Research On The Quality Standardization Of Turmeric And Toad Venom

Turmeric and toad venom are traditional Chinese medicines and have a wide range of medicinal properties.Both are recorded in Chinese Pharmacopoeia(2015).Turmeric has the effects of breaking blood and relieving pain.Therefore,it is commonly used for the treatment of thoracic tingling,chest pain,dysmenorrhea,rheumatism,and other diseases.In Chinese pharmacopoeia(2015),quality control was carried out for turmeric by TLC identification,examinations(water content,total ash content),quantitative analysis of curcumin and volatile oil.The existing quality control indicators can not fully evaluate the quality of turmeric.Therefore,a more comprehensive quality evaluation method of turmeric was established in this study,including chemical fingerprinting,quantitative analysis of curcumin,demethoxycurcumin and bisdemethoxycurcumin,determination of volatile oil,determination of heavy metal,tests of moisture and ash,and so on.The turmeric quality grade standard was established.Toad venom is a precious and toxic Chinese medicine.It is the dry secretion of the Bufo bufo gargarigans and B.melanostictus.According to Chinese pharmacopoeia(2015),the quality control of toad venom include identification,inspection(water content,total ash content,acid insoluble ash content)and quantitative analysis(the total content of cinobufagin and resibufogenin is not less than 6.0%).In addition to cinobufagin,resibufogenin,bufalin has better anti-tumor and cardiac effects.In view of the high activity,high toxicity,narrow safety window and easy poisoning of steroidal components of toad venom,it is urgent to establish a comprehensive quality evaluation system.1 Study on quality standardization of turmeric1.1 Thin-layer chromatography identificationTLC method was developed for qualitative analysis of the four Curcuma species(Curcuma phaeocaulis,C.kwangsiensis,C.wenyujin,and C.longa).The chromatography was performed on silica gel 60F254 plate eluted with chloroform-methanol-formic acid(96:4:0.7,v/v/v).Through comparison of the reference herbs,92 batches of turmeric and 39 batches of turmeric prepared slices were identified as authentic samples.1.2 Quantitative analysis of curcuminoids componentsA total of 92 batches of turmeric and 39 batches of turmeric slices were collected from different cultivation areas,which were extracted by methanol in ultrasonic bath for 45 min,and the contents of three major curcuminoids(curcumin,demethoxycurcumin,and bisdemethoxycurcumin)were analyzed by HPLC.The samples were separated on a Zorbax SB-C18 column at 30 0C,eluted with a gradient of CH3CN and 0.1%formic acid at 1.0 mL/min,and detected at 430 nm.The results showed the contents of three curcuminoids varied remarkably.In samples collected from Guangxi and Yunnan,the average content of curcumin in turmeric is 0.54%,the average content of three curcuminoids is 1.31%;the content of curcumin in the prepared slices of turmeric is 0.44%,and content of three curcuminoids is 0.67%.In samples collected from Sichuan and Guizhou,the average content of curcumin in turmeric is 1.5%,the average content of three curcuminoids is 2.66%;the average content of curcumin in prepared slices is 1.49%,and the content of three curcuminoids is 2.53%.Chinese Pharmacopoeia specifies the content of curcumin in turmeric is not less than 1.0%,and the content of curcumin in the prepared slices is not less than 0.9%.In order to further improve the quality of turmeric,the turmeric grade standard was established.Turmeric crude drugs:first-class,curcumin content?1.5%,content of three curcuminoids?2.5%;second-class,curcumin content?1.5%,2.0%?content of three curcuminoids ?2.5%;1.0%?curcumin content<1.5%,content of three curcuminoids?2.0%.The prepared slices of turmeric:first-class,curcumin content?1.4%,content of three curcuminoids ?2.2%;second-class,0.9%?curcumin content,1.5?content of three curcuminoids<2.2%.1.3 Fingerprinting analysisHPLC-DAD method was used to establish the fingerprint analysis method for turmeric,and systematic methodological investigation was carried out.Fingerprint analysis was carried out on different origins of medicinal materials,and standard fingerprints were established accordingly.There are 7 common peaks in the fingerprint of turmeric,and 4 of them were identified as curcumin,demethoxycurcumin,bisdemethoxycurcumin,and ar-Turmerone.The similarity index of different batches of samples were calculated.The index for turmeric crude drugs was between 0.7 and 1,and the index for turmeric prepared slices was above 0.9,indicating the quality was stable.1.4 Determination of heavy metal contentsThe contents of heavy metals Cu,As,Cd and Pb in turmeric and prepared slices were determined by inductively coupled plasma mass spectrometry(ICPMS).For 57.89%of the samples,the cadmium concentrations were above the required limit,and samples from Sichuan and Guangxi were seriously polluted.The contents of the other elements meet the limit standard.1.5 Other detection itemsAccording to the "Chinese Pharmacopoeia"method to detect the moisture,total ash,and volatile oil of turmeric and prepared slices,the test results show that the moisture and total ash in the sample are in compliance with the relevant standards of the pharmacopoeia;in the detection of volatile oil content,60%of the turmeric crude drug samples meet the requirements,and 96%of the prepared slices meet the requirements.2 Study on quality standardization of toad venom2.1 Microscopic identification studyMicroscopic identification was used to identify adulterated substances in toad venom.When 60%sulfuric acid was used,37 batches of samples were observed under the lens with a small amount of insoluble matter(skin,plant fiber,sandstone,etc.).Using iodine liquid loading,6 batches of samples were observed to contain starch.2.2 Thin-layer chromatography identificationThe thin-layer chromatography method established in this study can identify the authenticity of the medicinal materials.The chromatography was performed on silica gel 60F254 plate with cyclohexane-trichloromethane-acetone(3:3:2,v/v/v).As a results,cinobufagin,resibufogenin and bufalin were well separated.37 batches of toad venom samples were recognized as authentic.2.3 Quantitative analysis of toad venomIn this study,37 batches of samples were collected and analyzed by HPLC.The samples were separated on a Zorbax SB-C18 column,eluted with a gradient of CH3CN and 0.1%formic acid at a flow rate of 1.0 mL/min.The multi-evaluation method was used to determine the components of toad venom.The contents of cinobufagin and resibufogenin ranged from 0.03%-1.49%;the content of bufalin ranged from 0.48%-1.99%.According to the Chinese pharmacopoeia,the total content of cinobufagin and resibufogenin in toad venom is not less than 6.0%.5 batches of samples comply with the pharmacopoeia requirements.Finally,the following quality standard for toad venom was proposed:the total content of cinobufagin and resibufogenin is not less than 6.0%,and the content of bufalin is not less than 1.0%.2.4 Fingerprinting analysisThe fingerprints of different origins were analyzed,and the standard fingerprints were established.There were 10 common peaks in the fingerprints,and 8 peaks were identified by comparing with reference standards(11?--hydroxybufalin,arenobufagin,5-hydroxybufalin,bufotaline,5-hydroxycinobufagin,bufalin,cinobufagin,resibufogenin).2.5 Other detection itemsAccording to the Chinese Pharmacopoeia,the total ash content of the samples was determined.The results showed that the total ash content in the samples met the relevant standards of the pharmacopoeia.The inductively coupled plasma mass spectrometry was used to determine the contents of heavy metals Cu,As,Cd and Pb.Among the 13 batches,only one batch contained higher amounts of copper,and contents of the other elements meet the requirements.