Study on the good agricultural practice for Shanghai Pheretima / 药学实践杂志

Objective To establish the Good Agricultural Practice（GAP）for standard operation procedure of Shanghai Pheretima. Methods According to the comprehensive technical requirements of GAP standardized production of Chinese herbal medicine, the key technologies of standardized cultivation and processing were studied in Sangxin Town, Chongming District, Shanghai. Results The production area environment, variety characteristics, breeding technology, pest control, harvesting and processing, packaging, storage and transportation, quality monitoring and other technical links of Shanghai Pheretima were clearly defined and standardized. Conclusion This study provides practical basis for the standardization and modernization quality management of Shanghai Pheretima.

The impact of cow dung augmentation on soil restoration and bio-accumulation of metals (Lead and Cadmium) in Pheretima posthuma (Annelida: Clitellata) / O impacto do aumento de esterco de vaca na restauração do solo e bioacumulação de metais (chumbo e cádmio) em Pheretima posthuma (Annelida: Clitellata)

To investigate the role of cow dung in soil reclamation and bio assimilation along with bio accumulation of heavy metals in earthworm (P. posthuma) (N=900) earthworms were used and treatment groups of CD-soil mixture of different proportion of cow dung were designed. Nonlethal doses of lead acetate and cadmium chloride were added in treatment groups. Mature P. posthuma were released in each experimental pot maintaining the favorable conditions. The pH, carbon, nitrogen, phosphorus, exchangeable cations, and heavy metal level of each mixture was evaluated. The results indicated that bio-assimilation of Pb and Cd by P. posthuma were significantly (P ˂ 0.01) higher in different soil-CD treatments compared to control. Highest bio-assimilation of both metals was observed in T¹ of both groups (Pb = 563.8 mg/kg and Cd = 42.95 mg/kg). The contents of both metals were significantly (P ˂ 0.05) lowered in casting. The nutrient concentration in the final castings of all soil-CD treatments were also equally transformed from less or insoluble to more soluble and available for plants, except for carbon level which increased with CD proportion. It is concluded that cow dung as organic matter has a positive effect on soil reclamation and bio-assimilation of metals by P. posthuma.

Para investigar o papel do esterco de vaca na recuperação do solo e bioassimilação, juntamente com a bioacumulação de metais pesados em minhocas (P. posthuma) (N = 900), minhocas foram usadas e grupos de tratamento de mistura CD-solo de diferentes proporções de esterco de vaca foram projetados. Doses não letais de acetato de chumbo e cloreto de cádmio foram adicionadas aos grupos de tratamento. P. posthuma maduros foram liberados em cada vaso experimental, mantendo as condições favoráveis. Foram avaliados o pH, carbono, nitrogênio, fósforo, cátions trocáveis e nível de metais pesados de cada mistura. Os resultados indicaram que a bioassimilação de Pb e Cd por P. posthuma foi significativamente (P ˂ 0,01) maior em diferentes tratamentos de solo-CD em relação ao controle. A maior bioassimilação de ambos os metais foi observada em T1 de ambos os grupos (Pb = 563,8 mg / kg e Cd = 42,95 mg / kg). O conteúdo de ambos os metais foi significativamente (P ˂ 0,05) reduzido na fundição. A concentração de nutrientes nas fundições finais de todos os tratamentos de solo-CD também foi igualmente transformada de menos ou insolúvel para mais solúvel e disponível para as plantas, exceto o nível de carbono que aumenta com a proporção de CD. Conclui-se que o esterco de vaca como matéria orgânica tem um efeito positivo na recuperação do solo e na bioassimilação de metais por P. posthuma.

The impact of cow dung augmentation on soil restoration and bio-accumulation of metals (Lead and Cadmium) in Pheretima posthuma (Annelida: Clitellata)

Abstract To investigate the role of cow dung in soil reclamation and bio assimilation along with bio accumulation of heavy metals in earthworm (P. posthuma) (N=900) earthworms were used and treatment groups of CD-soil mixture of different proportion of cow dung were designed. Nonlethal doses of lead acetate and cadmium chloride were added in treatment groups. Mature P. posthuma were released in each experimental pot maintaining the favorable conditions. The pH, carbon, nitrogen, phosphorus, exchangeable cations, and heavy metal level of each mixture was evaluated. The results indicated that bio-assimilation of Pb and Cd by P. posthuma were significantly (P 0.01) higher in different soil-CD treatments compared to control. Highest bio-assimilation of both metals was observed in T1 of both groups (Pb = 563.8 mg/kg and Cd = 42.95 mg/kg). The contents of both metals were significantly (P 0.05) lowered in casting. The nutrient concentration in the final castings of all soil-CD treatments were also equally transformed from less or insoluble to more soluble and available for plants, except for carbon level which increased with CD proportion. It is concluded that cow dung as organic matter has a positive effect on soil reclamation and bio-assimilation of metals by P. posthuma.

RESUMO Para investigar o papel do esterco de vaca na recuperação do solo e bioassimilação, juntamente com a bioacumulação de metais pesados em minhocas (P. posthuma) (N = 900), minhocas foram usadas e grupos de tratamento de mistura CD-solo de diferentes proporções de esterco de vaca foram projetados. Doses não letais de acetato de chumbo e cloreto de cádmio foram adicionadas aos grupos de tratamento. P. posthuma maduros foram liberados em cada vaso experimental, mantendo as condições favoráveis. Foram avaliados o pH, carbono, nitrogênio, fósforo, cátions trocáveis e nível de metais pesados de cada mistura. Os resultados indicaram que a bioassimilação de Pb e Cd por P. posthuma foi significativamente (P 0,01) maior em diferentes tratamentos de solo-CD em relação ao controle. A maior bioassimilação de ambos os metais foi observada em T1 de ambos os grupos (Pb = 563,8 mg / kg e Cd = 42,95 mg / kg). O conteúdo de ambos os metais foi significativamente (P 0,05) reduzido na fundição. A concentração de nutrientes nas fundições finais de todos os tratamentos de solo-CD também foi igualmente transformada de menos ou insolúvel para mais solúvel e disponível para as plantas, exceto o nível de carbono que aumenta com a proporção de CD. Conclui-se que o esterco de vaca como matéria orgânica tem um efeito positivo na recuperação do solo e na bioassimilação de metais por P. posthuma.

The impact of cow dung augmentation on soil restoration and bio-accumulation of metals (Lead and Cadmium) in Pheretima posthuma (Annelida: Clitellata) / O impacto do aumento de esterco de vaca na restauração do solo e bioacumulação de metais (chumbo e cádmio) em Pheretima posthuma (Annelida: Clitellata)

Abstract To investigate the role of cow dung in soil reclamation and bio assimilation along with bio accumulation of heavy metals in earthworm (P. posthuma) (N=900) earthworms were used and treatment groups of CD-soil mixture of different proportion of cow dung were designed. Nonlethal doses of lead acetate and cadmium chloride were added in treatment groups. Mature P. posthuma were released in each experimental pot maintaining the favorable conditions. The pH, carbon, nitrogen, phosphorus, exchangeable cations, and heavy metal level of each mixture was evaluated. The results indicated that bio-assimilation of Pb and Cd by P. posthuma were significantly (P ˂ 0.01) higher in different soil-CD treatments compared to control. Highest bio-assimilation of both metals was observed in T1 of both groups (Pb = 563.8 mg/kg and Cd = 42.95 mg/kg). The contents of both metals were significantly (P ˂ 0.05) lowered in casting. The nutrient concentration in the final castings of all soil-CD treatments were also equally transformed from less or insoluble to more soluble and available for plants, except for carbon level which increased with CD proportion. It is concluded that cow dung as organic matter has a positive effect on soil reclamation and bio-assimilation of metals by P. posthuma.

RESUMO Para investigar o papel do esterco de vaca na recuperação do solo e bioassimilação, juntamente com a bioacumulação de metais pesados ​​em minhocas (P. posthuma) (N = 900), minhocas foram usadas e grupos de tratamento de mistura CD-solo de diferentes proporções de esterco de vaca foram projetados. Doses não letais de acetato de chumbo e cloreto de cádmio foram adicionadas aos grupos de tratamento. P. posthuma maduros foram liberados em cada vaso experimental, mantendo as condições favoráveis. Foram avaliados o pH, carbono, nitrogênio, fósforo, cátions trocáveis ​​e nível de metais pesados ​​de cada mistura. Os resultados indicaram que a bioassimilação de Pb e Cd por P. posthuma foi significativamente (P ˂ 0,01) maior em diferentes tratamentos de solo-CD em relação ao controle. A maior bioassimilação de ambos os metais foi observada em T1 de ambos os grupos (Pb = 563,8 mg / kg e Cd = 42,95 mg / kg). O conteúdo de ambos os metais foi significativamente (P ˂ 0,05) reduzido na fundição. A concentração de nutrientes nas fundições finais de todos os tratamentos de solo-CD também foi igualmente transformada de menos ou insolúvel para mais solúvel e disponível para as plantas, exceto o nível de carbono que aumenta com a proporção de CD. Conclui-se que o esterco de vaca como matéria orgânica tem um efeito positivo na recuperação do solo e na bioassimilação de metais por P. posthuma.

Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice / 南方医科大学学报

OBJECTIVE@#To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.@*METHODS@#The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.@*RESULTS@#We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.@*CONCLUSION@#The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.

Herbal Textual Research on Pheretima in Famous Classical Formulas / 中国实验方剂学杂志

By consulting ancient herbal medicines and medical books， combined with modern documents and field investigations， the textual research of Pheretima has been conducted to verify the name， origin， producing area， quality， harvesting and processing changes， and sort out the relationship of origin between ancient and modern times， so as to provide reference and basis for the development and utilization of the related famous classical formulas. Through textual research， it is known that there are many aliases for Pheretima， the rectification of name was "Qiuyin" or "Baijing Qiuyin" in materia medica books. In the Song dynasty and later prescription books， the prescription name is mostly Dilong. From the beginning of Yaowu Chuchanbian （《药物出产辨》）， Dilong was used as the rectification of name. It is widely distributed in our country， which is produced all over the country and mostly wild. According to ancient Pheretima with "Baijing Dilong"， "Jingbai Shenzi" and "Datiao" as the principles of medicine， combined with historical origin， producing area and easy access， it is confirmed that Pheretima used in ancient times to the present is mainly Pheretima aspergillum， and it also has many other Qiuyin as Pheretima for medicinal purposes. Chinese Pharmacopoeia has unified the origin of the Pheretima since the 1995 edition based on historical origins and actual harvesting conditions. The medicinal material processed by P. aspergillum was called Guangdilong， and the medicinal materials processed by P. vulgaris， P. guillelmi and P. pectinifera were called Hudilong. Since then， all the herbal books published in the future are in line with Chinese Pharmacopoeia that was implemented at that time. The authentic production areas of Guangdilong are Guangdong and Guangxi， and the authentic production areas of Hudilong is Jiangsu， Shanghai， Zhejiang and Anhui. The Guangdilong produced in Guangdong and Guangxi has the best quality. After harvesting， remove the soil and offal， wash and dry. Clinically cut into sections for medicine， or prepare medicine according to prescription. The Pheretima in ancient used "Baijing Dilong"， "Jingbai Shenzi" and "Datiao" as the mainstream quality evaluation standards. According to historical origins， P. aspergillum should be the main source of Pheretima， and its quality is better than other species. Therefore， it is recommended that Pheretima in Shentong Zhuyutang use P. aspergillum， which is produced in Guangdong， Guangxi and other places. After harvest， the abdomen was opened in time to remove the viscera and sediment， washed and dried.

Rapid Enrichment and Activity Research of Antithrombotic Proteins from Pheretima vulgaris / 中国实验方剂学杂志

Objective:A strong antithrombotic protein component， named PvQ， was purified and enriched from total protein of <italic>Pheretima vulgaris</italic>，<italic> </italic>a<italic> </italic>traditional Chinese medicine. Moreover， we evaluated its fibrinolytic and anticoagulant activity， and expected to provide reference for the research on antithrombotic substances of Pheretima. Method:A rapid <italic>in</italic> <italic>vitro</italic> activity-oriented separation combined with the AKTA-Pure protein purification system conducted on <italic>P. vulgaris</italic>. Meanwhile， the fibrinolytic and anticoagulant activities of PvQ were measured by fibrin plate method and fibrinogen-thrombin time （Fibg-TT） method. And the <italic>in vitro</italic> thrombolysis assay was used for evaluating the lysis ability of PvQ to thrombus. Then the stability of PvQ was also analyzed for its anticoagulant activity at different pH and temperature. Result:The PvQ was successfully enriched and its activity was determined to have significant fibrinolytic and anticoagulant activities. And the result of <italic>in vitro</italic> thrombolysis assay revealed that PvQ could hydrolyze more than 80% of thrombus after 5 h of incubation at 37 ℃. In addition， the changes of temperature and pH had significant effects on antithrombotic activity， and this study showed that PvQ was rapidly inactivated at ≥60 ℃ or in acidic conditions （pH<7）. While， the activity of PvQ was unaffected or less affected at ≤50 ℃ and under alkaline conditions. Conclusion:A feasible preparation method of PvQ is established， and it can affect fibrin and fibrinogen at the same time， thus exerting a dual fibrinolytic effect and possessing significant fibrinolytic and anticoagulant activities. It provides a scientific interpretation for the treatment of thrombotic diseases by PvQ and a reference for the development of antithrombotic protein products of Pheretima.

Pheretima extract ameliorates nucleus pulposus cell degeneration in the intervertebral disc by inhibiting nuclear factor-kappa B pathway / 中国组织工程研究

BACKGROUND: Inflammatory microenvironment of nucleus pulposus cells is an important factor to promote nucleus pulposus degeneration. Inflammatory factors amplify the cascade of inflammation by activating the nuclear factor-κB signaling pathway to form a vicious cycle and accelerate the process of nucleus pulposus degeneration. It could delay the progression of intervertebral disc degeneration by inhibiting the activity of nuclear factor-κB signaling pathway. OBJECTIVE: To investigate the mechanism of Pheretima extract on nucleus pulposus cell degeneration. METHODS: Pheretima active ingredients were extracted by cold dipping. The cell counting kit-8 method was used to detect the effects of 0, 25, 50, 100, 200, 400 mg/L Pheretima extract on rat nucleus pulposus cell activity, and then suitable drug concentrations were chosen for following research. Tumor necrosis factor-α (TNF-α) was used to stimulate nucleus pulposus cells to induce the nucleus pulposus cell degeneration model. Pheretima extracts (50, 100, 200 mg/L) were used to treat the cell model. Western blot was used to detect the expression of Aggrecan, Collagen II, TNF-α and interleukin-6. RT-qPCR was used to detect mRNA expression of TNF-α and interleukin-6. Immunofluorescence was used to detect the nuclear expression of P65 in the nucleus pulposus cells. RESULTS AND CONCLUSION: Compared with the blank group (0 mg/L), 400 mg/L Pheretima extract inhibited the activity of nucleus pulposus cells, and 50, 100, 200 mg/L extracts of Pheretima were used for subsequent experimental research. Compared with the normal group, the expression of Aggrecan and Collagen II in the model group was reduced, the protein and mRNA expression levels of TNF-α and interleukin-6 were up-regulated, and P65 expression increased in the nucleus. Compared with the model group, Pheretima extract could increase the expression of Aggrecan and Collagen II, down-regulate the protein and mRNA levels of TNF-α and interleukin-6, and reduce P65 expression in the nucleus. To conclude, Pheretima extract can reduce the expression of inflammatory factors, increase the synthesis of extracellular matrix and inhibit nuclear factor-κB pathway activity. Pheretima extract may ameliorate nucleus pulposus cell degeneration in the intervertebral disc via the nuclear factor-κB signaling pathway.

Research progress on Pheretima and earthworms with related origin / 中国中药杂志

Through literature analysis of Pheretima and its origin-related earthworm,this study summarized the progress on Pheretima in textual criticism of origin,origin identification,effective components,detection of harmful components,and pharmacological effects,which can lay a basis for further research on Pheretima. Through literature research,the authors found that Pheretima was first recorded in Secret Formulary for Traumatology and Fracture Taught by Immortal written by LIN Daoren in Tang Dynasty rather than the Taiping Holy Prescriptions for Universal Relief in Song Dynasty. The latest techniques for origin identification include microscopic trait identification,DNA barcoding,and HPLC. The main effective components of Pheretima are proteins,polypeptides,enzymes,nucleotides,amino acids,and trace elements. According to recent studies,Pheretima has anti-pulmonary and anti-renal interstitial fibrosis,respiratory syncytial virus-inhibiting,human hypertrophic scar fibroblast proliferation-suppressing,and mouse embryonic fibroblast proliferation-promoting effects. Moreover,Pheretima can prevent colitis-induced colon cancer by inhibiting the activation of COX-2/PGE2/β-catenin signaling pathway. METHODS:: for detecting the harmful components and their residues( organic pollutant polychlorinated biphenyl,heavy metals) and bacteria in Pheretima,have been established. Pheretima,mainly derived from wild earthworms,has remarkable clinical efficacy. However,the wild resource is in short supply and artificial culture is expected to be a promising solution.

In-vitro anthelmintic activity of Boswellia serrata and Aloe barbadensis extracts on Pheretima posthuma: Indian earthworm

Background: The development of anthelmintic resistance and the high cost of conventional anthelmintic drugs led to the evaluation of medicinal plants as an alternative source of anthelmintics. In the current study, in- vitro experiments were conducted to determine the possible anthelmintic effects of crude aqueous and alcoholic extracts of the resins of Boswellia serrata and leaves of Aloe barbadensis on adult Indian earthworm (Pheretima posthuma).Methods: Various concentrations (50, 100, 150 mg/ml) of each extracts were tested and results were expressed in terms of time for paralysis and time for death of worms. The activities are well compared with the standard drug Albendazole as a positive control and saline water as negative control.Results: Anthelmintic activity was observed as dose dependent manner. It was found that alcoholic  extract exhibited maximum anthelmintic activity at concentration 100 and 150 mg/ml compared to standard drug Albendazole (10mg/ml) while aqueous extract show modest significant activity at concentration 150 mg/ml against worm Pheretima posthuma. All results was statistically analysed by using ‘Dunnett’s test’ one- way ANOVA; the p<0.001 were significant when compared with control and standard group.Conclusions: The present study proves the potential of combination of B. serrata and A. barbadensis as an anthelmintic drugs. Further studies are necessary to isolate and reveal the active compounds and to establish the mechanism of action.

Morphological and DNA double barcode identification of Pheretima and its adulterants / 中草药

Objective: To develop an accurate and effective method to identify the original animals of Chinese medicine Pheretima (Dilong in Chinese) and its adulterants by using mitochondrial genes, cytochrome coxidase I (CO I) and 16 S rRNA as DNA barcoding sequences to certify and improve morphological analysis. Methods: A total of 66 samples were initially identified according to their morphological characteristics, and then their CO I and 16 S rRNA sequences were simultaneously amplified for DNA barcoding. The two gene sequences were amplified with improved primers. One-step double PCR was used and the experimental conditions were optimized. The genetic distance among and within species of Pheretima and its hybrids was calculated by MEGA 5.1. The N-J tree was constructed based on K2P model. Results: Combined with morphological identification, CO I and 16S rRNA double DNA barcoding identification can accurately identify Pheretima and its adulterants. Conclusion: Morphological identification is the basis of molecular identification. Molecular identification can be a powerful supplement to the morphological identification. The combination of molecular identification and morphological identification can increase the accuracy of the identification of the original animals of Pheretima and its adulterants. Double DNA barcoding can also provide a solution for the identification of the medicinal materials of Pheretima and other animal medicinal materials.

Analysis on rule of first group of Chinese medicine masters in treating acute apoplexy / 中草药

Objective: To explore the prescription medication rule of the first batch of Chinese medicine masters in treating acute stage of stroke by using the data mining method. Methods: The experience books and published journal articles of Chinese medicine masters and the shared medical records of national famous TCM studios in the cloud platform V1.5 of ancient and modern medical records were searched; The first batch of national physician master data for the treatment of acute phase of stroke was selected; A standardized basis database, standardization of medicines were established after using statistical analysis of system integration, association rules analysis, methods of complex networks; The formula frequency, medicinal, and core prescription drugs were analyzed. Results: A total of 142 medical cases were eventually included. The frequency results of traditional Chinese medicine showed that 14 kinds of high-frequency traditional Chinese medicine were obtained, including Pheretima, Chuanxiong Rhizoma, Persicae Semen, etc., and most of the drugs were warm, smooth, bitter, sweet, and return to liver and lung meristem. The results showed that the frequency of chordal pulse and tongue red was the most. A total of 24 association rules and 18 TCM association rules were obtained by association rule analysis. Conclusion: In the treatment of acute apoplexy, the Chinese medicine master usually adopted the methods of clearing heat and relieving wind, promoting blood circulation and removing blood stasis, eliminating phlegm and resuscitative medicinal, moving qi, and dredging collaterals, and stopping convulsions.

Herbal Textual Research and Modern Analysis of Pheretima / 中国实验方剂学杂志

Pheretima has a long history of medication, its original name was earthworm, and it was first recorded in Shennong Bencaojing, which was listed as inferior product. In the 2015 edition of Chinese Pharmacopoeia, two varieties of Pheretima were included. However, due to the variety of Pheretima, there are mixed non-pharmacopoeia collection of Pheretima family status. Based on the systematic review of ancient and modern literature, this paper conducts herbal textual research on Pheretima in terms of name, origin, distribution of origin, genuine production area, harvesting time, processing methods, etc. Based on the analysis of various ancient books and modern research documents of herbal medicine and their accompanying drawings, it is found that the Pheretima and white-necked Pheretima mentioned in ancient books are the general names of the genus Pheretima. The ancient people thought that white-necked Pheretima was a good medicinal material, which was the same as the opinion that Guangdilong was better in quality than Tudilong in modern research. In ancient and modern literature, the origin of earthworm is relatively consistent, but due to the change of environment, the output of wild Pheretima is reduced, and now the output of Pheretima is mainly artificial breeding. In ancient times, harvesting should be as far as possible in spring, summer and autumn. However, in modern times, the best harvest time is autumn. Different processing methods of earthworm in different ages and regions are different. Attention should be paid to following and inheriting the ancient processing methods, combining with modern research techniques, the quantitative standard of processing of Pheretima should be formulated, so that Pheretima medicinal materials can be applied comprehensively and effectively. This research provides the basis for the original source, resource development, correct use, genuine producing area and processing method determination of Pheretima.

Study on Material Basis and Processing Principle of Fishy Smell of Pheretima aspergillum by Electronic Nose and HS-GC-MS / 中国实验方剂学杂志

Objective:To analyze and compare the fishy components in raw, stir-fried, liquorice-processed, vinegar-processed and wine-processed products of Pheretima aspergillum, and explore the material basis and processing principle of fishy smell of P. aspergillum. Method:Heracles Ⅱ ultra-fasted gas chromatography electronic nose technology combined with chemometrics was used for the overall analysis of volatile components in raw P. aspergillum and its processed products. Headspace gas chromatography-mass spectrometry (HS-GC-MS) was used to analyze and identify the volatile compositions in the raw products and processed products. Gas chromatographic conditions were as following：temperature program (initial temperature at 60 ℃, kept for 5 min, up to 120 ℃ with the heating rate of 3 ℃·min-1, and then up to 230 ℃ with the heating rate of 10 ℃·min-1 and finished), the inlet temperature at 280 ℃, high purity helium as the carrier gas, the flow rate of 1.0 mL·min-1, the split ratio of 20∶1. Mass spectrum conditions were as following：electron impact ionization (EI), electron collision energy of 70 eV, ion source temperature of 230 ℃, quadrupole temperature at 150 ℃, scanning range of m/z 50-550. The relative content of each component was calculated by peak area normalization. Result:Principal component analysis (PCA) and discriminant factor analysis (DFA) of the electronic nose showed that the raw products and its processed products could be clearly distinguished from each other. Among them, the difference between raw products and stir-fried, liquorice-processed products was small, but the difference between raw products and vinegar-processed, wine-processed products was large. A total of 25, 27, 22, 26 and 33 components were respectively identified from raw, stir-fried, liquorice-processed, vinegar-processed and wine-processed products of P. aspergillum, there were 13 common components in these products, including 4 aldehydes (isovaleraldehyde, 2-methylbutyraldehyde, hexanal, benzaldehyde), 2 ketones (2-heptanone, 2-tridecanone), 1 carboxylic acid (lauric acid), 4 heterocyclic compounds (2-methylpyrazine, 2,5-dimethyl pyrazine, 2-pentylfuran, 2-ethyl-6-methyl pyrazine), 1 amine (trimethylamine) and 1 alcohol (1-octen-3-ol). Conclusion:The odorous components in the raw products are mainly derived from aldehydes (isovaleraldehyde, 2-methylbutyraldehyde, isobutyraldehyde, 2-ethylhexanal, hexanal) and amines (trimethylamine). Odorous components of P. aspergillum can be reduced effectively by stir-fried and liquorice, vinegar, wine processing, while flavoring substances can be increased by wine processing to cover its ugly odor. This paper can provide scientific basis for the deodorization of P. aspergillum by processing, and also provide reference for the analysis and correction of ugly odor of other animal medicines.

Identification of Pheretima by Multiplex Allele-specific PCR / 中国实验方剂学杂志

Objective: In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market. Besides,the medicinal materials have mostly lost the main identification features, and are difficult to distinguish. Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima. Method: According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR) method for identification was explored and verified in terms of tolerance and feasibility in this study. The specific primers were combined to build multiple PCR systems. An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants. Result: Through the established multiplex PCR reaction system, 366,487,487 and 475 bp of fragments were amplified from DNA templates of P. aspergillum,P. vulgaris,P. guillelmi and P.pectinifera respectively. All of the adulterants were negative by the multiplex PCR assay. The PCR amplification of specific alleles method established in this paper can accurately identify pheretima. Conclusion: The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.

Study on Quality Standard Improvement of Pheretima / 中国药房

OBJECTIVE： To provide reference for improving the quality standard of Pheretima. METHODS： The contents of hypoxanthine and inosine in medicinal material samples were determined by HPLC. HPLC fingerprint of Pheretima was established according to “Similarity evaluation system for TCM chromatogramtic fingerprint” （2012 edition） software， and similarity evaluation was conducted. The determination was performed on Purospher STAR RP-18 endcapped with mobile phase consisted of methanol-water （gradient elution） at the flow rate of 1 mL/min. The detection wavelength was 248 nm， and the column temperature was set at 30 ℃. The sample size was 20 μL. RESULTS： The results of methodological investigation of content determination showed that the linear range of hypoxanthine and inosine were 1.58-31.6 μg/mL （r＝0.999 9）， 5.52-110.4 μg/mL（r＝0.999 8）， respectively. limits of quantify were 0.316， 0.552 μg/mL， respectively； limits of detection were 0.158， 0.110 μg/mL， respectively； RSDs of precision， stability （24 h） and repeatability tests were all less than 2.0％ （n＝6）. Average recovery rates were 103.0％ （RSD＝1.7％， n＝6） and 101.2％ （RSD＝1.2％， n＝6）， respectively. HPLC fingerprint for 15 batches of samples were established， and 8 common peaks were identified. The similarity of HPLC fingerprint of 14 batches of sample with control fingerprint R was higher than 0.900. CONCLUSIONS： The established method for content determination of hypoxanthine and inosine and HPLC fingerprint of Pheretima are simple， accurate and reproducible， and can be used for quality control of Pheretima.

Analysis on chemical constituents of Pheretima aspergillum by UPLC-Q-TOF-MS / 中草药

Objective: To establish a method for the analysis of constituents of Pheretima aspergillum by UPLC-Q-TOF-MS. Methods: The analysis was performed on an Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution. The mobile phase consist of 0.1% formic acid-acetonitrile and 0.1% formic acid-water at a flow rate of 0.3 mL/min. The column temperature was at 35 ℃. The information of the compounds was acquired in positive and negative mode. Results: Total 83 compounds were inferred, contains 11 free amino acids, 26 organic acids, 11 necleosides, 5 dipeptides and cyclic dipeptideds and other 21 nitrogen-containing substances, the rest on a total of 10. Conclusion: The method is accurate, rapid, and sensitive, and provide a basis for further clarifying the material basis of its efficacy and the selection of quality control indicators.

Cloning of MT-2 gene and its promoter in Pheretima aspergillum / 中草药

Objective: To lay a foundation for attenuating the heavy metal accumulation in Pheretima aspergillum by means of genetic engineering technology in further research, we revealed the transcriptional regulation mechanism of MT-2 gene. Methods: The coding sequence of MT-2 gene was amplified by PCR with specific primers, which were designed according to their known cDNA sequences, and the outcomes were contrastively analyzed after the sequencing process. Prior to the isolation of 5' promoter sequence by genome walking technology, three specific primers were designed based on MT-2 cDNA sequence. Meanwhile, the cis-acting elements of MT-2 gene were analyzed by Promoter Prediction online software. Results: After PCR and sequencing processes, a 2 826 bp coding sequence of MT-2 gene were obtained, four exons and four introns were found to compose the coding area by comparing with the known MT-2 cDNA sequence (accession No.KC787373.1). Besides, after genome walking and Promoter Prediction online analysing, a 1 534 bp promoter region of MT-2 was isolated, which contained not only CAAT box, TAAT box, and other core promoter elements, but also three MRE elements which specifically response to heavy metal involved in regulating the MT-2 expression. Conclusion: The expression of MT-2 gene in P. aspergillum can be induced by heavy metal, and the transcriptional level is achieved by MRE regulatory elements located in MT-2 gene promoter region.

Progress in Pharmacological Effects and Clinical Research of Pheretima / 中国药师

As a traditional Chinese medicine,pheretima is widely used for thousands of years in China. According to the studies in recent years,the pharmacological effects and clinical researches of pheretima were summarized based on the introduction of chemical constituents, which could provide information for the further study.

Study on the HPLC Characteristic Chromatogram of Pheretima / 中国药房

OBJECTIVE:To establish the HPLC characteristic chromatogram of pheretima,and compare the differences of the main ingredient contents of Guangdong pheretima and Shanghai pheretima and the chromatogram differences among pheretima and 3 other animal drugs. METHODS:Pheretima HPLC characteristic chromatogram method was adopted to determine the characteris-tic chromatograms of 16 Guangdong pheretima,8 Shanghai pheretima,3 eupolyphaga,3 hirudo and 3 catharsius. Similarity evalua-tion and t test were used to analyze the differences of chromatogram data of 5 animal drugs. RESULTS:The established HPLC char-acteristic chromatogram method firstly identified 11 common characteristic peaks,including 6 nucleosides,4 nucleobase and 1 ami-no acid;and it could be used for the identification of pheretima from eupolyphaga,hirudo and catharsius;the differences of main ingredient contents in the characteristic chromatogram of Guangdong pheretima and Shanghai pheretima were firstly studied. The contents of xanthine and adenosine in Guangdong pheretima were higher than Shanghai pheretima,while the contents of uridine, guanosine and 2′-deoxy guanosine in Shanghai pheretima were higher than Guangdong pheretima. A new index S,calculated by these 5 constituents,was successfully applied to distinguish the 2 kinds of pheretima. CONCLUSIONS:The characteristic chro-matogram can be used for the identification of pheretima,and can provide reference for the pharmacodynamic differences study of Guangdong pheretima and Shanghai pheretima.