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        Citrus Hystrix Leaf is the dried leaf of Citrus hystrix DC. (C. papeda Miq.,  C. tuberoides J. W. Benn.) (Family Rutaceae), Herbarium Specimen Number:  DMSC 342. 

Constituents  Citrus Hystrix Leaf contains volatile oil, of which citronellal, citronellol and citronellol acetate are its major components; sabinene, α- and β-pinene, α-phellandrene, limonene, α- and γ-terpinene, cymene, linalool, indole alkaloids are also found.  It also contains rutin, hesperidin and diosmin.

Description of the plant  (Figs. 1a, 1b)  Shrub or small tree 2 to 12 m tall; branchlets compressed-angular when young; spine solitary, 0.5 to 1.5 cm long.  Leaves alternate or spirally arranged, very young leaves light reddish purple; winged petioles obovate to obcordate-oblong, 1 to 8 cm long, 1 to 4.5 cm wide; lamina orbicular-ovate or ovate-oblong, 3 to 15 cm long, 2.5 to 6 cm wide, apex obtuse to obtusely acuminate or slightly emarginate, base cuneate or rounded, margin more or less crenate. Inflorescence axillary or terminal, 1- to 15-flowered, fragrant; pedicel 2 to 5 mm long; calyx cupular, 1 to 1.5 mm  long, 4-lobed; petals 4 to 5, oval-oblong, 6 to 10 mm long, 3 to 5 mm wide, obtusely acuminate, yellowish white or suffused with violet, with scattered pellucid dots; stamens 24 to 30, free, filaments 2 to 6 mm long; ovary superior, subglobose.  Fruit ellipsoid, ovoid or globose,  5 to 7 cm in diameter, irregular bumpy; fruit-pulp yellowish green, very sour, slightly bitter.  Seeds ovoid-oblong, 1 to 1.5 cm long, 3 to 5 mm wide. 

Description  Odour, characteristic, aromatic; taste, slightly bitter.

      Macroscopical  (Fig. 1a)  Citrus Hystrix Leaf occurs as a mixture of entire and broken, greenish brown to brown dried leaves.  Petioles obovate, 1 to 8 cm long, 1 to 4.5 cm wide.  Lamina ovate, 3 to 15 cm long, 2.5 to 6 cm wide; apex obtuse or retuse or slighty emarginate, undulate at margin.

      Microscopical  (Figs. 2a, 2b)  Transverse section of the leaf shows upper epidermis, a layer of rectangular cells in sectional view or slightly wavy-walled cells in surface view, stomata absent.  Mesophyll composed of 2 to 3 layers of palisade and several layers of spongy cells with idioblasts containing calcium oxalate prismatic crystals; fibrovascular bundle and schizolysigenous oil cavity containing oil droplets located in spongy tissues.  Fibrovascular bundle composed of fibre surrounding amphicribal vascular bundle; xylem composed of spiral, reticulate, and bordered-pitted vessels and xylem parenchyma some of which  containing prismatic crystals of calcium oxalate.  Collenchyma, 2 to 3 layers of cells, located in the central part of midrib beneath lower epidermis.  Lower epidermis, a layer of rectangular cells in sectional view or wavy-walled cells in surface view with anomocytic type of stomata.

     Citrus Hystrix Leaf in powder possesses the diagnostic microscopical characters of the unground drug.

Packaging and storage  Citrus Hystrix Leaf shall be kept in well-closed containers,  preferably of metal or glass, protected from light and stored in a cool and dry place.  It should be used within 1 year and air-dried every 2 to 3 months.

Identification

      A. Reflux 1 g of the sample, in powder, with 30 mL of ethanol for 15 minutes, and filter (solution 1).  To 2 mL of solution 1, add a few drops of ammonium molybdate TS:  a yellowish green precipitate is produced.

      B. To 2 mL of solution 1, add a few drops of a freshly prepared 1 per cent w/v solution of iron(III) chloride:  a deep greenish brown colour is produced.

      C. Extract 1 g of the sample, in powder, with 10 mL of chloroform, shake occasionally for 20 minutes, and filter.  Evaporate 2 mL of the filtrate to dryness, dissolve the residue in 2 mL of acetic anhydride, and add slowly 1 mL of sulfuric acid to make two layers:  a brown colour forms at the zone of contact and the upper layer is green.

      D. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance and chloroform as the mobile phase.  Apply to the plate, 10 μL of the test solution prepared by refluxing 1 g of the sample, in powder, with 30 ml of ethanol for 15 minutes, filtering, and concentrating the filtrate to 5 mL.  After removal of the plate, allow it to dry in air, and examine under ultraviolet light (254 nm), marking the quenching spots.  Examine the plate under ultraviolet light (366 nm) (Table 1); see also Fig. 3.  Spray the plate with anisaldehyde TS and heat at 105° for 5 minutes.  Several spots of different colours are observed (Table 1); see also Fig. 3.

Table 1 hR_f Values of Components in Ethanolic Extract of the Leaves of Citrus hystrix DC.

Water  Not more than 11.0 per cent v/w (Azeotropic Distillation Method, Appendix 4.12).

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 17.0 per cent w/w (Appendix 7.7).

Ethanol-soluble extractive  Not less than 6.0 per cent w/w (Appendix 7.12).

Water-soluble extractive  Not less than 18.0 per cent w/w (Appendix 7.12).

Chloroform-soluble extractive  Not less than 5.0 per cent w/w (Appendix 7.12H).

Volatile oil  Not less than 0.6 per cent v/w (Appendix 7.3H).  Use 25 g of hand-crushed leaves, accurately weighed.  Use 250 mL of water as the distillation liquid and a 500-mL round-bottomed flask.  Distil at a rate of 2 to 3 mL per minute for 5 hours.  Use 2.0 mL of xylene in the graduated tube.