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Category  Antipyretic.

       Uncaria Hook is the dried hook-bearing branch of Uncaria rhynchophylla (Miq.) Miq. ex Havil. and possibly other species of Uncaria (Family Rubiaceae).  It contains not less than 0.2 per cent of total alkaloids calculated as isorhynchophylline (C₂₂H₂₈N₂O₄), on the dried basis.

Origin of plant  Uncaria Hook is distributed throughout East Asia from South China to Vietnam, Taiwan, and Japan.

Consituents  Uncaria Hook contains indole alkaloids such as isorhynchophylline and rhynchophylline.  It also contains flavonoids, quinic acids and tannins.

Description  Odour mild; taste, bland. 
Macroscopical  Fragments of stem bearing paired hooks; stem cylindrical or sub-square, 2 to 3 cm long, 2 to 5 mm wide, externally reddish brown and glabrous, longitudinally finely striated, petiole scars and arc-shaped stipule scars usually present; hook slightly flattened or rounded and curved downwards, 1 to 5 cm long, apex acute, base broad, mostly occur in paired, opposite to each other along the stem, occasionally single.  
Microscopical  Transverse section of the stem shows a central cavity or a spongy, whitish pith. 
Transverse section of the hook shows vascular bundles arranged in a ring and unevenly distributed throughout the cortex; and parenchyma containing sandy crystals of calcium oxalate in the secondary cortex.
Powdered drug of Uncaria Hook shows dermal tissues, phloem parenchyma, pericyclic fibres, sclereids of cortical parenchyma, xylems, medullary rays, and pith. Dermal tissue: irregular polygonal epidermal cells covered with thick cuticle, and collenchyma.  Phloem parenchyma, some containing sandy crystals of calcium oxalate.  Pericyclic fibres, numerous, with thick and unchannelled walls.  Sclereids of cortical parenchyma, slightly thickening walled and obliquely pitted cells varying greatly in shape and size, isolated or in small groups.  Xylem: large bordered-pitted vessels, bordered-pitted tracheids and annular vessels, all of which often associated with the outer layer of pith with channel-walled cells.  Medullary rays, numerous bordered pitted thick-walled rectangular cells.  Pith, rounded to slightly pitted-walled ovoid cells.

Packaging and storage  Uncaria Hook shall be kept in well-closed containers and stored in a dry place.

Identification
     A.  To 1 g of the powdered drug add 20 mL of methanol, boil under a reflux condenser on a water-bath for 5 minutes, and filter.  Evaporate the filtrate to dryness, add 5 mL of dilute acetic acid to the residue, warm the mixture on a water-bath for 1 minute, and filter after cooling.  Spot one drop of the filtrate on a filter paper, air-dry, spray Dragendorff’s TS for spraying on it, and allow to stand for a while:  a yellow-red colour develops.
     B.  Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1).
          Standard solution  Dissolve 1 mg of isorhynchophylline and 1 mg of rhynchophylline in 2 mL of methanol.
          Test solution  To 1.0 g of the powdered drug add 1 mL of strong ammonia solution.  After 30 minutes, add 25 mL of ethyl acetate and sonicate for 20 minutes.  Filter and evaporate the filtrate to dryness under reduced pressure.  Dissolve the residue in 1 mL of methanol, centrifuge and use the supernatant.
          Adsorbent  Silica gel F254 (HPTLC plate)
          Mobile phase  Water, methanol, and dichloromethane (1:15:125).
          Application  Apply 5 µL each of Standard solution and Test solution as 8-mm bands.
          Development and drying  Allow the solvent front to ascend 6 cm above the line of application.  Dry the developed plate in air.
          Detection  Examine the plate under ultraviolet light (254 nm).  
          Results  When examined under ultraviolet light (254 nm), the test solution shows a quenching band due to isorhynchophylline at about the middle and a quenching band due to rhynchophylline in the lower third of the chromatogram, corresponding in R_f value to the bands shown by the standard solution.

Loss on drying  Not more than 10.0 per cent w/w after drying at 105° for 2 hours (Appendix 4.15).

Total ash  Not more than 3.0 per cent w/w (Appendix 7.7).

Assay  Carry out the test as described in the “Liquid Chromatography” (Appendix 3.5).  (Note  Use freshly prepared solutions.  Store and inject them at 5°, using a cooled autosampler.)
     Standard preparation (a)  Dissolve about 5 mg of Isorhynchophylline RS, accurately weighed, in a mixture of equal volumes of methanol and dichloromethane and dilute to 25.0 mL with the same mixture of solvents.  Dilute 1.0 mL of the solution to 10.0 mL with methanol. 
     Standard preparation (b)  Dissolve about 5 mg of Uncaria Stem with Hooks Dry Extract for System Suitability RS, accurately weighed, in 1.0 mL of methanol.  Filter through a 0.45-µm membrane filter. 
     Assay preparation  Weigh accurately about 500 mg of the powdered Uncaria Hook and add 3.0 mL of strong ammonia solution.  After 30 minutes, add 50.0 mL of a mixture of equal volumes of methanol and dichloromethane.  Sonicate for 30 minutes.  Filter and evaporate the filtrate to dryness under reduced pressure.  Dissolve the residue in methanol and dilute to 10.0 mL with the same solvent.  Filter through a 0.45-µm membrane filter. 
     Mobile Phase:
     MOBILE PHASE A  Dilute 0.15 mL of diethylamine to 1000 mL with water. 
     MOBILE PHASE B  Acetonitrile.
The step gradient of mobile phases is as follows:

       Chromatographic system
             DETECTOR  Ultraviolet light (245 nm) 
             COLUMN A stainless steel column (15 cm × 4.6 mm), packed with octadecylsilyl silica gel for chromatography with extended pH range (5 µm), end-capped.
              TEMPERATURE  
                    Column 5°
             FLOW RATE  1.0 mL per minute
          System suitability
             SAMPLE  Standard preparation (b)
          Suitability requirements
             RESOLUTION  Not less than 1.5 between alkaloid and isorhynchophylline peaks.
             RELATIVE STANDARD DEVIATION  Not more than 0.2 per cent for total alkaloids.
         Procedure  Inject a portion (about 10 mL) of Standard preparation (a), Standard preparation (b) and Assay preparation into the chromatograph.  Measure the areas of the principal peaks in each chromatogram.  Identify the peaks due to alkaloids 1, 2, 3, 4, 6, 7, 8, and 9 and isorhynchophylline in the assay preparation using the retention time of isorhynchophylline in the standard preparation (a) and the approximate relative retention times of 0.50, 0.58, 0.94, 0.97, 1.05, 1.13, 1.22, and 1.27 for alkaloids 1, 2, 3, 4, 6, 7, 8, and 9 in the standard preparation (b), respectively, with reference to the isorhynchophylline peak.
         Calculation  Calculate the percentage content of total alkaloids, calculated as isorhynchophylline, in the portion of the Uncaria Hook taken by the expression,

A(WP)/25aw

in which A is the sum of the areas of the peaks due to alkaloids 1, 2, 3, 4, 6, 7, 8, and 9 and the peak due to isorhynchophylline obtained from Assay preparation; W is the weight, in g, of Isorhynchophylline RS in Standard preparation (a); P is the declared content of C₂₂H₂₈N₂O₄ in Isorhynchophylline RS; a is the peak area due to isorhynchophylline obtained from Standard preparation (a); and w is the weight, in g, of Uncaria Hook in Assay preparation.