A Preliminary Study On The Quality Control Of The Traditional Chinese Medicine Huangsi Yujin And Its Anti-inflammatory Activity

Curcuma Longa Radix is the dry roots of Zingiberaceae Curcuma longa L.,which has the effect of promoting blood circulation and relieving pain,relieving qi stagnation,clearing away the heart and cooling blood,and relieving gallbladder and removing yellow,Curcuma Longa Radix is mainly distributed in Shuangliu,Chongzhou,Wenjiang,Xinjin and Qianwei,and is Sichuan genuine medicinal material.Modern pharmacological research showed that Curcuma Longa Radix played an important role in anti-tumor,anti-inflammatory,blood activating and stasis removing,hemostasis,lipid-lowering,antioxidation,antipyretic,analgesic,liver protection and other pharmacological effects.Objective:"Chinese Pharmacopoeia"(2015 Edition)only specified the characteristics,identification,moisture and total ash content of Curcuma Longa Radix,but the extract,content determination,fingerprint and safety check were not included to guarante the quality control of Curcuma Longa Radix.Therefore,it is necessary to establish a comprehensive and reasonable quality control methods,so as to provide basis for improving the quality standard of Curcuma Longa Radix in the future.Methods:1.Dichloromethane-methanol-formic acid(12:0.5:0.1)was used as the developing solvent to identify Curcuma Longa Radix.Toluene method was used to check the moisture content of Curcuma Longa Radix,and total ash method was applied to determine the total ash content of Curcuma Longa Radix.The dilute ethanol was used as solvent to determine the ethanol soluble extract of Curcuma Longa Radix,The content of volatile oil was determined by steam distillation.High performance liquid chromatography(HPLC)was used to simultaneously determine the content of curcumin,demethoxycurcumin and bisdemethoxy-curcumin in Curcuma Longa Radix,meanwhile,the methodological study was conducted to provide scientific methods for improving the quality standard of Curcuma Longa Radix,Gas chromatography(GC)combined with similarity evaluation,cluster analysis and principal component analysis were used to establish the fingerprint of volatile oil of Curcuma Longa Radix.2.Inductively Coupled Plasma-Mass Spectrometry(ICP-MS)was used to determine the contents of 5 heavy metal elements,including lead,cadmium,mercury,arsenic and copper in Curcuma Longa Radix.Meanwhile,gas chromatography(GC)was used to detect benzene hexachloride,dichlorodiphenyltrichloroethane,pentachloronitrobenz-ene,hexachlorobenzene,heptachlor,cis heptachlor epoxy,trans-heptachlor epoxy,aldrin,cis chlordane,trans chlordane,and chlordane oxide.The residues of organochlorine pesticides were evaluated for safety of Curcuma Longa Radix.3.On the basis of the conventional methods of Yu Jin medicinal materials,the modern evaluation methods in this paper was used to make a comprehensive evaluation of Curcuma Longa Radix,and initially establishes its grade standard.4.In this experiment,the anti-inflammatory activity of the water extract of Curcuma Longa Radix was preliminarily studied through LPS-induced RAW264.7 cell inflammation model by adopting preventive action mode and therapeutic action mode.In the former mode,RAW264.7 cells was pretreat with different concentrations of water extracts of Curcuma Longa Radix and then treated with LPS.While,the later mode,the RAW264.7 cells was induced by LPS firstly,and then treated with different concentrations of water extracts of Curcuma Longa Radix,The concentration of NO in each group was detected at different experimental exposure endpoints.Results:1.In the TLC identification experiment,curcumin,demethoxycurcumin and bisdemethoxycurcumin in Curcuma Longa Radix could be separated perfectly by using the developing solvent dichloromethane-methanol-formic acid(12:0.5:0.1).Toluene method was used to check the moisture content of Curcuma Longa Radix,and the result showed that all samples collected from the different places were conformed to the standard of Chinese Pharmacopeias.The total ash content of Curcuma Longa Radix was determined by total ash method,which is consistented with the Pharmacopoeia requirements.The dilute ethanol was used as solvent to determine the ethanol soluble extract of Curcuma Longa Radix,It was suggested that the ethanol soluble extract should not be less than 11.0%.Steam distillation method was used to determine the content of volatile oil in Curcuma Longa Radix.In the experiment,the extraction time and the ratio of material to liquid were investigated.Finally,12.5 times water volume was determined and the volatile oil was extracted completely after 5 hours of boiling.It was suggested that volatile oil of Curcuma Longa Radix can not be less than 2%(mL/g).The determination method of three active components of curcumin,demethoxycurcumin and bisdemethoxycurcumin in Curcuma Longa Radix was established,and the content of 3 components in 32 batches of Curcuma Longa Radix in different producing areas of Sichuan province was determined.The result showed that the contents of curcumin,demethoxy-curcumin and bisdemethoxycurcumin were 0.223?2.753 mg/g,0.059?0.564 mg/g,0.018?0.595 mg/g,respectively.The results of methodological investigation showed that the precision,repeatability,stability and sample recovery of this method were in accordance with the relevant regulations of Chinese pharmacopoeia,which can be used as a method for the determination of three kinds of components of Curcuma Longa Radix.The gas chromatography fingerprint of volatile oil from Curcuma Longa Radix was established in this experiment.20 common characteristic peaks have been identified in the gas chromatography fingerprint.Furthermore,10common peaks of terpinolene,?-Caryophllene,(E)-?-Famesene,a-curcumene,zingiberene,?-bisabolene,?-sesquiphellandrenendrene,Curlone,(6R,7R)-Bisabolone,(E)-a-Atlantone have been identified by gas chromatography-mass spectrometry(GC-MS).The similarity evaluation,cluster analysis and principal component analysis were used to comprehensively evaluate the quality of 32 batches of Curcuma Longa Radix and the results of three methods were consistent and verified each other.The results of the analysis showed that the similarity of 27 batches from the 32 batches of Curcuma Longa Radix were higher than 0.80,and the similarity of 78.13%of the herbs higher than 0.98.2.In terms of safety,ICP-MS was used to determine the contents of lead,cadmium,mercury,arsenic and copper in Curcuma Longa Radix.The results showed that the Hg,As and Cu heavy metal elements of the 19 batches of Curcuma Longa Radix in different producing areas of Sichuan were lower than the heavy metal limitation standard documented in the "Green industry standard for the import and export of medicinal plants and preparations"(WM2-2001).While the Pb element in the S2 sample reached 11.21mg/kg and Cd element of 10 batches of Curcuma Longa Radix have gone beyond the current limit standard,so it is recommended that the content of P'b and Cd elements of medicinal materials must be strictly detected in planting base of Curcuma Longa Radix;Meanwhile,GC was used to detect benzene hexachloride,dichlorodiphenyltrichloroethane,pentachloronitrobenzene,hexachlorobenzene,hept-achlor,cis heptachlor epoxy,trans-heptachlor epoxy,aldrin,cis chlordane,trans chlordane,and chlordane oxide in Curcuma Longa Radix.Residues of organochlorine pesticides were not detected in 32 batches of samples,indicating that the samples were qualified.3.On the basis of the conventional methods,the modern evaluation methods was used in this paper to make a comprehensive evaluation of Curcuma Longa Radix,according to the shape,size,color,surface characteristics,cross section,texture,odor,flavor,extract,volatile oil,curcumin,normethoxycurcumin and bisdemethoxycurcumin content.The result showed that the first class of Curcuma Longa Radix should meet the followed critical,including content of extract(?11%),volatile oil(?2.0%),curcumin content(?0.10%),and for the second class,content of extract(<11%),volatile oil(<2.0%),curcumin content(<0.10%).Thus,the grade standard was preliminarily established.4.In this study,the anti-inflammatory effect of aqueous extract of Curcuma Longa Radix was studied at the cell level for the first time.The RAW264.7cell inflammation model was induced by LPS.The prophylactic anti-inflammatory activity and therapeutic anti-inflammatory activity of the aqueous extract of Curcuma Longa Radix were preliminarily investigated by using the quantity of nitric oxide as an indicator.The results showed that no obvious toxicity in RAW264.7was induced by water extract of Curcuma Longa Radix(2000?g/mL)after 24 h treatment.The results showed that in the prophylactic anti-inflammatory mode,the 24 h treatment of aqueous extract of Curcuma Longa Radix(2000?g/mL)could inhibite the release of NO and the inhibition rate on NO release was31.69%.In the therapeutic anti-inflammatory mode,after LPS exposure 3 h,2000 ?g/mL of Curcuma Longa Radix aqueous extract for 24 hours showed the greatest impact on NO release and the inhibition rate on NO release was 31.76%.While LPS induced 6h,2000/?g/mL of Curcuma Longa Radix aqueous extract treatment for 3 hours could reduce the NO release to 21.52%.It showed that the extract of Curcuma Longa Radix has certain preventive and curative effects on LPS induced inflammatory in RAW264.7cells.Conclusion:In this experiment,we firstly examined the moisture and total ash content of Curcuma Longa Radix according to the Chinese Pharmacopoeia(2015 Edition),established the method for the determination of the extract,volatile oil,three kinds of curcumin active ingredients and the fingerprint of volatile oil.Furthermore,the safety test was carried out in order to provide a more comprehensive and effective evaluation for the quality control of Curcuma Longa Radix.Also,the modern evaluation methods in this paper was used to make a comprehensive evaluation of Curcuma Longa Radix,and its grading standard is initially established to ensure the quality of circulating Curcuma Longa Radix in the market.The experimental results showed that the Curcuma Longa Radix has certain preventive and therapeutic anti-inflammatory effects on the inflammation in RAW264.7 cells induced by LPS.It provides some scientific and theoretical basis for the research,further rational development and utilization of Curcuma Longa Radix.