Pharmacognostic evaluation of leaves of certain Phyllanthus species used as a botanical source of Bhumyamalaki in Ayurveda.

Today, World over, there is a great deal of interest in Ayurvedic system of medicine and thus the demand for various medicinal plants in the production of Ayurvedic medicines is ever increasing. Due to varied geographical locations where these plants grow, a great deal of adulteration or substitution is encountered in the commercial markets. Histological studies of the plant drugs are not only to study the adulterants but also are indispensable in accurate identification. Microscopic observations of the Phyllanthus species revealed the occurrence of anisocytic and paracytic type of stomata in Phyllanthus amarus , while only anisocytic type of stomata is present in P. fraternus and P. maderaspatensis. Epidermal cell walls of P. amarus and P. fraternus are wavy and straight walled epidermal walls are observed in P. maderaspatensis. In India all the above-mentioned species of Phyllanthus are called "Bhumyamalaki" and they are being used in the treatment of various liver disorders. However, all the species of Phyllanthus doesn't have the active constituents responsible for the treatment of liver disorders. In the present investigation by using simple micro techniques accurate identification of different species of Phyllanthus has been established.

Introduction

The World Health Assembly in resolutions has emphasized the need to ensure the quality of medicinal plant products by using current control techniques and applying appropriate standards.[1] Typical pharmacognostical studies are normally quite adequate for quality control of herbal drugs.[2] Pharmacognostical standardization of herbal drugs include macroscopic, microscopic, physio-chemical constants and fluorescence analysis of investigated parts, and to evolve standards for single drugs and compound preparations in order to validate genuineness of the crude drugs of plant, mineral and animal origin. The study includes to highlight the macroscopical and microscopical characters as distinctive features for authentication and identification purposes.[34] According to WHO (1998), the macroscopic and microscopic description of a medicinal plant is the first step toward establishing its identity and purity and should be carried out before any test are undertaken.[5] In this direction, the plant drug “Bhumyamalaki,” one of the promising herbal drugs used in Indian system of medicine for various liver disorders is attributable to Phyllanthus niruriLin.[6] belongs to the family Euphorbiaceae. P. niruri is endemic to West Indies and not found in India.[7] Various Phyllanthus species like P. amarus Schum and Thonn, P. fraternus Web., P. maderaspatensis Linn., P. simplex Retz., P. urinaria Linn. are being termed as “Bhumyamalaki.”[8] Literature survey reveals that “Bhumyamalaki” has been used to treat jaundice, gonorrhea, frequent menstruation, dysentery, and diabetes.[9-11] It has also been used as a treatment for skin ulcers, sores, swelling, etc.[12] Phyllanthus focuses on its potential for fighting viruses specifically with hepatitis B virus.[1314] Studies suggest that Phyllanthus may suppress the growth and replication of the virus and may decrease the amount of hepatitis B virus found in the blood stream. It has not been reported to remove the viruses, but it is effective in suppressing the symptoms in addition fighting with hepatitis B virus. Phyllanthus may also support the overall health of the liver.[15]

Review of literature revealed that only few handful workers studied the structural details of P. fraternus. Saha and Krishna Murthy (1959) studied the structural details of P. fraternus Web.[16] Later Yelene et al., carried out the leaf structural studies.[17] Khatoon et al., studied three species of Phyllanthus.[18] So far there is no published report about the comparative microscopic diagnostic characters of all different species of Phyllanthus known for its hepatoprotective activity. Histological studies of the plant drugs are not only to study the adulterants but also are indispensable in the accurate identification.

The present paper deals with the distinguishing characters of different species of Phyllanthus by using simple microscopic techniques.[19-21]

Materials and Methods

The fresh plant materials of Phyllanthus amarus Schum and Thonn., Phyllanthus fraternus Web., Phyllanthus maderaspatensis Linn., Phyllanthus simplex Retz., and Phyllanthus urinaria Linn. were collected from the foot hills of Western Ghats, India, and the voucher samples were deposited at Department of Pharmaceutical Sciences, Guru Jambheshwar University, Hisar, Haryana, India. All the samples were dried in shade. To study the epidermal structural studies for the dried leaf structural studies, first the epidermal imprints of the leaf lamina were prepared. A domestic adhesive (Quick fix) was applied uniformly on both the upper and lower epidermis of the dried leaves. It is then dried at room temperature. The dried transparent film of “Quick fix” is then stripped off carefully from the surface of the leaf and placed on a clean dry glass slide with the imprint surface uppermost. Cover slip is placed over it and lightly tapped to flatten the film. It is then observed under a compound microscope. Line drawings were made by using mirror-type camera lucida.[1322]

Results

Epidermal cell walls of P. amarus, P. fraternus, and P. urinaria are wavy but P. amarus consist of paracytic and anisocytic type of stomata [Figure 1a], while P. fraternus [Figure 1b] and P. urinaria [Figure 1e] possess anisocytic and paracytic type of stomata, respectively. In the case of P. maderaspatensis [Figure 1c] and P. simplex [Figure 1d] the epidermal cell walls are straight and they showed anisocytic and paracytic type of stomata respectively. In P. amarus at some place the margin of leaf lamina possess papillae like out growths [Figure 2a]. In P. fraternus club-shaped cells are seen lying in an overlapping manner [Figure 2b]. The plant P. simplex showed dentate type cells all along the margins of leaf lamina [Figure 2d]. Trichome like two-celled out growths is observed all along the lamina margin of P. urinaria [Figure 2e], whereas P. maderaspatensis have regular cells along the lamina margin [Figure 2c].

Figure 1

(A-E) Lower epidermis of the lamina in surface view 583 x - P. amarus, P. fraternus, P. maderaspatensis, P. simplex, and P. urinaria, respectively, Ac: Anisocytic type of stomata, Le: Lower epidermis, Lm: Lamina margin, Pa: Parenchyma tissue, Pc: Paracytic type of stomata, Ps: Palisade cells, Ue: Upper epidermis, Vb: Vascular bundle

Figure 2 (A-E)

Leaf margin in surface view 583 x - P. amarus., P. fraternus, P. maderaspatensis, P. simplex, and P. urinaria, respectively

[Figure 3 (A-D)]: Crystals of calcium oxalate 583 x - P. amarus, P. maderaspatensis, P. simplex, and P. urinaria, respectively

(Figure 4): Transverse section of midrib showing (arrowed) discontinuous palisade cells 583 x - P. amarus, Ac: Anisocytic type of stomata, Le: Lower epidermis, Lm: Lamina margin, Pa: Parenchyma tissue, Pc: Paracytic type of stomata, Ps: Palisade cells, Ue: Upper epidermis, Vb: Vascular bundle

A cluster crystals of calcium oxalate are observed in the ground tissue of P. amarus [Figure 3a], P. maderaspatensis [Figure 3b], P. urinaria [Figure 3d], and P. simplex [Figure 3c]. Rarely prismatic crystals of calcium oxalate are observed in P. amarus. Crystals are found to be absent in P. fraternus.

In P. maderaspatensis and P. urinaria, the palisade tissue is continuous even in the midrib region while it is discontinuous and restricted to only lamina blade in P. amarus [Figure 4], P. fraternus, and P. simplex.

Table 1 illustrates the distinguishing characters of various species of Phyllanthus.

Table 1

Comparative microscopic diagnostic characters of various Phyllanthus species

Discussion

Phyllanthus niruri Linn. mentioned in the flora of British India (Hooker, 1887) and found on the labels of number of Indian Herbal formulations is a mixture of five distinct species, namely P. amarus Schum and Thonn, P. fraternus Web., P. maderaspatensis Linn., P. simplex Retz., P. urinaria Linn. In recent times based on the clinical efficacy, P. niruri mentioned in the flora of British India and “Bhumyamalaki” mentioned in the classical literature are equated with P. amarus.[23] However, due to the morphological similarities, all the above-mentioned five species of Phyllanthus are mixed up and being sold in herbal drug markets of the country by the same vernacular name. From the present investigation by using very simple practical methodology diagnostic characters of all the five studied species of Phyllanthus are developed.

Conclusion

The microscopic diagnostic characters drawn from the present investigation by using simple techniques will help in authenticate genuine samples of Phyllanthus used in liver disorders. This is a first such report on the comparative microscopic diagnostic characteristics on “Bhumyamalaki.”