| Anemarrhena is the dry plant rhizome of the Anemarrhena asphodeloides Bge.in Liliaceae which has the effects of clearing heat and fire,nourishing yin,and moistening.Anemarrhena Rhizoma could be found around the country,especially in the Heibei and Shanxi Provinces.Although the Zhangjiakou Area,Hebei Province,is rich in the resource of Anemarrhena Rhizoma,it still lacks of the unified standard for quality evaluation currently.Therefore,to ensure the medication safety and availability,it is of great significance to identify the Anemarrhena Rhizoma produced from the Zhangjiakou Area.The identify and measure of content should be done.Meanwhile,the fingerprint,mutual mode,as well as the unified standard for quality control also should be established.Modern pharmacological studies show the compositions of steroidal saponins and diphenypyridone in the Anemarrhena rhizome could inhibit ?-glucosidase in the small intestine effectively,decreasing the absorption area of the small intestine on the sugar,and delaying the absorption time,resulting in reducing postprandial blood sugar.Therefore,basing on the pharmacological action of Anemarrhena Rhizoma could decreases the blood sugar,the inhibitory effect of the Anemarrhena Rhizoma extractives on the ?-glucosidase in vitro was investigated to make perfect evaluated the quality of the Anemarrhena Rhizoma produced from the Zhangjiakou Area.Through compared the differences on the identification of medicinal materials,moisture,ash,acid-insoluble ash,active ingredient content,fingerprints,and drug efficacy between the Zhangjiakou region and other areas of Anemarrhena Rhizome to control the quality of Anemarrhena Rhizome in Zhangjiakou and also to provided an effective method for the identification of Anemarrhena Rhizome in Zhangjiakou.1.Identification of the Anemarrhenae RhizomaThe Anemarrhena Rhizoma produced from different areas can be identified by the following two methods.One is the morphological identification: 25 samples of known hard texture,internal cross-sectional color yellow-white,shiny,slightly gas,slightly sweet taste,slightly bitter,sticky,Mao Anemarrhena Rhizoma have roots,Anemarrhena Rhizoma Meat without roots,different origin of Anemarrhena Rhizoma not obvious differences are in line with the provisions of the national pharmacopoeia.The other is the thin-layer identification on the basis of the thin layer chromatography(TLC,appendix VI B).The chromatogram of the test product showed the same fluorescent spots at the positions corresponding to the chromatogram of the reference substance.There is no significant difference in the chromatogram of Anemarrhena Rhizoma from different origins,but it can be used as a qualitative identification of Anemarrhena Rhizoma.2.Determination of moisture,total ash,and acid-insoluble ash contentsAccording to the second method of the appendix general rule(0832)in the Chinese Pharmacopoeia(2015 edition),the moisture content of the Anemarrhenae Rhizoma was determined,which indicates that the water content of wild Anemarrhena Rhizoma meat from Zhangjiakou is 7.77% ~ 8.21%,averaging 7.99%,the moisture content of planted Anemarrhena Rhizoma meat from Zhangjiakou is 7.33% and 8.76%,averaging 8.00%.The moisture content of planted Anemarrhena Rhizoma meat from other areas is 8.77% ~ 9.35%,averaging 9.07%,which is 1.07% higher than that in Zhangjiakou.The moisture content of the planting Anemarrhena Rhizoma roots from Zhangjiakou is 7.32%~8.05%,averaging 7.83%.The moisture content of Anemarrhena Rhizoma roots from other areas is 7.64% ~ 9.50%,averaging 8.51%,which is 0.68% higher than that in Zhangjiakou.Not more than 12.00% of the Pharmacopoeia requirements are met.According to the “Chinese Pharmacopoeia” Appendix General Principles(2302)method of the 2015 edition,the total ash content of wild Anemarrhena Rhizoma meat from Zhangjiakou is 4.12% ~ 5.40%,averaging 4.67%.The total ash content of planted Anemarrhena Rhizoma meat from Zhangjiakou is 5.09% ~ 5.73%,averaging 5.39%.The total ash content of planted Anemarrhena Rhizoma meat from other areas is 4.72%~5.98%,averaging 5.39%.The total ash content of the planting Anemarrhena Rhizoma roots from Zhangjiakou is 3.84% ~ 5.96%,averaging 5.20%.The total ash content of Anemarrhena Rhizoma roots from other areas is 4.73% ~ 5.67%,averaging 5.20%.Are in compliance with the provisions of the pharmacopoeia can not exceed 9.00%.According to the “Chinese Pharmacopoeia” Appendix General Principles(2302)method of the 2015 edition,acid-insoluble ash determination of Anemarrhena Rhizoma showed that the insoluble ash content of wild Anemarrhena Rhizoma meat from Zhangjiakou is 1.35% ~ 1.62%,averaging 1.47%.The acid-insoluble ash content of planted Anemarrhena Rhizoma meat from Zhangjiakou is 1.48%~1.64%,averaging 1.58%.The content of acid-insoluble ash of planted Anemarrhena Rhizoma meat from other areas is 1.52% ~ 1.72%,averaging 1.58%.The content of acid-insoluble ash of the planting Anemarrhena Rhizoma roots from Zhangjiakou is 1.54%~1.78%,averaging 1.70%.The content of acid-insoluble ash of Anemarrhena Rhizoma roots from other areas is 1.53%~1.89%,averaging 1.72%.Are in compliance with the provisions of the pharmacopoeia must not exceed 4.00%.3.Determination of the total flavonoids and total saponins contentsAfter ultrasonic extraction for 30 min with 70% alcohol,centrifuging,add 5% sodium nitrite solution 0.5 m L,shake,place 6 min,10% aluminum nitrate solution 0.5 m L,shake,place 6 min,add 5% sodium hydroxide solution 5 m L,70% ethanol to 25 m L and obtaining the supernatant solution,the absorbance is measured at the wave length of 258 nm,which indicates that the total flavonoids content of wild Anemarrhena Rhizoma meat from Zhangjiakou is 3.56% ~ 4.92%,averaging 4.16%.The total flavonoids content of planted Anemarrhena Rhizoma meat from Zhangjiakou is 2.01% ~ 2.65%,averaging 2.35%.The total flavonoids content of planted Anemarrhena Rhizoma meat from other areas is 1.29%~3.35%,averaging 2.29%,which is 0.06% lower than that in Zhangjiakou.The total flavonoids content of the planting Anemarrhena Rhizoma roots from Zhangjiakou is 1.03% ~ 4.92%,averaging 2.92%.The content of total flavonoids of Anemarrhena Rhizoma roots from other areas is 1.57%~3.83%,averaging 2.88%,which is 0.04% lower than that in Zhangjiakou.The samples were extracted ultrasonically for 30 min with 30% acetone,centrifuging,and obtaining the supernatant solution;then adding the phosphoric acid 6.0 m L and 1.0% p-dimethylamino benzaldehyde ethanol solution 2.0 m L,and cooling for 5 min in ice water bath after heating for 60 min in 60?water bath;finally adding absolute ethyl alcohol 4.0 m L,30% acetone to 25 m L,shaking up and measuring the absorbance at the wave length of 520 nm.The result shows that the total saponins content of wild Anemarrhena Rhizoma meat from Zhangjiakou is 7.42% ~ 8.12%,averaging 7.74%.The total saponins content of planted Anemarrhena Rhizoma meat from Zhangjiakou is 6.78% ~ 7.58%,averaging 7.18%.The total saponins content of planted Anemarrhena Rhizoma meat from other areas is 6.45% ~ 7.57%.averaging 6.97%,which is 0.21% lower than that in Zhangjiakou.The total saponins content of the planting Anemarrhena Rhizoma roots from Zhangjiakou is 6.65%~8.24%,averaging 7.59%.The content of total saponins of Anemarrhena Rhizoma roots from other areas is 6.76%~8.32%,averaging 7.57%,which is 0.02% lower than that in Zhangjiakou.4.Determination of neomangiferin and mangiferin contentsThe neomangiferin and mangiferin contents were measured with the chromatographic column of the Syncronis C18(50 mm × 2.1 mm,1.7 ?m)and acetonitrile and water(13:87)as the mobile phase under the conditions of the 0.25 m L·min-1 fluid rate,258 nm wave length,35 ? column temperature,5 ?L injection volume,and 6 min running time.The results indicate that the linear ranges of neomangiferin and mangiferin are 1.25 ~ 80 ?g·m L-1and 1.875~120 ?g·m L-1,respectively.The content of neomangiferin in wild Anemarrhena Rhizoma meat from Zhangjiakou area is 1.44%~2.90%,averaging 2.22%,and the content of neomangiferin planted in Anemarrhena Rhizoma meat is 0.94%~1.65%,averaging 1.28%.The content of neomangiferin in the other producing areas is 0.49%~1.86%,averaging 1.12%,which is 0.16% lower than that in Zhangjiakou.The content of neomangiferin in planted Anemarrhena Rhizoma roots from Zhangjiakou is 0.98%~2.61%,averaging 1.68%.The content of neomangiferin in the other producing areas is 0.66%~1.88%,averaging 1.29%,which is 0.39% lower than that in Zhangjiakou.The content of mangiferin in wild Anemarrhena Rhizoma meat from Zhangjiakou area is 1.45%~2.58%,averaging 1.73%,the planted mangiferin content is 0.83%~1.20%,averaging 0.96%.In other areas,the content of Anemarrhena Rhizomae meat mangiferin is 0.33%~1.71%,averaging 0.97%,which is 0.01% higher than that in Zhangjiakou.The content of mangiferin planting roots in Zhangjiakou area is 0.93%~2.09%,averaging 1.40%.The content of Anemarrhena Rhizomae in plants of other production areas is 0.70%~1.79%,and the average content of mangiferin is 1.43%,which is 0.03% higher than that in Zhangjiakou.5.Establishment the fingerprint of Anemarrhena RhizomaThe fingerprint analysis was performed on ACQUITY UPLC? HSS T3 (100 mm × 2.1 mm,1.8 ?m),The mobile phase was consisted of water(A)and acetonitrile(B)in gradient elution(0~2 min,5%~20%B;2~5 min,20%~25%B;5 ~ 10 min,25%~100%B),The flow rate was 0.25 m L· min-1.The determined wavelength was 210 nm.The temperature of column was 35?.Sample injection volume was 5?L and the run time was 10 min.The ultra high performance liquid chromatography(UPLC)fingerprint and common pattern were established from the 10 samples of Zhangjiakou area and 5 samples of other areas of Anemarrhena Rhizoma,and 16 chromatographic peaks were identified as the basic characteristic peaks.15 samples were subjected to cluster analysis.The results was divided into 2 groups.The fingerprints of Zhangjiakou samples from No.1 to No.11 in No.1were taken to establish the fingerprint and common pattern of Zhangjiakou region,and 20 peaks were identified as common peak for 11 samples.The similarity analysis results of the fingerprint common patterns of the Zhangjiakou region and the 15 fingerprints of the Anemarrhena Rhizoma samples showed that the similarities of the common patterns and the fingerprints of No.1 to No.11 samples were all above 0.95,reflecting that the Anemarrhena Rhizoma of Zhangjiakou areas were very similar in the overall chemical structure,and there is no significant difference between wild and planted Anemarrhena Rhizoma,which provides a basis for artificial planting.7.Inhibitory of Anemarrhena Rhizoma extractives on the ?-glucosidaseThe inhibitory of Anemarrhena Rhizoma extractives on the ?-glucosidase was in vestigated with the 4-Nitrophenyl ?-D-glucopyranoside(PNPG)method.Results show that the inhibitory rate of wild Anemarrhena Rhizoma meat from Zhangjiakou area is 90.35%~95.44%,averaging 91.80%.The inhibitory rate of planted Anemarrhena Rhizoma meat is 73.71%~86.54%,averaging 80.68%.The Anemarrhenae Rhizoma from other producing areas which indicates that the inhibitory rate is 59.16%~89.86%,averaging 78.80%,which is 1.88% lower than that in Zhangjiakou.The inhibition rate of planting roots of Anemarrhena Rhizoma in Zhangjiakou area is 85.37%~93.76%,and the average inhibition rate is 90.00%.The inhibitory rate of planting Anemarrhena Rhizoma from other areas is 81.74% to 90.69%,averaging is 87.2%,which is 2.8% lower than that of Zhangjiakou area.By the correlation analysis of the inhibitory rate of the ?-glucosidase,it could be found that the mangiferin and total saponins have a great inhibitory effect on the ?-glucosidase while the inhibitory effect from the neomangiferin is not obvious.Thus,it is suggested that mangiferin could be used as the the quality of traditional Chinese medicine(Q-Marker)of Anemarrhena Rhizoma,which is helpful to establish its whole quality control and quality traceability system. |
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