Pharmacognostic evaluation of the leaves and roots of Cassia sieberiana DC

Cassia sieberiana DC. (Leguminosae Caesalpinioideae), commonly known as drumstick and ‘aridantooro’ in Yoruba, is a perennial tree native to Africa. It is used in ethno-medicine to manage arthritis and rheumatism. Pharmacological activities such as myorelaxant, antispasmodic, antiinflammatory, and antimicrobial have been reported in literature. Pharmacognostic investigation including microscopy, chemomicroscopy, physicochemical analysis and phytochemical investigations including thin layer chromatographic finger printing were conducted on fresh and powdered leaf and root samples of this plant. The macro and microscopic studies revealed the leaves to be simple, petiolated, glabrous and pinnately veinnated. The lower epidermal surface is characterized by abundant anomocytic stomata, polygonal epidermal cells and numerous uniseriate, unicellular trichomes. Quantitative leaf analysis revealed the following: stomatal number (163.8), stomatal index (19.04), palisade ratio (17.01), vein islet number (56.45) and vein termination number (61.45). Chemomicroscopic characters present include lignins, tannins, mucilage, starch, oils and calcium oxalate crystals. The physicochemical parameters evaluated are moisture content of 6.3%, total ash of 4.2%, acid-insoluble ash of 3.4%, sulphated ash of 11.0%, water-soluble ash of 0.8%, alcohol-soluble extractive of 21.3%, and water-soluble extractive of 16.7%. Chromatographic fingerprints of ethanol 70% extracts show major spots at Rf = 0.18 daylight (brown), UV366 (deep brown), spray reagent at 100°C (brown); Rf = 0.57 daylight (brown), UV366 (deep brown), spray reagent at 100°C (brown); Rf = 0.89 daylight (green), UV366 (red), spray reagent at 100°C (brown). The pharmacognostic evaluation of the leaves of C. sieberiana is reported here for the first time. The results of this research provide information which can be included in official monograph of the plant for its proper identification and quality control.


INTRODUCTION
Cassia sieberiana DC. (Leguminosae -Caesalpinioideae) is a shrub or small tree, 15 to 20 m tall; bole short, twisted; bark fissured, grey to brown, with blackish stripes and young branches densely hairy.It is wide spread in India and tropical Africa including northern and southern Nigeria, especially in cultivated or old clearings by the *Corresponding author.E-mail: omololafatokun@gmail.com.Tel: +234803-069-1346.
The major secondary metabolites present in C. sieberiana are phenols, anthraquinones, alkaloids, glycosides, flavonoids and saponins (Hafiza et al., 2002).The anthraquinone glycosides are responsible for many of the medicinal properties observed in the plant, although anthraquinones have not been isolated from the plant (Akomolafe et al., 2003).
The evaluation of pharmacognostic and proximate parameters is very essential in establishing the quality, identity and purity of crude drugs.Pharmacognostic standards must be set for every crude drug to be included in a herbal pharmacopoeia.Moisture content is among the most essential and commonly used measurements in the processing, preservation and storage of medicinal plants ( African Pharmacopoeia, 1986).
Ash values and extractive values are reliable tools in detecting adulteration and also help in establishing the purity of crude drugs.Ash from medicinal plants is the total sum of the residue remaining after all moisture has been removed as well as the organic material (such as fat, protein, carbohydrates, vitamins and organic acid) have been incinerated at a temperature of about 500°C.Extractive values are primarily useful for the determination and evaluation of the chemical constituents present in the crude drug and also help in estimation of specific constituents soluble in particular solvents (African Pharmacopoeia, 1986).Similar studies have been carried out on root samples collected outside Nigeria (Sam et al., 2013).

Collection
Leaves and roots of C. sieberiana were collected from the Suleja Local Government Area of Niger State, Nigeria in January, 2016.The plant specimens were authenticated and a herbarium specimen was deposited at NIPRD Herbarium with Voucher number NIPRD/H/6736.

Chemicals, reagents and solvents
All chemicals, reagents and solvents used during the experimentation were of analytical grade.

Morphological evaluation
Leaf and root samples of C. sieberiana were subjected to macroscopic analysis, viz., organoleptic characteristics such as appearance, taste, colour, odour, shape, texture, fracture, etc., of the drug.These parameters are considered to be quite useful in quality control of the crude drug and were evaluated as specified by WHO guidelines (African Pharmacopoeia, 1986;WHO, 1992).

Microscopy
Microscopic analysis was carried out on the pulverized root, the adaxial and abaxial epidermal surfaces of leaves and the pulverized leaf samples.A quantity of each pulverized sample was cleared in chloral hydrate, mounted in glycerin-water (1:1) and viewed under the microscope at different magnifications.The method of Ugbabe and Ayodele (2008) was used to prepare epidermal surfaces of leaves.About 5 mm 2 to 1 cm 2 leaf fragments were obtained from the standard median portion of leaves and macerated in concentrated nitric acid in Petri-dish for 18 to 24 h.The appearance of air bubbles indicated the readiness of the epidermises to be separated.The fragments were transferred into water in a Petri-dish with a pair of forceps.The upper, lower epidermises and mesophyll were separated and cleaned using forceps and carmel hair brush.Each surface was transferred into 50% ethanol to harden and later stained with safranin O for 5 min.The excess stain was washed off in water and the epidermal peel was mounted on a slide with glycerin.

Chemomicroscopic studies
Chemomicroscopic studies of the pulverized leaf and root samples were carried out using reagents and stains like iodine, concentrated sulphuric acid, concentrated hydrochloric acid, ferric chloride, Sudan III, ruthenium red and phloroglucinol with concentrated HCl (1:1) to test for the presence of various metabolites (African Pharmacopoeia, 1986;Ugbabe and Ayodele, 2008).

Quantitative microscopy
The quantitative examinations of leaf samples such as vein islet number, vein termination number, palisade ratio, stomatal number and stomatal index were carried out using standard methods (African Pharmacopoeia, 1986;Ugbabe and Ayodele, 2008).

Physicochemical evaluation
Various physicochemical parameters such as moisture content, total and sulphated ash values, acid-insoluble and water-soluble ash values and water and alcohol extractive values were determined following WHO guidelines (African Pharmacopoeia, 1986;WHO, 1992).

Photomicrography
Photomicrographs of different sections were taken at different magnifications (×100 and 400) using a Leica CM E microscope with a Digital Microscope Eyepiece attachment and Photo Explorer 8.0 SE Basic software.

Statistical analysis
The data obtained were expressed as mean ± standard error of mean (SEM), and n represents the number of replicates in an experiment.

RESULTS AND DISCUSSION
Macroscopic and microscopic methods are central to the identification of different parts of medicinal plants.Different morphological characters were observed on macroscopic examination of the leaf and root samples (Table 1).
The transverse section (TS), of the leaf showed the presence of covering trichomes on the upper epidermis; well-developed collenchyma cells were seen below the lower epidermis.The mid-region showed the vascular bundles (phloem and xylem) separated by the cambium, and also the pith parenchymatous cells (Figures 1 and 2).
Microscopy of C. sieberiana upper leaf surface showed polygonal epidermal walls and a few trichomes but no stomata.The lower epidermal surface was characterized by abundant anomocytic stomata, polygonal epidermal cells and numerous uniseriate, unicellular trichomes with large base and tapering ends.The presence of numerous stomata on just one surface of the leaf (hypostomatic) implies that transpiration takes place on the abaxial (lower) surface for photosynthesis and water loss.The leaf surface also showed the presence of palisade cells, vein-islets and vein-terminations (Figure 3).Leaf constants such as stomatal number, stomatal index, palisade ratio, vein-islet number and veinlet termination number were measured.These parameters, especially stomatal index, are important in the identification of different plants as they vary from plant to plant (Table 3).Chemomicroscopic evaluation of the comminuted leaf  and root of C. sieberiana indicated the presence of lignin, tannins, cellulose, starch and oils.Protein was absent in the leaves but present in the roots (Table 4).
Microscopy of comminuted leaf and root samples indicated the presence of tetragonal prism/rosette types of calcium oxalate crystals in leaf and prism type in root samples, respectively (Figures 4 and 5).Rosette crystals are formed from tetragonal calcium oxalate crystals (Trease and Evans, 2003).Other characteristic features of the comminuted roots are starch grains, pericyclic fibres reticulate xylem vessels and cork cells (Figure 6).
Moisture content obtained for C. sieberiana leaf and root were 6.34 and 4.54%, respectively.This suggests a low moisture content as it is lower than the limit for water content (8 to 14%) for vegetable drugs (African Pharmacopoeia, 1986).Results for ash analysis on dry matter of the leaves showed that total, acid-insoluble, water-soluble and sulphated ash were 4.17, 3.36, 0.85 and 11%, respectively while for values for the roots were: 7.4, 1.47, 2.6 and 8.9%, respectively (Table 2).Results are indicative of low inorganic contents though the values are subject primarily to the soil type/mineral composition of soil used to cultivate the plant.The extractive values obtained for the roots indicated that constituents were more efficiently extracted into 70% ethanol than aqueous solvent whilst the reverse was obtained for the leaves as shown in Table 2.It can be said that there are more constituents soluble in alcohol in roots than in leaves (Table 5).Results obtained for the physicochemical properties of roots vary from that reported by Ajayi et al. (2015), who reported a higher moisture content, 9.5% and much lower total, acid-insoluble and water-soluble ash values of 2.2, 0.4 and 0.5%, respectively.The results also vary slightly with those reported by Bello et al. (2016), who reported a higher moisture content, 6.2% and lower total, acid-insoluble and water soluble ash values of 5.8, 1.0 and 3.5%, respectively.This could be due to differences in geographical location (samples were collected from Jos, Plateau State and Giwa, Kaduna State, Nigeria, respectively), time of collection and varying mineral contents in the soil.

Result of chromatographic fingerprinting
Detection was in daylight, UV 366nm and 10% v/v aqueous H 2 SO 4 spray reagent plates were dried at 100°C after spraying.Major spots were obtained as shown in Table 5.
Results obtained for the physicochemical properties of roots vary from that reported by Ajayi et al. (2015), who reported a higher moisture content, 9.5% and much lower  total, acid-insoluble and water-soluble ash values of 2.2, 0.4 and 0.5%, respectively.The results also vary slightly with those reported by Bello et al. (2016), who reported a higher moisture content, 6.2% and lower total, acidinsoluble and water soluble ash values of 5.8, 1.0 and 3.5%, respectively.This could be due to differences in geographical location (samples were collected from Jos, Plateau State and Giwa, Kaduna State, Nigeria, respectively), time of collection and varying mineral contents in the soil.

Conclusion
The pharmacognostic evaluation of C. sieberiana leaf is being reported for the first time and results from this study have provided information on the morphological and anatomical features and the physicochemical parameters of C. sieberiana leaf and root.These parameters can be used for identification and quality control of the plant drug and provide information which may be incorporated into the Nigeria Herbal Pharmacopoeia (NHP) and the West African Herbal Pharmacopoeia (WAHP).

Figure 4 .
Figure 4. Microscopy of leaf powder of C. sieberiana (x400): A and B showing calcium oxalate crystals-rosette (r) and tetragonal crystals (c); C showing epidermal cells (e) and fibre (f) and D showing uniseriate trichome (t).

Table 1 .
Macroscopic and organoleptic characteristics of C. sieberiana leaf and root.

Table 2 .
Physicochemical evaluation of C. sieberiana leaf and root (dry matter).

Table 4 .
Chemomicroscopic evaluation of C. sieberiana leaf and root.

Table 5 .
Chromatographic fingerprinting of C. sieberiana leaf and root powder.