1、1 cmFigure 1 A photograph of Cortex Moutan12Cortex Moutan1. NAMESOfficial Name: Cortex MoutanChinese Name: .nullnonmarkingreturn Chinese Phonetic Name: Mudanpi2. SOURCECortex Moutan is the dried root bark of Paeonia suffruticosa Andr. (Fam. Ranunculaceae). The root iscollected in autumn, the rootlet
2、s removed, the bark stripped off, then dried in the sun to obtain CortexMoutan.3. DESCRIPTIONQuailed or semi-quailed shape, longitudinally fissured, both edges of the cortex usually curved inwards,varying in size and up to 20 cm in length, 516 mm in diameter, 14.5 mm in thickness. Externally darkbro
3、wn or yellowish-brown, showing transverse lenticels, outer surface reddish-brown, pink or yellow-white after the removal of cork; the inner surface pale greyish-yellow or pale brown, with obvious finelongitudinal striation, usually showing bright crystals. Texture hard and fragile, easily broken, fr
4、acturerelatively even, pale pink or greyish-white, and mealy. Odour, aromatic; taste, slightly bitter and astringent.(Fig. 1)4. IDENTIFICATION4.1 Microscopic Identification (Appendix III)Transverse sectionThe transverse section shows cork consisting of several rows of cells. Cortex consisting of 102
5、1rows of parenchyma, which are prolonged tangentially. Pholem broad, occupying up to 80% inradial direction of transverse section; phloem rays 13 cells wide. Clusters of calcium oxalate, 650 m in diameter, sometimes crystal cells joined, arranged in rows, or one to several clusters inone cell. (Fig.
6、 2)13Cortex MoutanPowderWhite to greyish-brown. Fairly abundant starch grains present in parenchymatous cells; simplegranules suborbicular or polygonal, 327 m in diameter, with dotted, cleft or V-shaped hilum;compound granules composed of 26 units, showing a black, cross shape when examined under ap
7、olarizing microscope. Clusters of calcium oxalate 650 m in diameter, crystal cells sometimesjoined, arranged in rows or one to several clusters in one cell; showing a polychrome when examinedunder a polarizing microscope. Cork cells rectangular, slightly thick-walled. (Fig. 3)4.2 Physicochemical Ide
8、ntificationProcedureWeigh 1.0 g of the powdered sample and put into a 100-mL conical flask, then add 20 mL ofdiethyl ether. Shake the mixture for 2 min. Filter and transfer 5 mL of the filtrate to a test tube.Evaporate to dryness on a water bath at about 60 C, then cool to room temperature. Cautious
9、lyadd about 1 mL of nitric acid to the test tube, a clear greenish-blue solution is observed.4.3 Thin-Layer Chromatographic Identification Appendix IV(A)Standard solutionsPaeoniflorin standard solutionWeigh 2.0 mg of paeoniflorin and dissolve in 1 mL of methanol.Paeonol standard solutionWeigh 2.0 mg
10、 of paeonol and dissolve in 1 mL of ethyl acetate.Developing solvent systemPrepare a mixture of chloroform, ethyl acetate, methanol and formic acid (40:5:10:0.2, v/v).Spray reagentMix 1 mL of dilute sulphuric acid (50%, v/v) and 10 mL of p-hydroxybenzaldehyde in methanol(2%, w/v). Freshly prepare th
11、e reagent.Test solutionWeigh 1.0 g of the powdered sample and put into a 100-mL conical flask, then add 20 mL ofmethanol. Sonicate the mixture for 30 min, filter and then evaporate to dryness at reduced pressurein a rotary evaporator. Dissolve the residue in 1 mL of ethanol.Cortex Moutan14Figure 2 M
12、icroscopic features of transverse section of Cortex MoutanA. Sketch B. Section illustration C. Clusters of calcium oxalate scatteredD. Clusters of calcium oxalate arranged in a row1. Cork 2. Cortex 3. Phloem 4. Clusters of calcium oxalateACDB1234123450 m50 m 100 mCortex Moutan15Figure 3 Microscopic
13、features of powder of Cortex Moutan1. Starch grains 2. Starch grains in parenchymatous cells 3. Cork cells4. Clusters of calcium oxalate 5. Clusters of calcium oxalate arranged in a rowa. Features under a light microscope b. Features under a polarizing microscope1a 1b2a 3a4a 4b5a 5b50 m 50 m50 m 50
14、m50 m50 m50 m 50 m16Cortex MoutanProcedureCarry out the method by using a HPTLC silica gel F254plate and a freshly prepared developingsolvent system as described above. Develop over a path of about 4 cm. Apply separately paeoniflorinstandard solution (3 L), paeonol standard solution (5 L) and the te
15、st solution (2 L) to the plate.After the development, remove the plate from the chamber, mark the solvent front and dry inair. Spray the plate evenly with the spray reagent and heat at about 125 C for about 15 min.Examine the plate in visible light. Calculate the Rfvalue by using the equation as ind
16、icated inAppendix IV(A).For positive identification, the sample must give spots or bands with chromatographiccharacteristics, including the colour and the Rfvalue, corresponding to those of paeoniflorin andpaeonol.4.4 High-Performance Liquid Chromatographic Fingerprinting (Appendix XII)Standard solu
17、tionPaeonol standard stock solution, Std-Stock (2000 mg/L)Weigh 4.0 mg of paeonol and dissolve in 2 mL of methanol.Paeonol standard solution for fingerprinting, Std-FP (200 mg/L)Pipette 0.5 mL of paeonol Std-Stock to a 5-mL volumetric flask and make up to the mark withmethanol.Test solutionWeigh 0.2
18、 g of the powdered sample and put into a 50-mL centrifugal tube, then add 10 mL ofmethanol and weigh. Sonicate the mixture for 30 min and weigh again. Add an appropriate amountof methanol to compensate the weight loss, if any. Mix and centrifuge at about 3000 x g for 5 min.Filter through a 0.45-m PT
19、FE filter.Chromatographic systemThe liquid chromatograph is equipped with a detector (254 nm) and a column (4.6 x 250 mm)packed with ODS bonded silica gel (5 m particle size). The flow rate is about 0.8 mL/min.Programme the chromatographic system as follows 17Cortex MoutanTime Water AcetonitrileElut
20、ion(min) (%, v/v) (%, v/v)020 10080 020 linear gradient2040 8070 2030 linear gradient4060 7050 3050 linear gradientSystem suitability requirementsPerform at least five replicate injections each with 10 L of paeonol Std-FP. The requirements ofthe system suitability parameters are as follows: the RSD
21、of the peak area of paeonol should benot more than 5.0%; the RSD of the retention time of paeonol peak should be not more than 2.0%;the column efficiency determined from paeonol peak should be not less than 50,000 theoreticalplates.The R value between peaks 3 and 4 (Fig. 4) in the test solution shou
22、ld be not less than 2.0.ProcedureSeparately inject paeonol Std-FP and the test solution (10 L each) into the HPLC system andrecord the chromatograms. Measure the retention time of paeonol peak in the chromatogram ofpaeonol Std-FP and the retention times of the five characteristic peaks (Fig. 4) in t
23、he chromatogramof the test solution. Under the same HPLC conditions, identify paeonol peaks in the chromatogramof the test solution by comparing its retention time with that in the chromatogram of paeonol Std-FP. The retention times of paeonol peaks from the two chromatograms should not differ by mo
24、rethan 2.0%. Calculate the RRTs of the characteristic peaks by using the equation as indicated inAppendix XII.The RRTs and acceptable ranges of the five characteristic peaks of Cortex Moutan extract arelisted in Table 3.Table 3 The RRTs and acceptable ranges of the five characteristic peaks of Corte
25、x Moutan extractPeak No. RRT Acceptable Range1 0.36 0.032 (paeoniflorin) 0.45 0.033 0.71 0.034 0.77 0.035 (marker, paeonol) 1.00 -18Cortex MoutanFigure 4 A reference fingerprint chromatogram of Cortex Moutan extractFor positive identification, the sample must give the above five characteristic peaks
26、 with RRTsfalling within the acceptable range of the corresponding peaks in the reference fingerprintchromatogram (Fig. 4).5. TESTS5.1 Heavy Metals (Appendix V): meet the requirements.5.2 Pesticide Residues (Appendix VI): meet the requirements.5.3 Mycotoxins (Appendix VII): meet the requirements.5.4
27、 Foreign Matter (Appendix VIII): not more than 1.0%.5.5 Ash (Appendix IX)Total ash: not more than 5.0%.Acid-insoluble ash: not more than 1.0%.5.6 Water Content (Appendix X): not more than 13.0%.19Cortex Moutan6. EXTRACTIVES (Appendix XI)Water-soluble extractives (cold extraction method): not less th
28、an 19.0%.Ethanol-soluble extractives (cold extraction method): not less than 18.0%.7. ASSAYCarry out the method as directed in Appendix IV(B).Standard solutionMixed paeoniflorin and paeonol standard stock solution, Std-Stock (1000 mg/L each)Weigh accurately 10.0 mg of paeoniflorin and 10.0 mg of pae
29、onol, and dissolve in 10 mL of methanol.Mixed paeoniflorin and paeonol standard solution for assay, Std-ASMeasure accurately the volume of the mixed paeoniflorin and paeonol Std-Stock, dilute with methanolto produce a series of solutions of 5, 25, 100, 200, 400 mg/L for both paeoniflorin and paeonol
30、.Test solutionWeigh accurately 0.2 g of the powdered sample and put into a 50-mL centrifugal tube, then add accurately10 mL of methanol and weigh. Sonicate the mixture for 30 min and weigh again. Add an appropriateamount of methanol to compensate the weight loss, if any. Mix and centrifuge at about
31、3000 x g for 5min. Filter through a 0.45-m PTFE filter.Chromatographic systemThe liquid chromatograph is equipped with a detector (230 nm) and a column (4.6 x 250 mm) packedwith ODS bonded silica gel (5 m particle size). The flow rate is about 0.8 mL/min. Programme thechromatographic system as follo
32、ws Time Water AcetonitrileElution(min) (%, v/v) (%, v/v)010 9070 1030 linear gradient1020 7020 3080 linear gradientSystem suitability requirementsPerform at least five replicate injections each with 10 L of the mixed paeoniflorin and paeonol Std-AS(200 mg/L). The requirements of the system suitabili
33、ty parameters are as follows: the RSD of the peakareas of paeoniflorin and paeonol should be not more than 5.0%; the RSD of the retention times of20Cortex Moutanpaeoniflorin peak and paeonol peak should be not more than 2.0%; the column efficiency determinedfrom paeoniflorin peak and paeonol peak sh
34、ould be not less than 50,000 theoretical plates.The R value between paeoniflorin peak and the closest peak in the test solution should be not less than1.5.Calibration curvesInject a series of the mixed paeoniflorin and paeonol Std-AS (10 L each) into the HPLC system andrecord the chromatograms. Plot
35、 the peak areas of both paeoniflorin and paeonol against the correspondingconcentrations of the mixed paeoniflorin and paeonol Std-AS. Obtain the slopes, y-intercepts and the r2values from the corresponding 5-point calibration curves.ProcedureInject 10 L of the test solution into the HPLC system and
36、 record the chromatogram. Identify paeoniflorinpeak and paeonol peak in the chromatogram of the test solution by comparing their retention times withthose in chromatogram of the mixed paeoniflorin and paeonol Std-AS. The retention times of paeoniflorinpeaks and paeonol peaks in both chromatograms sh
37、ould not differ from their counterparts by more than5.0%. Measure the peak areas and calculate the concentrations (in milligram per litre) of paeoniflorinand paeonol in the test solution, and calculate the percentage contents of paeoniflorin and paeonol in thesample by using the equations indicated in Appendix IV(B).LimitsThe sample contains not less than 0.49% of paeoniflorin (C23H28O11) and not less than 1.1% of paeonol(C9H10O3), calculated with reference to the dried substance.
