Pharmacognostic Studies of the Leaves, Stem and Root of Capparis erythrocarpos Isert (Capparaceae)

Introduction: The roots of Capparis erythrocarpos are used traditionally across Africa for the management of pain, arthritis and other forms of inflammatory conditions. Owing to its proven efficacy, it has gained commercial value, particularly as a key ingredient in several herbal products and alcoholic beverages. The increased scarcity owing to demand outstripping supply lend the roots of C. erythrocarpos to adulteration. This paper presents a detailed pharmacognostic evaluation of the leaf, stem and root of C. erythrocarpos which will be used in its identification and consequent standardization. Methods: The leaf, stem and root were evaluated for their macroscopic and microscopic features as were the physicochemical parameters and phytochemical screening done. Results: Leaves are alternately arranged, have collateral vascular bundle, crystal sheaths and a pericyclic fibre. Actinocytic stomata and secretory cells were contained in powdered leaves. The stem showed lenticels and thorns, stellate and branched trichomes which leave off cicatrices in older stems. The powdered stem and roots contained stone cells, secretory cells and scalariform vessels. However, the roots lacked thorns, trichomes and had smaller secretory cells. Aqueous and ethanolic extracts of the leaves, stem and roots were slightly acidic to neutral. Ash values of leaves, stem and roots are (16.58 ± 0.09) % w/w, (5.01 ± 0.09) % w/w and (6.53 ± 0.19) % w/w respectively. Preliminary phytochemical screening of the leaves, stem and roots showed the presence of glycosides, flavonoids and tannins. Conclusion: The determined parameters for the leaf, stem and root of C. erythrocarpos constitute quality parameters for their unequivocal identification


INTRODUCTION
Natural products hold potential sources of novel compounds with varied bioactivities.Bioactivity studies should be preceded with a clear cut standardization of the plant to avoid intentional or non-intentional adulteration as this can lead to treatment failure, sub therapeutic effect or even death.Capparis erythrocarpos, an important medicinal plant in herbal medicine used globally in the management of pain and other inflammatory conditions lack standards for quality control.Globally, there are about 650 species present in the family and 250 species present in the genus Capparis.2] Among these species, C. erythrocarpos, commonly referred to as capers 3 has been found to be the most commonly used for the management of several ailments.A mention of the plant in Ayurveda states the use of the stem bark and root for its analgesic and other inflammatory disorders. 4Across Africa, the roots are used for the treatment of conjunctivitis, headache 5 and chronic diarrhoea. 6In Tanzania, capers is used in the management of child convulsive fever and inflammation of the connective tissue of the eye. 7n Uganda, the whole shrub is used for the management of menstrual pains, infertility and anemia. 8In Ethiopia, it is used traditionally for the management of symptoms of cyst and skin infections.In Kenya, it is used as an antidiarrheal and anthelmintic agent. 4n Ghana, it is used for the management of pain, arthritis and other forms of inflammatory conditions. 5armacological investigations have been conducted on C. erythrocarpos and it possesses anti-arthritic effects [7][8][9][10] without any organ specific toxic effects. 11hytochemical analysis of the ethanol extract of C. erythrocarpos root revealed the presence of alkaloids and flavonoids. 9 Ghana, Capparis erythrocarpos roots and its formulations are in high demand commercially.It is used by Kasapreko Group of Companies for their Alomo bitters brand variants which have received global acclaim 12 and is marketed as an alcoholic beverage and appetite stimulant.The Centre for Plant Medicine Research (CPMR) uses the root in an oral formulation, Sirrapac, as an antiarthritic medication. 11Due to their popularity and high value, C. erythrocarpos containing products are a target for economically driven adulteration.In the face of demand outstripping supply and the economic motives for adulteration being considerable, Capparis erythrocarpos is at risk of an admixture or complete substitution with closely related species.This is made worse, by the use of the roots (which requires the destruction of the whole plant).A study in 2007 highlighted the unnoticed threatened state of C. erythrocarpos and the need for its sustainable use and conservation. 13espite its wide use and potential for adulteration, no report exists of a pharmacognostic study aimed at unequivocally identifying C. erythrocarpos from its closely related species.Authentication of C. erythrocarpos is of primary importance for both consumers and the industries that manufacture products containing C. erythrocarpos.For the industries, product authentication is essential to maintain trust of consumers and avoid unfair competition that can eventually create a destabilized market and disrupt regional or national economies.Consumers also will have the full benefit of quality products that promote their health and well-being.Clearly, a study that addresses this gap in knowledge holds numerous benefits for the herbal medicine industry.Consequently, this paper aims at establishing quality standards for the unequivocal identification of Capparis erythrocarpos.The macro-and micro morphological features, physicochemical properties and fluorescence characteristics were determined using standard methods.

Materials
Reagents and chemicals of analytical grade were purchased from Sigma Aldrich Co. Ltd.A Leica DM. 700 light microscope fitted with camera (Leica ICC50 HD) was used for all the microscopy.Fluorescence analysis was performed with UVGL58 UV lamp (Cambridge, UK).

Plant Collection and Authentication
Leaves, stem and roots of Capparis erythrocarpos were collected from Ayikuma, Ghana (5°55'2.384''N,0°0'40.3092''W).Parts collected were authenticated by Dr. George Henry Sam of the Department of Herbal Medicine.Leaf samples were pressed while the stem and root samples were stored in formaldehyde (37%) solution.They were deposited at the herbarium of the Herbal Medicine Department, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), Kwame Nkrumah University of Science and Technology (KNUST) with the identification codes KNUST/HM1/2017/ L002, KNUST/HM1/2017/S012 and KNUST/HM1/2017/R004 for leaf, stem and root respectively.

Macromorphology and micromorphology of leaf
The color, taste, odor and texture of the leaves of Capparis erythrocarpos were determined and recorded as per standard procedures. 14he leaves of C. erythrocarpos were macro-morphologically characterized. 14] Powdered leaves was mounted in appropriate reagents.][16] Quantitative evaluations (stomatal number, stomatal index, palisade ratio, vein-islet number and veinlet termination numbers) and measurements were done.Area measurements and particle counting were achieved using Image J (National Institute of Health, USA, http://imagejnih.gov/ij) and Microsoft Excel, Microsoft Office Professional Plus 2013.

Macromorphology and micromorphology of stem
Similar standard procedures were performed as described for leaves.Quantitative evaluations were however not performed.Powdered stem was mounted in appropriate reagents and observed under the microscope.

Macromorphology and micromorphology of roots
Similar standard procedures were performed as described for the leaves.Powdered roots was mounted in appropriate reagents and observed under the microscope.Quantitative evaluations were not performed.

Physicochemical and phytochemical characters
5] Test samples were run in triplicates.

Fluorescence analysis
The leaves, stem and root powders were observed for characteristic colors.Strong acids, alkalis and organic solvents were added and thereafter observed under the visible, short (254 nm) and long wavelengths (366 nm).

Plant extraction
The method of extraction was carried out as per the protocol employed by Centre for Plant Medicine Research (CPMR), Akuapim-Mampong, Eastern Region.Plant material (roots, stem and leaves) was warm macerated in water for three hours.It was filtered, concentrated under low temperature (40°C) for 6 h and freeze dried to obtain a powdery mass.The yield obtained for the roots, stem and leaves were 0.79 %, 0.59 % and 2.73 % respectively.

Fourier Transform Infrared (FTIR) fingerprint
The spectra of the extracts were measured as a solid film using a PerkinElmer Spectrum Two Universal Attenuated Total Reflectance (UATR) FTIR spectrometer.They were recorded with 32 scans at a resolution of 4 cm -1 over a wavenumber range of 4000 -400 cm -1 .

Macromorphology and micromorphology of leaf
The fresh matured leaves were green colored, elliptical to ovate shaped, bitter to taste and musty in odour.The simple leaves were alternately arranged with pinnate venation, sinuate margin, acute apex, short stalked petiole and has two spiny stipules on the symmetrical base (Figure 1A).They are fleshy and have a glossy appearance.The striking difference between the young and old leaves is the light green lanceolate shaped young leaves (4.2×0.8)cm (Figure 1A) as opposed to dark green elliptical to ovate old leaves (7.6×3.8)cm.
The transverse section of the midrib of C. erythrocarpos young leaf showed a simple arrangement consisting of a layer of upper and lower epidermal cells each respectively overlaid with a thick cuticle.The abaxial surface had no palisade cells (Figure 2A).Dispersed in the collenchyma and parenchyma cells are crystal sheaths and starch grains.Also present was an arc-shaped unicollateral vascular bundle (Figure 2A).The lamina showed wavy epidermal cells with stomata scattered in them.Attached to the lamina were branched and stellate trichomes (Figure 3).Worth noting is the more advanced cellular structures: higher degree of lignification and pericyclic fibers (Figure 2B) in the older leaf.Also present in the older leaves were branched and/or unicellular clothing trichomes.
In the section of the young and old leaves, dark blue stained starch grains and pink lignified xylem vessels were observed.Safranin stained the cuticle bright red and the lignified pericyclic vessel purplish violet (Figure 2B).
Powdered leaves have prismatic calcium oxalate crystals, crystal sheaths, stomata with straight to wavy walled epidermal cells, unicellular and branched trichomes, secretory cells, lignified vessels and fibers.

Leaf surface data determinations
Epidermal cells on both surfaces of C. erythrocarpos leaf are slightly wavy walled.C. erythrocarpos has a palisade ratio of 6.The main type of stomata present on both surfaces is the anomocytic stomata.Other types of stomata present include anisocytic and actinocytic.The leaf is amphistomatic with the adaxial surface bearing a lower number (12.4 to 29.7 to 49.5) than the abaxial surface (24.8 to 42.1 to 49.5).The stomatal index ranges for the adaxial and the abaxial surfaces are 4.8 to 14.2 to 25.0 and 11.1 to 15.5 to 25.0 respectively.The stomata is 0.018 ± 0.004 mm long and 0.019 ± 0.003 mm wide.The vein-islet number and the veinlet terminations were 3.96 to 4.46 and 1.86 to 3.47 respectively.The vein islets are polygonal and the vein islet terminations are fork shaped (Figure 3).

Macromorphology and micromorphology of stem
The woody stem of C. erythrocarpos organoleptically assessed has bitter taste and a musty odor.The young stems are green, old stems are greenish brown (Figure 1B) and the inner parts are creamish white.The stem showed white patches, lenticels and wrinkles which were well distributed all over the outer bark.The pattern of fracture is splintery and it assumes a channeled curvature.
A transverse section of the stem of C. erythrocarpos reveals a lot of branched and stellate trichomes attached to a thick layer of cuticle.Sections from older stems have cicatrices (stumps from broken trichomes).Present was a single layer of epidermal cells (Figure 4), isodiametric parenchyma cells and a pith containing prismatic calcium oxalate crystals and numerous starch grains.The vascular bundle has lignified vessels with outer phloem (Figure 4).
The powdered stem contains a lot of starch grains, numerous vessels and lignified sclerenchymatous cells.

Macromorphology and microscopy of root
The root of C. erythrocarpos has bitter taste.Macromorphological features of the root are similar to the stem (Figure 1C).
The root has one to two layer(s) of epidermal cells overlaid with a cork with no trichome and four to seven layers of cortical collenchyma cells.Highly dispersed through the isodiametric parenchyma cells are large starch grains and stone cells.Present is a collateral vascular bundle and lignified medullary rays consisting of 15-25 rows of parenchyma cells (Figure 5).In the middle lies a pith.All throughout the cells in the section (from the epidermis to the pith) are oil deposits (Figure 5).
The powdered root showed similar structures as the stem but it lacked trichomes and had smaller secretory cells.

Physicochemical principles
Results on ash values have been presented in Table 1 while that of extractive values and pH readings have been presented in Table 2.

Characteristic fluorescence of C. erythrocarpos
This result is as presented in Table 3.

Fourier Transform Infrared fingerprint (FTIR)
The FTIR spectra of the leaf, stem and roots extracts of C. erythrocarpos has been presented in Figure 6.

DISCUSSION
This paper focused on filling the gap in knowledge of standards that guides the correct collection and quality control of C. erythrocarpos parts.

Macromorphology and micromorphology of leaf
Leaf dimensions of C. erythrocarpos are macroscopically similar to those of another member in the genus; Capparis zeylanica.However, C. zeylanica leaves have a rounded base and a mucronate apex. 17C. erythrocarpos has small amphistomatic leaves [18][19] which shorten travel distance of carbon dioxide to mesophyll cells in the leaves. 202] Thick outer epidermal walls, a feature of xerophytes which was confirmed in C. erythrocarpos [23][24][25][26] protects it against drought. 26These observed features are essential for easy identification and authentication of the plant.Safranin, a stain for detecting different types of cells; lignified, cutinized or suberized cell wall 25 confirmed the presence of lignin and suberin in C. erythrocarpos leaves.

Leaf surface data determinations
The stomata of C. erythrocarpos is shorter (0.018 ± 0.004 mm) but broader (0.019 ± 0.003 mm) than another member of the genus, C. spinosa (0.028mm long, 0.010 mm wide). 26Another distinctive feature about C. erythrocarpos leaves is its hyperstomata.Stomatal index, a distinct feature which shows less variability for a given species over stomatal number was determined to aid in C. erythrocarpos' identification. 14The ratio of stomatal number on the adaxial and abaxial surfaces was determined since it is of diagnostic importance. 14The presence of different types of stomata in C. erythrocarpos is essential for unique physiological regulation of water as these stomatal types respond appropriately to changing climatic conditions. 27he vein-islet number and the veinlet termination values were determined as part of identification parameters of C. erythrocarpos. 14Polygonal shaped vein islets and the fork shaped vein islet terminations seen for C. erythrocarpos are similar to those observed in C. sepieria. 24cromorphology and micromorphology of stem C. erythrocarpos stem, similar to C. spinosa possesses thorns. 3,23Also, comparable to C. sepieria, it has thick fibre, vessels arranged within the collateral vascular bundle and small, compact parenchymatous cells. 24imilar to C. erythrocarpos, C spinosa has cortical sclereids but not pericyclic fibres. 24umerous starch grains in stem serves as energy reserves for the plant in adverse weather conditions. 28

Macromorphology and microscopy of root
The observed features are essential for easy identification and authentication of the plant.
The powdered root has smaller secretory cells as compared to the stem.

Physicochemical principles
Ash values of C. erythrocarpos (Table 1) are essential in determination of quality; which focuses on grade, presence of earthy matter and purity of drug. 29pH readings of water and alcohol extracts are weakly acidic to neutral (4.0 -6.54) (Table 2).Extractive values are useful in crude drug evaluation.Values are useful as quality parameters and detection of adulteration in a claimed C. erythrocarpos sample.Physicochemical characterization revealed that water extraction gave a better yield as compared to ethanol (Table 2).

Examination of Phytochemical Constituents
Secondary metabolites present agrees with documented literature of members of this genus and species, C. erythrocarpos. 10,30Capparis erythrocarpos have been reported to possess analgesic, anti-inflammatory, antimicrobial, anthelminthic and hepatoprotective properties. 31This is mainly due to the secondary metabolites present.
Characteristic fluorescence of C. erythrocarpos Characteristic color produced due to the presence of fluorochromes can be used in characterization and subsequent evaluation of crude drug samples (Table 3).

Fourier Transform Infrared Fingerprinting
Fourier transform Infrared was done to obtain fingerprint for quality control of leaves, stem and roots of C. erythrocarpos.

CONCLUSION
This research work has laid down morphological and physicochemical standards that can be used in the quality control of the leaves, stems and roots of C. erythrocarpos.

Figure 2 :
Figure 2: Transverse section of the midrib of a leaf of C. erythrocarpos showing A (X 400): section with Cu=cuticle, Ec=epidermal cell, Cs=Crystal sheath, Cc=collenchyma cell, Pc=Parenchyma cell, X=Xylem, P=Phloem, Pf=Pericyclic fiber, B (X 400)=Safranin stained section; Midrib of leaf of C. erythrocarpos Figure 6 reveals unique fingerprints which could be used as reference standard in the authentication of the leaf, stem and root of C. erythrocarpos.