| Dilong(Earthworm)is the dried body of Pheretima aspergillwn(E.Perrier), Pheretima vulgaris Chen,Pheretima guillelmi(Michaelsen)or Pheretima pectinifera Michaelsen(Fam.Megascolecidae).The former is known as "Guang Dilong",the latter three are known as "Hu Dilong".Dilong is an animal drug in common use,which has functions of clearing away heat,tranquilizing the mind,antiasthmatic and diuretic effects.Modern research also indicates that earthworm has many kinds of pharmacological effects such as descending blood pressure,anti-thrombus,anti-arrhythmia,anticancer, anti-ulcer,allaying fever and analgesia,sedation,antiasthmatic,antibacterial and so on, involving almost all the body systems.Therefore the demand for earthworm grew day by day in recent years.Since long ago the earthworm grows in the nature condition,the raw materials for medicine use have been mainly collected in wild,and the artificial cultivation of Dilong has not yet large-scaly developed.Being affected by the gathering method,environment and weather,the raw material output and quality of Dilong was unstable.Because there are many kinds of earthworms,but no effective species identification method,the earthworm was often collected by mistake.Therefore the medicine varieties in the markets were in a mess,the price of Dilong has changed greatly and its' quality was unstable.As a result the production and the development of earthworm has been affected enormously.To ensure the sustainable development and the use of the earthworm resources,we established the earthworm(Pheretima aspergillum)GAP base in Huizhou of Guangdong province,carried out the study of investigation of Dilong' germplasm resources,the biology characteristic,heredity multiplicity and its geography background system.A quality evaluating system of have been established.The research summary is as follows:1.Literature Review After consulting a large amount of related data both in China and abroad,we systematically summarized the research status of Dilong.In view of the weak links and problems among the previous studies on germplasm resources,identification,quality control and estimation,and safety research of Dilong,we raised the study content and the object.2.Investigaition of Dilong' germplasm resourcesWe investigated the medicine source and studied the taxonomy of earthworm,and found that there are earthworms distributed all over the country."Guang Dilong" is mainly produced in Guangdong province and Guangxi province;"Hu Dilong" is mainly produced in Shanghai,Zhejiang province and Jiangsu province;and there is "Tu Dilong" used habitually in other regions.Dilong in the market over the country are mainly "Guang Dilong".There are totally 14 kinds of earthworms used as Dilong in China,belonging to 3 genera of Megascolecidae and Lumbricidae.3.Species identification of Dilong' medical animal resourcesWe collected 351 earthworm samples from 16 different places in the domestic earthworm production areas,and identified the samples by classical systematic zoology method.We found that a larger part of samples in Guangdong province were Pheretima aspergillum(E.Perrier),and a small part of which were Pheretima californica(Kinberg) and P.robusto;There were not only Pheretima aspergillum(E.Perrier)in Guangxi province,but also quite many Pheretima.vulgaris(Chen.)and Pheretima californica,as well as few P.guillelmi(Michaelsen)and P.robusto;The samples in Hubei province were mainly Allolbophora caliginoa erapezoides(Ant.Duges),Eisenia foetida and P.vulgaris (Chen.),as well as few P.guillelmi(Michaelsen);There were many P.robusto,in Fujian province.This finding further confirmed that Guangdong and Guangxi were the main production area of Guang Dilong.After comparing several different earthworm processing and preservation methods, we suggested that if the samples were used for the character distinction,it should be processed and preserved in formalin;samples used for physics and chemistry analysis could be seasoned directly;samples used in the molecular biology experiment must be kept in 70%ethyl alcohol or 0.9%of NaCl solution,-20℃.Using the stereoscope to observe the sample body surface characteristic and the internal structure,and comparing with the reference,we could identify the species accurately.According to the result,we established a retrieval table of 14 kinds of earthworms used for medicinal purposes,and the table could be helpful in the species identification of Dilong. This finding indicated that by using the classical systematic zoology method,we could identify the earthworm with good characteristic preservation.4.Molecular Identification of Dilong' germplasm resources by ISSRISSR was applied to identify the DNA varieties of Dilong for the first time.The ISSR amplification of 7 materials by 8 ISSR premiers screened out resulted in 137 electrophoresis strips with 127 strips(92.7%)polymorphic.The NTSYSpc-2.10e software was applied to cluster analysis to the ISSR phenotype data matrix.The minimal value of similarity coefficient among Dilong' germplasm resources was 0.43,while the maximal was 0.66,which demonstrated greater genetic variance and DNA polymorphism of Dilong' germplasm resources.The cluster analysis result indicated that the materials were classified to 4 groups in the similarity factor 0.56.The 3rd and the 4th class groups presented closer genetic relationship,while the genetic relationship between 1stand 2ndgroups was far.The genetic relationship of individuals in the same genera was closer than those in different genera.The result was the same with the finding by using the classical systematic zoology method.5.Determination of multi chemical constituents and the HPLC fingerprinting of Dilong(1)Determination of uracil,hypoxanthine,uridine and inoine in Dilong by HPLCWe established an accurate and stable method for the determination of uracil, hypoxanthine,uridine and inoine in Dilong from different places as to efficaciously control the quality of medicinal material.Method:The samples were extracted with 0.9%NaCl solution by ultrasonic and determined by RP-HPLC method,0.01mol/L KH2PO4 solution and 50%MeOH as flowing,gradient elution,examination wave length was 254nm,column temperature was 27℃,10ul sample was injected.Result:The calibration curve of uracil was linear in the quality range of 0.0075-0.24/μg(r=0.9993),the average recovery was 99.82%,RSD was 2.34%; hypoxanthine was 0.0250-0.8000μg(r=0.9992),average recovery was 101.93%,RSD was 1.45%;uridine was 0.0125-0.4000/μg(r=0.9992),average recovery was 97.53%,RSD was 1.73%;inoine was 0.0500-1.6000μg(r=0.9991),average recovery was 102.06%,RSD was 2.44%.This method reproduction quality,the returns-ratio is good,may use in the broad earthworm uracil and so on 4 ingredients the content determination.The method displayed good reproducibility and high recovery,and it provides an effective way for the study of Dilong.(2)HPLC fingerprints of 0.9%NaCl solution extracts from Dilong We established the HPLC fingerprints of 0.9%NaCl solution extracts from Dilong to evaluate its quality.Gradient elution HPLC was used to analyze 0.9%NaCl solution extracts from Dilong,and the data of their fingerprints were analyzed by similarity.17 samples of Dilong from different places were analyzed to test the HPLC fingerprint.The relative retention time and relative area of 11 main common peaks were obtained as correlation technical parameters of Dilong.The repeatability of the relative retention times was good for the main common peaks and the relative peak areas had significant differences.From the above results,we suggested that the HPLC fingerprinting could be used to evaluate the quality and identification of Dilong.The HPLC fingerprint data of Dilong were cluster analyzed using the SPSS software. 17 samples were classified to 4 groups basically.The cluster analysis result indicated that the chemical contents of Dilong from Guangdong and Guangxi province were quite similar,which presented closer genetic relationship;the samples in Hainan province were similar;while Hu Dilong was obviously different from them.This result also verified that Dilong from different places varied form their chemical composition.So we suggest that the species and origin must be fixed in the processing.6.Determination and analysis of heavy metals and hazardous elements in DilongWe established an UV method to determine the content of heavy metals in Dilong and supply the basis for standardizing the content limits.The determination wave length was 215nm.The regression equation was:y=0.7231x+0.043,correlation coefficient R2=0.9968.The linear range of the calibration curve was 0～50μg/g,and the average recovery was 99.97%.The method was simple,rapid,reliable and had good reproducibility,and could be used to determine the content of most heavy metals in Dilong.Due to a great distinction between 9 batches of Dilong samples in the heavy metal element content,which mostly beyond the limit of not surpass 30/1000000(Chinese Pharmacopoeia,Volume 1,2005),we should pay attention to the heavy metal problem in Dilong.7.Study on the correlation between ecological environmental and quality of Dilong(1)Investigation and analyzing on the ecological environment factors of DilongWe investigated the distribution and environment of Dilong,and collected 11 soil samples,planning to inspected Dilong' habitat suitability and the main environment factors concerned with the quality by analyzing on the ecological environment factors and the growth soil(moisture content,pH value,heavy metal and so on).The results would provide as a technical support for the standardized cultivation of Dilong.The investigation on the ecological environment factors of Dilong' habitats indicated that Guangdong Province,Guangxi Province,Fujian Province and Hainan Province situating at the east China coast,their' climate,topography condition were superior and suitable for P.aspergillum to grow.P.aspergillum could grow better in the soil with 25%water content.The appropriate pH value was 6.8-7.6.P.aspergillum' tolerance to heavy metal was similarly to the other earthworms,and it could survive in the soil with As(4.929-16.000mg/kg), Cd(0.999-8.604mg/kg),Cr(26.830-110.168mg/kg),Cu(44.900-349.356mg/kg),Ni (94.400-480.088mg/kg),Pb(15.084-114.715mg/kg),and the Zn(11.163-601.859mg/kg). Industrial development in Dilong habitats had close relationship with the quality of Dilong. The more heavy metals emissioned to the environment due to industrial development,the worse Dilong' quality would be.(2)Study on the correlation between ecological environmental and heavy metal content in Dilong①The heavy metal concentration dynamics characteristic of P.aspergillumThe double box dynamics model under half static condition was applied in this experiment.P.aspergillum were raised in soil with different heavy metal conditions firstly, been digested with HNO3-H2O2 at low temperature,then the heavy metal in the P. aspergillum been determined.According to the results,we could study the dynamics characteristic of P.aspergillum to the Cd,Hg,and Pb in the soil.Results:The biology concentration ratio of earthworm P.aspergillum in Pb high, middle and low concentration was k1=1.232,K2=1.325,K3=1.058 respectively;In Cd high, middle and low concentration the ratio was k1=0.1792,K2=0.1753,K3=0.1382 respectively; In Hg,the ratio was undulatory.Conclusion:The concentration ability of P.aspergillum in Pb was higher than in Cd obviously,and increases with the lapse of time.Concentration characteristic in Hg was absorbed quickly at first,discharged quickly when accumulated to certain density,and then absorbed gain.②Study on the heavy metal inducing mechanism of P.aspergillum wMT byMT(Metallothionein)is a low-molecular-weight protein in the organism.It is rich in cysteinyl residues and contains no aromatic amino acids.MT shows high affinity for bivalent heavy metals.Because the earthworm contains a special MT(wMT),it could concentrate heavy metals in the soil,and heavy metal content in its vivo is much high.P.aspergillum were raised in soil with different heavy metal conditions for 30 days. The wMT in P.aspergillum was extracted and determined by the silver saturation method combined with ASS.According to the results,we could study the induction mechanism of P.aspergillum wMT to the heavy metals in the soil.Results:The P.aspergillum wMT content could be induced by different heavy metal, the induce ability was in turn:Cu＞Cd＞Zn＞Pb;And generally speaking wMT content in the internal organs was higher than in the muscle,this result also indicated that wMT mainly expressed in earthworm's internal organs.8.Study on quality standard of DilongBased on the experimental and analytical study in this paper,some substantial modifications have been made to Chinese Pharmacopoeia(Volume 1,2005)concerning the quality of Dilong.Determination item of Dilong have been added,choosing hypoxanthine and inoine as determination target,the minimal limited quantity of hypoxanthine in the dry drug should be no less than 0.2 mg/g;Inoine should be no less than 0.3 mg/g.In addition,some revision have been made in the character and processing items.The words "to clean the soil in the two ends" have been added in the processing method.The words "the abdomen has already been cut open except two ends,the internal organs have been cleaned or occasionally remain" have been added in the character description.The words "to clean the impurity and soil in the body" have been added in the preparation.This study has established the determination method for Dilong,and modified the quality standard.The modifications would promote the quality control of Dilong.9.Conclusion and further studyA systematic research has been made on polymorphism of Dilong' germplasm resources,which will provide the reference for the further use of Dilong.We have established a HPLC fingerprinting and determination method,as well as an UV method to determine the content of heavy metals in Dilong.This study has modified the quality standard to promote the quality control of Dilong.Based on the investigation and analyzing of the ecological environment factors of Dilong,the research will serve as a reference to promote the qualiy of Dilong.Insufficient research has been made on the induction mechanism of P.aspergillum wMT by heavy metal,and it's worth making the further research in the future.
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