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                        Enhalus Acoroides Rhizome is the dried rhizome, with remnant of leaves, of Enhalus acoroides (L. f.) Royle (E. koenigii Rich., Stratiotes acoroides L. f.) (Family Hydrocharitaceae), Herbarium Specimen Number: DMSC 5382, Crude Drug Number: DMSc 1257.

Constituents  Enhalus Acoroides Rhizome contains flavonoids and their derivatives (e.g., apigenin, luteolin, and rutin). It also contains phenolic acids, tannins, sterols, etc.

Description of the plant  (Fig. 1)  Aquatic herb, dioecious, submerged; rhizome creeping, up to 1.5 cm in diameter, covered with black fibrous remaining of old leaves; roots whitish, numerous, up to 30 cm long, up to 6 mm in diameter. Leaves simple, distichous, mostly 2 to 6, at apex of rhizome, strap-like, 0.3 to 1.5 m long, 1.2 to 1.8 cm wide, apex rounded or obtuse, sheathing at base, margin entire, thickened by persistent, coarse, tough, parallel veins; leaf sheath compressed, up to 15 cm long, membranous, translucent. Male inflorescence cymose, axillary and/or terminal; peduncle 5 to 10 cm long; bracts 2, sessile, slightly keeled, midvein hairy; male flowers numerous, whitish; pedicel 0.3 to 1.3 cm long; sepals 3, reflexed, oblong, about 2 mm long; petals 3, erect, broader than sepal, about 1.8 mm long; stamens 3, 1.5 to 2 mm long. Female flower solitary, axillary; peduncle 40 to 50 cm long, coiling; bracts 2, sessile, 4 to 6 cm long, 1 to 2 cm wide, greenish red to reddish green, with rough red hairy along keels and nerves; sepals 3, reddish, margin recurved; petals 3, yellowish white with pink dots, 4 to 5 cm long, 3 to 4 mm wide, folded; ovary inferior, ovoid, 6-loculed, densely long fringe-like pubescent. Fruit fleshy, greenish to brownish, ovoid, 5 to 7 cm long, apex acuminate, pericarp rough hairy, with 2 persistent bracts and elongate-coiling peduncle. Seeds 4 to 12, angular, 1 to 1.5 cm long and wide, greenish.

Description  Odour, salty; taste, salty.

                        Macroscopical  (Fig. 1)  Cylindrical rhizomes, rarely branched, some slightly curved, 0.5 to 1.3 cm in diameter, covered with stiff black or light brown hairs, the remaining vein and veinlet, 3.2 to 18 cm long, and root scars; cut surface brown with dark brown to blackish circular ring in the centre.

                Microscopical  (Figs. 2a‒2c)  Transverse section of the rhizome shows epidermis, cortex, and vascular tissue. Epidermis: a layer of rectangular cells. Cortex: several layers of thick-walled parenchyma, some containing darkbrown substance and/or orange to brown tannin and/or numerous large or small starch grains; aerenchyma; and pseudoendodermis, a layer of irregularly shaped cells. Vascular tissue: collateral vascular bundles, scattered in the
cortex, non-lignified fibres, and vessels.

                Transverse section of the hair shows numerous non-lignified bast fibres with brown substance and vascular tissue on one side.

                Enhalus Acoroides Rhizome in powder possesses the diagnostic microscopical characters of the unground drug. Bundle of non-lignified fibres of hair is characteristic. The combination of parenchyma containing orange to brown tannin, parenchyma containing various sizes of starch grains, and aerenchyma should be characteristic.

Packaging and storage  Enhalus Acoroides Rhizome shall be kept in well-closed containers, protected from light, and stored in a dry place.

Identification

A. Reflux 2 g of the sample, in fine powder, with 25 mL of ethanol for 10 minutes and filter. To 2 mL of the filtrate, add 2 to 3 pieces of magnesium ribbon, shake well, and mix with a few drops of hydrochloric acid: a pinkish red colour is produced.

B. Boil 1 g of the sample, in fine powder, with 20 mL of water for a few minutes and filter. To 1 mL of the filtrate, add a few drops of iron(III) chloride TS and shake well: a greenish brown colour is produced.

C. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance and a mixture of 45 volumes of toluene, 45 volumes of ethyl acetate, 6 volumes of formic acid, and 1.5 volumes of methanol as the mobile phase and allowing the solvent front to ascend 10 cm above the line of application. Apply separately to the plate as bands of 8 mm, 10 µL of solution (A) and 2 µL of solution (B). Prepare solution (A) by refluxing 2 g of the sample, in fine powder, with 25 mL of ethanol for 10 minutes and filtering. For solution (B), dissolve 1 mg of luteolin in 1 mL of ethanol. After removal of the plate, allow it to dry in air. Examine the plate under ultraviolet light (254 nm); marking the quenching bands. The chromatogram obtained from solution (A) shows a quenching band (hRfvalue 51 to 55) corresponding to the luteolin band obtained from solution (B). Subsequently examine the plate under ultraviolet light (366 nm); the band corresponding to luteolin shows a dark fluorescence. Other three blue fluorescent bands are observed. Spray the plate with anisaldehyde TS, heat at 105° for 10 minutes, and examine under ultraviolet light (366 nm); the band due to luteolin is green fluorescent. One dark, two blue, and two white fluorescent bands are also observed (Fig. 3).

                Repeat the same procedure on another plate. After removal of the plate, allow it to dry in air. Heat the plate at 80° for 10 minutes and then spray the plate with natural products (NP) TS while the plate is still warm. Subsequently spray the plate with polyethyleneglycol (PEG) TS and observe the colours of the bands under ultraviolet light (366 nm) within 5 to 15 minutes; the band due to luteolin is orange fluorescent. One blue and one yellow
fluorescent bands are also observed (Fig. 3).

Loss on drying  Not more than 10.0 per cent w/w after drying at 105° to constant weight (Appendix 4.15).

Foreign matter  Not more than 2.0 per cent w/w (Appendix 7.2).

Acid-insoluble ash  Not more than 1.0 per cent w/w (Appendix 7.6).

Total ash  Not more than 14.0 per cent w/w (Appendix 7.7).

Ethanol-soluble extractive  Not less than 2.0 per cent w/w (Appendix 7.12).

Water-soluble extractive  Not less than 8.0 per cent w/w (Appendix 7.12).