Equisetum crude drugs : Epidermal Characters for Identification

Epidermal characters of nine of the Central European Equisetum species were documented using both scanning electron and light microscopy. The arrangement of silica pilulae on the subsidiary cells of stomata as well as the type and the arrangement of papillae on the ridges of main stems and branches allow an unambiguous identification of the species even in the powdered state. A key for the identification is presented, and recommendations for an improved text for the monograph in the European Pharmacopeia are given.


Introduction
The unambiguous identification of individuals of the genus Equisetum sometimes is a challenge even when complete herbarium specimens are on hand.
Botanical keys [e.g.I] recommend the preparation of transverse sections of the stems: the proportion of the hollows in the stem in combination with morphological characters of the nodes and leaf sheaths should allow a determination of the species.
In the monograph of Equisetum arvense of the European Pharmacopoeia emphasis is laid on the paracytic stomata with typical ridges of the superimposed subsidiary cells and on U-shaped epidermal cells, which should be discernible in a transverse section.Our preliminary investigations revealed that the mentioned characters of the stomata are present in all species of the genus Equisetum.
Furthermore the U-shaped epidermal cells are characters of the ridges of the branches, which are typically seen in the surface view.Therefore a revision of the monograph seemed to be appropriate.
The powder of Equisetum anlense mainly consists of non typical parenchyma and vessels.Only the few fragments showing the epidermis with stomata and ridges with papillae1 may serve for identification.Promising data are published from investigations using scanning electron microscopy (SEM) [2, 3, 41 or ash preparations [5, 61, which focus on silica incrusted pilulae and mamillae.These structures are poorly visible in the light microscope.An attempt for the identification of powdered material was done by Schier [7], he presented a coarse overview of the papillae on the ridges.
In order to provide feasible characters for the proof of identity, the objective of this study was a systematic collection of characters of the stomata, the epidermal cells and of the structure of the ridges of Central European Equisetum species by means of light microscopy with support of SEM, leading to a proposal for the monograph in the European Pharmacopoeia.

Su bqenus Himochaete (E. varieaatum, E. hvemale, E. ramosissimum)
The three species are characterized by deepened stomata (app.30 pm below the level of the epidermis), which are arranged mostly in a single exact vertical row at ' Definition of terms Pilulae: silica incrusted hemispherical or globose bead-like objects, 1-2 pm in diameter and length.
Papillae: projections of the cell wall, typically at the ridges of the stems, partly forming horizontal bars reaching the flanks of the grooves.each flank of a ridge (= 2 single rows between the ridges, examples given in Figs.1-4).Moreover the surface of the superimposed subsidiary cells is completely smooth.Silica pilulae, which would be discernible as small black dots in the light microscope, are absent.The epidermis between the stomata is nearly planar in E.
hyemale, only flattish horizontal ridges with few mamillae in the grooves between are present.In E. variegatum and E. ramosissimum conspicuous mamillae are abundant, additionally the latter is densely covered with silica pilulae.The subsidiary cells of all investigated species of the subgenus Equisetum are in plane with the epidermal cells.Two main types of distribution of silica pilulae on the subsidiary cells are present: a) the pilulae form a distinct margin, the area in between is uniformly scattered with smaller pilulae [E.arvense (Fig. 5), E. sylvaticum, E. fluviatile]; b) the pilulae are uniformly scattered, dense at the pore, sometimes getting fewer towards the margin of the subsidiary cell, a distinct margin of pilulae is absent [E.telmateia, E. palustre (Fig. 6), E. pratense].Although these pilulae are,not rich in contrast the arrangement is discernible in the light microscope, too (Fig. 7, 8).
The type of the distribution of silica pilulae on the subsidiary cells is identical on the main stem and on the branches.
On larger fragments in a powder the arrangement of stomata may be discernible.Stomata in a single row or in two close rows per flank in the main stem are characteristic for E. sylvaticum and E. pratense, while in E. anlense the stomata occur in rows of (2)-3-4.Stomata in more than 4 axial rows are typical for E. palustre, particularly towards the ridges the stomata appear in horizontal rows too.All these species is common that the central part of the grooves is free of stomata.
In contrast, in E. fluviatile the stomata are scattered all over the grooves.On the main stem of E. telmateia stomata are nearly absent, the branches resemble in respect to the arrangement of stomata those of E. anlense.
The size of the guard cells is hardly discernible because of the superimposed subsidiary cells.Their size seems to be too variable within a species to serve as differential character.In surface view in the light microscope the area of ridges in the subsidiary cells is usually larger than the area covered with silica pilulae.The variability of the density of the ridges in the subsidiary cells seems to be too large to serve as character for differentiation.Unicellular papillae in the typical sense occur on the ridges of the main stem of E. sylvaticum, they stand apart and reach a length of about 80 pm (Fig. 11, k).In E. palustre (main stem) the papillae are linked to horizontal aggregates with a smooth surface (Fig. I I , f, Fig. 13 and Fig. 15).
The papillae of the main stem of E. pratense are bicellular, the two cells are arranged at right angle to the axis.Therefore these papillae seem to consist of a single cell only when observed in longitudinal lateral view in the light microscope (Fig. 11, g and Fig. 17).The length of the papillae varies considerably.
In powdered material the ridges of main stems will be seen frequently in surface view.The projections of the papillae may pretend the presence of cell borders.On the ridges of the main stem of E. aniense the papillae frequently form short elongated groups at right angle to the epidermal cells (Fig. 14).The smooth surface of the horizontal bands of papillae in E. palustre are clearly discernible in the light microscope (Fig. 15).[7] presented drawings of the papillae of nearly all relevant species, unfortunately no differentiation between main stem and branches was done.In addition the arrangement of the papillae on the ridges remained unconsidered, some drawings differ considerably from our material.The papillae in lateral longitudinal view seem to be highly characteristic, particularly when combined with their arrangement on the ridges.The most similar parts are the branches of E. anlense and E. fluviatile.These species differ in their pattern of flavonoids, the presence of E. fluviatile could be detected by TLC [5].
An improved monograph of the European Pharmacopoeia should consider in addition to the presence of ridges on the subsidiary cells also the details of the silica pilulae on the subsidiary cells (forming a distinct margin), the number of rows of stomata per flank (2-4), and the presence of two types of papillae (low 2-celled on main stem, rectangular 2-celled on branches).
Proposal for the European Pharmacopoeia: Fragments of the epidermis in surface view, composed of rectangular cells with wavy walls and paracytic stomata in 2-4 rows, the subsidiary cells are in plane with the epidermis and cover the guard cells, they have cell wall thickenings in the shape of conspicuous radial ridges, small silica pilulae are scattered on the sutface and form a distinct margin; papillae on the ridges 2-celled, on the main stem low, on the branches large and rectangular.Fragments of large-celled parenchyma, fibres and vessels with spiral or annular wall thickening.
However, the best description of a powder doesn't allow a proof of identity and purity with the same accuracy like a proof of unmilled material.Even when the Pharmacopoeia claims to powder the sample prior to microscopic examination the identity and purity of a sample should assured preferably prior to this step, because the highly characteristic papillae can be clearly seen even with a stereomicroscope without a specific sample preparation.