectively.

Female flower
The petals of the female flower are hypostomatic with anomocytic stomata only on the abaxial surface (Figs.3E  and 3F).The shape both abaxial and adaxial epidermal cells irregular and the anticlinal cell walls are undulating.

Trichome types and distribution
The trichomes present in this species are glandular and non-glandular with uniseriate stalk, clavate and multicellular gland heads (Fig. 4).The non-glandular trichome types (Figs.4a, 4b, 4c and 4d) are found on the adaxial and abaxial surfaces of the male flower petals while glandular trichome types (Figs. 4g,4h,4i,4j and 4k) occurred on the adaxial epidermal surface of the male flower petals.The petals of the female flowers have glandular (multicellular gland head) trichome (Fig. 4i) and all the forms of non-glandular trichome identified on the lower epidermal surface of the male flower.Also the nonglandular trichome (Fig. 4l) and glandular trichome (Fig. 4i) were recorded on the adaxial surface of the leaf with the non-glandular types occurring mainly on the veins while the abaxial surface had trichome types (Figs.4f and 4g).

Anatomical characteristics
The result of the anatomical characteristics of the midrib, petiole, stem, tendril, male and female flower stalks and tendril is presented in Table 2, Figs. 5, 6 and 7.

Trichome types
Trichomes are of certain systematic significance and sometimes common types are even used for diagnostic purposes in association with other characters (Cutler 1984).The range of trichome variation in the Cucurbitaceae is enormous.The important aspects of variation of trichomes include the degree of modification of the adjacent epidermal cells and the scars left by lost trichomes, in addition to its distribution and anatomy.Generally, trichomes are categorised into glandular and the eglandular types.The significance of trichomes in relation to taxonomy has been shown in angiosperm families earlier by Gupta and Murty (1977) and Rao (1991).Metcalfe and Chalk (1950) had made a survey of various types of trichomes in different genera of the Cucurbitaceae.According to them glandular hairs with multiseriate stalks is one of the major characteristics of this family, but simple unicellular or uniseriate types as well as wart-like or spiny trichomes are also present in the Cucurbitaceae.Both glandular and non-glandular trichomes were encountered during.Furthermore, other glandular trichomes identified among cucurbits include: club-shaped or globose with a multicellular head and a single stalk cell, club-shaped or elongate consisting of a multicellular head and two-celled stalk and multicellular clavate head with multicellular stalk.Also non-glandular trichomes such as uniseriate, 1-2 celled with a single basal cell, uniseriate and multicellular with a single basal cell, uniseriate and multicellular with biseriate basal cells and uniseriate and multicellular with a multicelled base trichomes are characteristics of Cucurbitaceae.Both glandular and eglandular trichomes were present in Benincasa hispida and noted that this wide range of trichome characters observed can be used for diagnostic purposes (Mohammad and Fahad, 2011).This is also consistent with the different types of glandular and eglandular trichomes that have been studied and described in cucurbits (Okoli, 1989;Agbagwa and Ndukwu, 2001;Kolb and Muller, 2004).

Anatomy of stem, tendril, petiole and flower stalks
The arrangement of the vascular bundles and hollow piths in the stem, tendril, petiole and the male flower stalk have been reported (Metcalfe and Chalk, 1950;Ekeke et al., 2015) and the concentric nature of the vascular bundles (Ekeke et al., 2017) have made similar observation in other members of cucurbits.The vascular bundles in cucurbits are bicollateral with varying sizes and mostly forming concentric rings in the stem, petiole and tendril (Metchalfe and Chalk, 1950;Agbagwa and Ndukwu, 2004;Ekeke et al., 2015;Agogbua et al., 2015a).Also, the tendril, female flower/fruit stalk and the stem showed remarkable layers of sclerenchymatous cells.The sclerenchymatous cells in the tendril, stem of B. hispida and male flower stalk are continuous while the ones in the female flower/fruit stalk are partly continuous or discontinuous.This is evident because they are used for anchoring the plant on other plants or to support the weight of the fruits (Ekeke et al., 2015).This report supports previous works by Metcalfe

Discussion
Plant morphology is an important character used in plant classification.It is always used in combination with floral and anatomical features.In Cucurbitaceae, morphological characteristics have been employed in their classification (Hutchinson and Dalziel, 1954;Jeffrey, 1980Jeffrey, , 2005;;Okoli, 2013) while the application of anatomical features have been emphasized by Agbagwa and Ndukwu (2004), Ajuru and Okoli (2013), Ekeke et al. (2015), Agogbua et al. (2015a).In this study, morphology and anatomy of fruit, flower stalks, tendril, leaf and stem anatomy were investigated.Also, the epidermal characteristics of the leaf, petals male and female flowers were studied.

Morphological attributes
The morphological attributes of the B. hispida studied conform to the existing information on the species.For instance, Alejando (1998) reported average fruit size and weight of two accessions of B. hispida 55.5 cm x 24.7 cm and 11.7 kg for green winter melon and 48.9 cm x 22.1 cm and 6.1 kg for fuzzy white gourd.Also Sudhakar et al. (2008) recorded an average fruit weight of 3.06 kg to 13.67 kg and equatorial diameter of 52.99 cm to 80.11 cm in 34 accessions of B. hispida in India and in our study we recorded fruit size of 58.6 -59.4 cm long, 14.0 -15.5 cm wide and 8.5 -9.0 kg (Table 3).These morphological characteristics are in line with the works of Jeffrey (1990), Alejandro (1998), Ahmed (2011) and suggest that this is a cultivar or an accession B. hispida (Alejandro, 1998;Sudhakar et al., 2008).

Epidermal characteristics
The epidermal cells in the leaves and the petals are polygonal to irregular with anomcytic, tetracytic and anisocytic stomata.The anticlinal cell wall patterns are slightly straight, curved or undulating.Also, the leaf is amphistomatic.Okoli (1989), Ekeke et al. (2015), Agbagwa and Ndukwu (2004), Ajuru and Okoli (2013), Agogbua et al. (2015a) opined the fact that most members of this family are amphistomatic with anomocytic, tetracytic and isotricytic stomata types.Also Adebooye et al. (2012) reported more stomata on the abaxial than the adaxial surface of Trichosanthes cucumerina.In the same way, the stomatal indices in this study were more on the abaxial surfaces of the leaf and petals.This finding supports the and Chalk (1950) and Ekeke et al. (2015) and a strong phylogenic bond between B. hispida and other members of Cucurbitaceae.

Chemical composition and uses
Regarding the chemical composition of B. hispida, Wills et al. (1984) noted that the pulp of B. hispida contains glucose and fructose and that their concentrations are 0.5 -0.9% and 0.5 -0.8 % respectively in matured fruits.Also, other authors from different parts of the world have analysed and reported the concentrations of moisture, carbohydrate, fibre, fat and ash in the pulp of mature fruit of B. hispida (Morton, 1971;FAO, 1972;Wills et al., 1984;Mingyu et al., 1995;Tee et al., 1997) in Australia, Florida, Malaysia and China (Table 3).Our result on the concentrations of these nutrients in the pulp of the specimen collected from the University of Port Harcourt Biodiversity Centre is similar to the works of these authors.However we further reported the concentration in the skin and seeds.The seeds had the highest concentrations of crude protein, ash, carbohydrate, lipid and fibre.This however suggests that the combination of the skin, pulp and seeds will be for medicinal and other purposes.
The fruits of Benincasa have been reported to contain different amino acids and several antioxidant properties (Mingyu et al., 1995;2010;Yoshizumi et al., 1998) and the oil for B. hispida seeds as the best cucurbit seed oil from the hot and humid tropics (Martin, 1984;Lee et al., 2005;Sew et al., 2010).The presence of high crude protein and lipid in the seeds compared to other parts of the fruit studied in this work upholds the works of these previous authors and could account for the health benefits, therapeutic uses, pharmacological, medicinal and food properties of B. hispida (Uchikoba et al., 1998;Shih et al., 2001;Lee et al., 2005;Akinmoladun et al., 2007;Roy et al., 2008;Bhalodia et al., 2009;Qadrie et al., 2009;Monson, 2010;Girdhar et al., 2010).

Conclusions
The morphological, anatomical and phytochemical characteristics of B. hispisda from Nigerian are described for the first time.This species share the same ancestral characteristics in common with other members of Cucurbitaceae and supports the placement of this species in the family and the phytochemical constituents supports the medicinal, anti-compulsive effect, angiotensin-converting enzyme (ACE) inhibitor activity in vitro, Alzheimer disease treatment, anti-ulcer, anti-inflammatory, anti-obesity and anti-diarrheal agent of this species.

Fig. 3 .
Fig. 3. Epidermal characteristic of B. hispida: Leaf (A and B); female flower (C and D) and male Flower (E and F) bar = 45µm

Fig. 4 .
Fig. 4. Trichome types in B. hispida (a, b, c, d, e, f and l) non-glandular trichomes and (g, h, I, j and k) glandular trichomes, (g, h and i) multicellular gland head and (j and k) clavate gland head

Table 1 .
Epidermal characteristics of leaf, male and female flowers of B. hispida

Table 2 .
Anatomical Characteristics of B. hispida Diameter of pith, TOC = Thickness of cortex, TS = Thickness of Sclerenchyma, NA = Not applicable and NVB = Number of vascular bundle