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                        Ziziphus Attopensis Vine is the dried stem of Ziziphus attopensis Pierre (Z. trichocarpa H. T. Chang) (Family Rhamnaceae), Herbarium Specimen Number: DMSC 5381, Crude Drug Number: DMSc 1269.

Constituents  Ziziphus Attopensis Vine contains triterpenoids such as betulinic acid. It also contains lupeol, etc. 

Description of the plant  (Fig. 1)  Climbing shrub, up to 6 m tall; branch subcylindrical, greenish grey to brownish, pubescent when young, becoming purplish grey or reddish brown pubescent when aged, often densely lenticellate; stipulary thorn 1 per node, recurved. Leaves simple, alternate, ovate to elliptic, 7 to 14 cm long, 3 to 6 cm wide, apex acute to acuminate, base rounded, slightly oblique, margin serrulate, chartaceous to subcoriaceous, abaxially pubescent, distinctly triplinerved, midrib adaxially sunken, glabrous; petiole slender, 0.4 to 1 cm long, glabrous. Inflorescence compound cyme or panicle, terminal and/or axillary, up to 25 cm long; peduncle dichotomously divided 2 to 3 times, densely brownish pubescent. Flower yellowish green, 3 to 4 mm in diameter; pedicel slender, 4 to 8 mm long; hypanthium shallow, disc-shaped, pubescent; sepals 5, triangular, densely pubescent, apex acute; petals 5, creamy, spathulate, shorter than sepal, shortly clawed; stamens 5, slightly shorter than petal; ovary superior, globose, densely pubescent, apically 2-clefted, basally connate. Fruit a drupe, ellipsoid to globose, 1.9 to 2.2 cm long, 1.3 to 1.8 cm wide; stipe 0.5 to 1 cm long, pubescent. Seed 1, oblong-ellipsoid, about 1.3 mm long, about 1.1 mm wide, reddish brown. 

Description  Odour, mild; taste, bland.

                        Macrosopical  (Fig. 1)  Entire or fragmented pieces of transverse or longitudinal sliced stems, varied in shape and size; externally, greenish brown to dark brown, rough; internally, yellowish brown to brown, smooth.

                        Microscopical  (Figs. 2a, 2b)  Transverse section of the stem shows periderm, cortex, phloem, xylem, and pith. Periderm: several layers of rectangular cork cells, some containing brown substances. Cortex: parenchyma cells with brown substances and a sclereid band. Phloem: fibres, phloem rays containing starch grains, phloem parenchyma, some containing brown substances and starch grains. Xylem: vessels, axial parenchyma, some containing brown substances and starch grains, xylem fibres, and xylem rays, containing starch grains. Pith: parenchyma, some containing brown substances and prismatic crystals, and secretory ducts, containing brown substances.

                Ziziphus Attopensis Vine in powder possesses the diagnostic microscopical of the unground drug. Cork showing pitted canals, some containing brown substances, is characteristic.

Additional information  The crude drug used in Thai traditional medicine as “Kamlang Suea Khrong” refers to two different plant sources and parts: Kamlang Suea Khrong, Plueak Ton (the bark of Betula alnoides Buch.-Ham. ex D. Don) and Kamlang Suea Khrong, Thao (the dried stem of Ziziphus attopensis Pierre). Traditionally, both sources are used under the  Thai name “กำลังเสือโคร่ง” (Kamlang Suea Khrong).

Packaging and storage  Ziziphus Attopensis Vine shall be kept in well-closed containers, protected from light, and stored in a dry place.

Identification

A. Sonicate 5 g of the sample, in powder, with 10 mL of methanol for 30 minutes and filter. Evaporate the filtrate to dryness. Dissolve the residue in 2 mL of acetic anhydride, shake well, and slowly add 1 mL of sulfuric acid to form a layer: a brownish red colour develops at the zone of contact.

B. 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 70 volumes of toluene, 20 volumes of ethyl acetate, and 2.5 volumes of glacial acetic acid as the mobile phase and allowing the solvent front to ascend 8 cm above the line of application. Apply separately to the plate as bands of 8 mm, 6 μL each of the following solutions. Prepare solution (A) by refluxing 5 g of the sample, in powder, with 100 mL of methanol for 2 hours, filtering, evaporating the filtrate to dryness, then, adding 1 mL of methanol to 25 mg of the residue, and sonicating for a few minutes. For solution (B), dissolve 1 mg of betulinic acid in 1 mL of methanol. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm), marking the quenching bands. Subsequently examine the plate under ultraviolet light (366 nm) through the cut-off filter; four red and five blue fluorescent bands are observed. Spray the plate with a 10 per cent v/v solution of sulfuric acid in methanol and heat at 105ºfor 15 minutes. The chromatogram obtained from solution (A) shows a purple band (hRf value 47 to 54) corresponding to the betulinic acid band from solution (B). One purple, two orange, and six brownish purple bands are also observed (Fig. 3).

Loss on drying  Not more than 9.0 per cent w/w after drying at 105º 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 3.0 per cent w/w (Appendix 7.6).

Total ash  Not more than 5.0 per cent w/w (Appendix 7.7).

Ethanol-soluble extractive  Not less than 1.0 per cent w/w (Appendix 7.12).

Water-soluble extractive  Not less than 4.0 per cent w/w (Appendix 7.12).