Pharmacognostical and phytochemical evaluation of the leaves of Ziziphus xylopyrus (Retz) Willd.

BACKGROUND: The fruit decoction of Ziziphus xylopyrus (Retz) Willd. is used toward increase sterility in woman intended birth control in some parts of Rajasthan, India. This plant is widely used in Turkish medicines as a potent sedative. One to two inches of the fresh stem bark of this species are chewed with 1-2 peppers, and the sap swallowed once a day for 5 days in the treatment of cough. Standardization is one of the challenges in herbal medicine. It is essential to evaluate the herbal plants scientifically and proper documentation should be made to know their medicinal properties.
MATERIALS AND METHODS: Leaf samples of Z. xylopyrus were studied as recommended by World Health Organization for morphological, microscopic, physicochemical, phytochemical, powder characteristics and other methods for standardization.
RESULTS: Morphologically the leaves are obovate or orbicular in shape, pinnate venation having aromatic odour and pungent taste. Microscopically leaves showed the presence of ground tissue, vascular strand, xylem and phloem. The crystals are mostly rosette type. Microscopic examination of powder showed the presence of stomata, covering trichomes, sclerenchyma, collenchyma, epidermal cells and vascular strands. Phytochemical screening of the plant part with various solvents revealed the presence of alkaloids, carbohydrates, steroids and sterol, glycosides, saponins, flavonoids, phenolic compounds, triterpenoids in it. Physicochemical parameters such as ash value extractive values were also determined and results showed that water soluble extractive value to be higher than alcohol soluble extractive value.
CONCLUSION: Results may be helpful for further confirmation of selected species and in future these characters may be compared with the new batch of the same plant materials.

INTRODUCTION

Ziziphus xylopyrus family Rhamnaceae is commonly known as Jujab in English.[1] It is a large, straggling shrub or a small tree, armed with spines, up to 4 m in height. Fruits are globose, 3-rarely 2 or 4-celled, with usually a seed in each cell, very hard and woody. It is found in Pakistan and China, North-Western India, Uttar Pradesh, Bihar, Central and South India.[2] The fruit decoction of this plant is used in birth control in some parts of Rajasthan, India.[3] This plant is widely used in Turkish medicine as a potent sedative.[4] Bark and leaf powder paste are applied externally for chest pain arising from cough. In stomach ache and indigestion, fruit powder (3-4 g) is administered with a pinch of ginger powder thrice in a day. The leaves contain quercetin and quecitrin. The bark contains tannins (7.2%) 7, 3, 4-trihydroxy flavan-3, 4-diol and oleanic acid.[5] Z. xylopyrus is used in Pyorrhoea and to check oogenesis.[6] The bark is used for its astringent activity and as dental sticks for teeth cleaning. In different parts of India this plant is also used in the treatment of diarrhoea.[7] Leaf paste is applied on pimples and it is ground along with latex of Ipomea carnea and applied on boils.[8] One to two inches of the fresh stem bark of this species are chewed with 1-2 peppers (Piper nigrum) and the sap swallowed once a day for 5 days in the treatment of cough.[9] Xylopyrine-A and Xylopyrine-B, the two new 13-membered cyclopeptide alkaloids are also present in Ziziphus species i.e. Z. xylopyra.[10] Available literature reveals that pharmacognostic study on leaf was reported,[11] but not extensive study hence the present investigation was undertaken.

Thus, establishment of the pharmacognostic profile of the leaves of Z. xylopyrus will assist in standardization of the latter that can assure quality, purity and identification of samples.

MATERIALS AND METHODS

Plant material

Fresh leaves of the plant were collected in the month of July from Tirupati district, Andhra Pradesh, India. The plant was authenticated by the botanist, Dr. K. Madhavachetty, Assistant Professor, Department of Botany, Sri Venkateshwara University, Tirupati, Andhra Pradesh, India.

Macroscopy

The following macroscopic characters for fresh leaves were noted: Size and shape, color, odor, surface, venation, apex, margin, base, lamina, texture, odor and taste.[1213]

Microscopy

During histological studies for studying venation pattern and trichome distribution, paradermal sections (section taken parallel to the surface of the leaf) as well as clearing of leaf with 5% of sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey's maceration fluid were prepared. Transverse sections of fresh leaves through lamina, midrib and lateral veins stained and observed under Nikon lab photo 2 microscopic unit at projection ×10 and ×40. In microscopy of leaf of Z. xylopyrus followed standard procedure.[1415] Descriptive terms of the anatomical features are as given in the standard anatomy books.[16]

Powder microscopic examination

The powdered leaves were examined for stomata, covering trichomes, sclerenchyma, collenchyma, epidermal cells, vascular strand and other following standard methods.[17]

Phytochemical investigation

Chemical tests were performed in the preliminary phytochemical screening to identify various secondary metabolites such as alkaloids, glycosides, saponins, flavonoids, sterols using the standard procedure described by Kokate et al. and Khandelwal.[1819]

Physicochemical investigations

Physical parameters determined for the powdered leaves such as a percentage of ash values and extractive values were determined according to the well-established official methods and procedure.[2021]

RESULTS

Macroscopic character of leaves of Z. xylopyrus (Retz) Willd

The observed macroscopical characters of leaves of Z. xylopyrus were as follows:

Size: 2-3 to 6-7 cm

Shape: Obovate or orbicular

Color: Green

Odor: Aromatic

Taste: Pungent

Apex: Obtuse

Margin: Serrulate

Base: Oblique, rounded

Venation: Pinnate.

Microscopical studies

The transverse sections of the leaf of Z. xylopyrus (Retz) Willd. Showed the following characters:

Lamina

The leaves consist of abaxially protruding thick and midrib abaxially ridged lamina [Figure 1a].

Figure 1

(a) T.S. of leaf through midrib of Ziziphus xylopyrus (×40). (b) T.S. of leaf through midrib of Z. xylopyrus (×10). (c) T.S. of midrib enlarged of Z. xylopyrus (×10). Ads-adaxial side, La-Lamina, Lv-Lateral vein, MR-Midrib, Vb-Vascular bundle, Ads-Adaxial side, GT-Ground tissue, Ep-Epidermis, Ph-Phloem, x-Xylem

Midrib

The midrib is planoconvex with flat adaxial side and semicircular abaxial part. The midrib is 550 μm thick and 550 μm wide. The epidermal layer of the midrib is thin, less conspicuous, the cells being small and thick walled. The ground tissue is parenchymatous. The cells are circular and compact. Some of the ground cells are dilated and are filled with mucilage. Other cells contain calcium oxalate druses. These cells are similar to normal cells. The vascular strand is single, it is slightly concave, arc shaped strand. It is collateral with adaxial xylem and abaxial phloem. The xylem elements are in long, narrow parallel compact lines [Figure 1b and c]. Pholem occurs in thick abaxial arc and comprises sieve elements and parenchyma cells.

Crystal distribution

Calcium oxalate crystals are abundant in the midrib and lateral veins [Figure 2a]. The crystals are mostly rosette type. The rosette type of crystals was circular plate with spiny margin and central core dark organic substance. The rosettes in the phloem parenchyma of the midrib is smaller while those in the ground parenchyma are larger [Figure 2b]. The crystals are 15-20 μm in diameter.

Figure 2

(a) Crystals in the mesophyll of the lamina (polarised light) of z. xylopyrus (×40). (b) Crystals in the ground tissue of phloem tissue of midrib (polarised light) of z. xylopyrus (×10). (c) T.S. of leaf through lateral veins of ziziphus xylopyrus (×10). cr-crystal, lv-lateral veins, x-xylem, ph-phloem, x-xylem, ade-adaxial epidermis, mt-mesophyll tissue, tc-tannin cells,vs-vascular strand

Lateral veins

The lateral veins are prominent on the abaxial side. They project into thick abaxial ridges and possess small, centrally placed collateral vascular strand [Figure 2c]. The ridges are 100 μm thick.

Powder microscopy

Powder microscopic examination showed the presence of stomata, covering trichomes, sclerenchyma, collenchyma, epidermal cells and vascular strand in Z. xylopyrus [Figure 3a–f].

Figure 3

(a) Covering Trichomes of Ziziphus xylopyrus (×10). CV-Covering trichomes. (b) Epidermal cells of Z. xylopyrus (×10). Ep-Epidermal cells. (c) Collenchyma of Z. xylopyrus (×10). Cc-Collenchyma. (d) Sclerenchyma of Z. xylopyrus (×10). Sc-Sclerenchyma. (e) Stomata of Z. xylopyrus (×10). St-Stomata. (f) Vascular strand of Z. xylopyrus (×10). Vs-Vascular strand

The results of phytochemical screening of the various extracts of Z. xylopyrus leaves revealed the presence of alkaloids, carbohydrates, steriods and sterol, glycoside, saponins, flavonoids, phenolic compounds and triterpenoids shown in [Table 1]. These secondary metabolites are known to possess various pharmacological effects and may be responsible for various actions of Z. xylopyrus.

Table 1

Preliminary phytochemical screening of various extracts of Ziziphus xylopyrus leaves

Physicochemical evaluation parameters

The calculated physicochemical parameters such as ash values, extractive values of the leaves of Z. xylopyrus are presented in Table 2.

Table 2

Physical parameters of the leaves of Ziziphus xylopyrus (Retz) Willd

DISCUSSION

Ethnobotonically, the leaves of this plant were used by local people of Tirupati district, Andhra Pradesh, India in the treatment of various disease conditions without proper standardization techniques. The standardization of crude drug is an integral part of establishing its correct identity. Before any crude drug can be included in herbal pharmacopoeia, pharmacognostic parameters and standards must be established. Microscopic method is one of the simplest and cheapest methods to start with for establishing the correct identity of the source materials.[2223242526] The pharmacognostic standards and phytochemical screening for leaves of Z. xylopyrus have been carried out in an extensive way in this study. The macroscopic characters of leaves can serve as diagnostic parameters. Microscopically leaves showed the presence of parenchymatous ground tissue, single vascular strand, the xylem elements are in long, narrow parallel compact lines. Pholem occurs in thick abaxial arc and comprises sieve elements and parenchyma cells. The crystals are mostly rosette type.

Ash values and extractive values can be used as a reliable aid to detect adulteration. These studies help in the identification of the plant material.[27] Percentage extractives and ash analysis were carried out and results showed that total ash value of leaves is higher than other ash values and water soluble extractive value of leaves was higher than alcohol soluble extractive value. Ash values of drug give an idea of earth matter or the inorganic composition and other impurities present along with drug. Extractive values are preliminarily useful for the determination of exhausted and adulterated drugs. Extractive values are also useful to evaluate chemical composition present in the crude drug and also help in the estimation of specific constituents soluble in particular solvents.[2829]

Preliminary phytochemical screening was carried out and result showed that the presence of alkaloids, carbohydrates, steriods and sterol, glycosides, saponins, flavonoids, phenolic compounds, triterpenoids, Insulin and lignin in ethanolic and aqueous extracts of leaves. This phytochemical screening suggests that the leaves, ethanolic and aqueous extracts of Z. xylopyrus probably contain active lead molecule and this provides the basis for their use as a cure for some human ailments in folklore.

CONCLUSION

These parameter which reported for first time, could be useful in setting the diagnostic characters for the identification and preparation of a monograph of the Z. xylopyrus plant.