Earthworm Protein Protects Vascular Endothelial Function in Spontaneously Hypertensive Rats via PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10-3 g·kg-1·d-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg-1·d-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe2+）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates / Potencial antibacteriano e bacteriostático do fluido celômico e pasta corporal de Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) contra isolados bacterianos clínicos resistentes à ampicilina

Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates

Abstract Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Resumo Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates / Potencial antibacteriano e bacteriostático do fluido celômico e pasta corporal de Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) contra isolados bacterianos clínicos resistentes à ampicilina

Abstract Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Resumo Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

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.

Allolobophora caliginosa coelomic fluid ameliorates gentamicin-induced hepatorenal toxicity in rats

Objective: To explore the efficacy of earthworm's coelomic fluid against gentamicin-induced hepatic and renal toxicity in rats. Methods: The animals were divided randomly into three groups (n= 6 per group): control, gentamicin, and Allolobophora caliginosa coelomic fluid-treated groups. Toxicity was established after injection of gentamicin daily for 8 days at a dose of 100 mg/kg. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total proteins, albumin, creatinine, urea, uric acid, malondialdehyde, glutathione, catalase and histopathology of tissues were investigated in the study. Results: Allolobophora caliginosa coelomic fluid significantly decreased urea, creatinine, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and malondialdehyde levels while significantly increasing levels of total proteins, albumin, glutathione and catalase. The histopathological investigation showed partial restoration of renal and hepatic architecture. Conclusions: This study shows the potency of Allolobophora caliginosa coelomic fluid in improving the biochemical and histopathological changes induced by gentamicin in the liver and kidney of the rats.

Allolobophora caliginosa coelomic fluid ameliorates gentamicin-induced hepatorenal toxicity in rats

Objective: To explore the efficacy of earthworm's coelomic fluid against gentamicin-induced hepatic and renal toxicity in rats. Methods: The animals were divided randomly into three groups (n = 6 per group): control, gentamicin, and Allolobophora caliginosa coelomic fluid-treated groups. Toxicity was established after injection of gentamicin daily for 8 days at a dose of 100 mg/kg. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total proteins, albumin, creatinine, urea, uric acid, malondialdehyde, glutathione, catalase and histopathology of tissues were investigated in the study. Results: Allolobophora caliginosa coelomic fluid significantly decreased urea, creatinine, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and malondialdehyde levels while significantly increasing levels of total proteins, albumin, glutathione and catalase. The histopathological investigation showed partial restoration of renal and hepatic architecture. Conclusions: This study shows the potency of Allolobophora caliginosa coelomic fluid in improving the biochemical and histopathological changes induced by gentamicin in the liver and kidney of the rats.

Determination of the bioaccessibility of cadmium and arsenic in earthworms by PBET digestion in vitro / MDCK cell model with risk assessment / 药学学报

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the content of cadmium (Cd) and arsenic (As) in earthworms. A physiologically-based extraction test (PBET) digestion in vitro /MDCK cell model was established to investigate the bioaccessibility of Cd and As in earthworms. The hazard index (HI) method and the margin of exposure (MOE) method were used to assess the risks of the total content and the bioaccessible content of Cd and As. The results showed that the total content of Cd and As in six batches of earthworms ranged from 8.319 to 33.606 mg·kg-1 and from 0.532 to 16.412 mg·kg-1, respectively. After uptake by MDCK cells, the bioaccessibility of Cd in earthworms ranged from 10.13% to 64.16%, and the bioaccessibility of As was from 2.72% to 46.57%. The results of risk assessment showed that before uptake by MDCK cells, the MOE values of As and HI values of Cd for all batches of earthworms were greater than 1, which suggests that the risks of As are acceptable but the risks of Cd are unacceptable. After transportation by MDCK cells, except for one batch of earthworms, the HI values of Cd in the other five batches were less than 1, which suggests that the risks are at a safe level. This study provides important technical support for a more objective and scientific assessment of the health risks of heavy metals in traditional Chinese medicines, and for a more scientific and reasonable standard limit of heavy metals.

Effect of dietary incorporation of natural attractants on growth and survival during seed rearing of Indian butter catfish, Ompok bimaculatus

Aim: The objective of the study was to assess the effect of dietary incorporation of different feed attractants viz. tubifex, earthworm and fish meal on growth and survival of O. bimaculatus (pabda fish) fry. Methodology: Fifty numbers of fifteen days weaned fry (0.105±0.008g; 2.14±0.14cm) were stocked into each aerated aquariums (30.0 x 15.0 x 15.0 cm) following a completely randomized design (CRD) consisting of four treatments including the control with three replicates each. Four iso-nitrogenous purified diets were prepared including three treatment diets with attractants (5%) by replacing an appropriate amount of casein and cellulose and fed to the fishes twice daily. Results: Results from the study showed that growth of fry as measured by final weight, weight gain, mean daily weight gain and specific growth rate of fries were significantly (p<0.05) higher in tubifex supplemented group and lower in control group. Survival was significantly higher in tubifex supplemented group (42.66±1.3%) and lower in control group. Moreover, other yield parameters such as total biomass, condition factor and performance index also followed the same trend as survival. Further, higher protease activity was found in control group (2.233±0.038 units mg protein-1 min-1) followed by earthworm (0.354±0.031 mg protein-1 min-1) supplemented group (p<0.05). The lipase and amylase activity were higher (p<0.05) in tubifex and control groups, respectively. Interpretation: It could be concluded that the dietary supplementation of tubifex at 5% inclusion level could be a promising aquaculture feeding strategy for pabda fish due to its rich nutrient content, increased feeding stimulation and more palatability.

Research progress on proteins and peptides from earthworm / 中草药

Earthworm is one of animal drugs in Chinese materia medica. It was commonly used in clinic with rich resources in China. Modern studies showed that proteins and peptides were the main components in earthworm. It has been used for treating cardiovascular and cerebrovascular diseases because of its various activities, such as anticoagulant, anti-stroke, antibacterial, and antifibrotic activities, etc. In this review, 48 proteins and peptides from different species of earthworm reported since 1983 were summarized, including their names, molecular weights, amino acid sequences, isoelectric points, and activities. In addition, its pharmacological effects of earthworm proteins and peptides were summarized. In all, it will provide a scientific basis for the further study and comprehensive utilization of proteins and peptides of earthworm.

Presence of Eisenia fetida enhanced phytoremediation of anthracene by Lolium perenne / Presença de Eisenia fetida aumentou a fitorremediação do antraceno por Lolium perene

The efficiency of co-application of Eisenia fetida and ryegrass was evaluated in a process called earthworm-assisted phytoremediation. Anthracene was used as a model compound for polycyclic aromatic hydrocarbons (PAHs). The experiments were conducted on a loamy soil in greenhouse conditions. At the end of the experiment, the soil samples were analyzed for residual anthracene by HPLC. Results showed that, phytoremediation using ryegrass could remove 81% of anthracene; however, the rate of removal was 92% when E. fetida was applied simultaneously. E. fetida alone could also remove the initial concentration of anthracene by 40%. Although ryegrass itself could remove anthracene significantly, the employment of earthworm, together with plant was more efficient than each of them individually. The application of E. fetida could also enhance the growth parameters of ryegrass significantly. In comparison to the control, the presence of E. fetida increased plant dry weight (7.8%), root length (47%), shoots length (32%), and root volume (12%). The number of live earthworms was also increased in the planted pots, indicating the helpfulness of the plant for survival of the earthworm in the PAH-contaminated soil. Although plant and earthworm use completely different mechanisms for anthracene degradation, they improve efficiency and survival of the three-component-system.

A eficiência da co-aplicação de Eisenia fetida e azevém foi avaliada em um processo denominado fitorremediação assistida por minhocas. O antraceno foi usado como um composto modelo para hidrocarbonetos aromáticos policíclicos (PAHs). Os experimentos foram conduzidos em um solo argiloso em condições de estufa. No final da experiência, as amostras de solo foram analisadas quanto ao antraceno residual por HPLC. Os resultados mostraram que, a fitorremediação com azevém pode remover 81% do antraceno; no entanto, a taxa de remoção foi de 92% quando E. fetida foi aplicada simultaneamente. E. fetida sozinha também foi capaz de remover a concentração inicial de antraceno em 40%. Embora o próprio azevém pudesse remover significativamente o antraceno, o emprego da minhoca, juntamente com a planta, foi mais eficiente do que cada um deles individualmente. A aplicação de E. fetida também pode melhorar significativamente os parâmetros de crescimento do azevém. Em comparação com o controle, a presença de E. fetida aumentou o peso seco da planta (7,8%), o comprimento da raiz (47%), o comprimento da parte aérea (32%) e o volume radicular (12%). O número de minhocas vivas também aumentou nos vasos plantados, indicando a utilidade da planta para a sobrevivência da minhoca no solo contaminado com PAH. Embora plantas e minhocas usem mecanismos completamente diferentes para a degradação do antraceno, eles melhoram a eficiência e a sobrevivência do sistema de três componentes.

Protective effect of earthworm active ingredients against endoplasmic reticulum stress induced L-02 hepatocyte apoptosis / 中国中药杂志

This study focused on the protective effect of earthworm active ingredients (EWAs) on hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS) in L-02 cells. The L-02 cells were cultured in vitro. The cell viability was measured with CCK-8, the apoptosis of L-02 cells was detected by flow cytometry, and the relevant protein and mRNA expressions were detected by Western blot and qPCR. According to the findings, tunicamycin (TM) could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner. Compared with normal group, the apoptosis rate in model group was significantly increased (<0.05 or <0.01). The protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, were significantly up-regulated (<0.05 or <0.01), while Bcl-2 was significantly down-regulated (<0.05 or <0.01). After the administration with different concentrations of EWAs, compared with model group, EWAs could significantly increase the survival rate ofL-02 hepatocyte and decrease the cell apoptosis rates. It could also reduce the protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, in a dose-dependent manner (<0.05 or <0.01) and increased the protein and mRNA expressions of Bcl-2(<0.05 or <0.01). These results showed that EWAs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells. Its mechanism may be related to the down-regulation of mRNA and protein expressions of GRP78, PERK, ATF4, eLF2α, CHOP and Bax, and the up-regulation, the relief of ERS and the promotion of the proliferation of impaired L-02 cells.

De novo transcriptome assembly, functional annotation and differential gene expression analysis of juvenile and adult E. fetida, a model oligochaete used in ecotoxicological studies

BACKGROUND: Earthworms are sensitive to toxic chemicals present in the soil and so are useful indicator organisms for soil health. Eisenia fetida are commonly used in ecotoxicological studies; therefore the assembly of a baseline transcriptome is important for subsequent analyses exploring the impact of toxin exposure on genome wide gene expression. RESULTS: This paper reports on the de novo transcriptome assembly of E. fetida using Trinity, a freely available software tool. Trinotate was used to carry out functional annotation of the Trinity generated transcriptome file and the transdecoder generated peptide sequence file along with BLASTX, BLASTP and HMMER searches and were loaded into a Sqlite3 database. To identify differentially expressed transcripts; each of the original sequence files were aligned to the de novo assembled transcriptome using Bowtie and then RSEM was used to estimate expression values based on the alignment. EdgeR was used to calculate differential expression between the two conditions, with an FDR corrected P value cut off of 0.001, this returned six significantly differentially expressed genes. Initial BLASTX hits of these putative genes included hits with annelid ferritin and lysozyme proteins, as well as fungal NADH cytochrome b5 reductase and senescence associated proteins. At a cut off of P = 0.01 there were a further 26 differentially expressed genes. CONCLUSION: These data have been made publicly available, and to our knowledge represent the most comprehensive available transcriptome for E. fetida assembled from RNA sequencing data. This provides important groundwork for subsequent ecotoxicogenomic studies exploring the impact of the environment on global gene expression in E. fetida and other earthworm species.

An Accelerated Solvent Extraction-Solid Phase Extraction-High Performance Liquid Chromatographic Method for Determination of Polycyclic Aromatic Hydrocarbons in Soil and Earthworm Samples / 分析化学

A hugh performance luquud chromatographuc ( HPLC) method was extablushed for determunatuon of the content of 7 polycycluc aromatuc hydrocarbons ( PAHs) un soul and earthworm samples based on accelerated solvent extractuon ( ASE) for extractuon and solud phase extractuon ( SPE) column for sample purufucatuon. In thus method, the samples were furst extracted by the muxed solutuon of n-hexane and acetone extractuon (4∶1, V/V) , and then purufued by SPE column ( suluca gel column for soul sample purufucatuon and Al2 O3-suluca gel column for earthworm sample purufucatuon ) , eluted by 10 mL of elutuon of n-hexane and duchloromethane (9∶1, V/V), and evaporated to dryness wuth rotary evaporator. After that, the pretreated samples were re-dussolved wuth acetonutrule to a constant volume, followed by a fultratuon wuth a 0. 22-μm organuc fulter membrane for the quantufucatuon by HPLC. The recoverues of the method for 7 PAHs were 83. 5%-110. 2% un soul samples and 81. 2%-97. 1% un earthworm samples. The detectuon lumuts of the method for 7 PAHs were 0. 15-0. 85 μg/kg. The method had good reproducubuluty and met the qualuty control requurements of sample analysus.

RESPUESTA DE FITONEMATODOS DE PLÁTANO Musa AAB SIMMONDS A ESTRATEGIAS DE MANEJO INTEGRADO DEL SUELO Y NUTRICIÓN / PLANTAIN Musa AAB SIMMONDS PHYTONEMATODES RESPONSE TO SOIL INTEGRATED MANAGEMENT STRATEGIES AND NUTRITION

En Colombia el cultivo de plátano ocupa uno de los principales renglones de la economía; hace parte de la canasta familiar y es una fuente de empleo en las zonas donde se cultiva. La producción de este frutal se ve amenazada por el ataque de nematodos fitoparásitos que afectan el sistema radical, disminuyen la absorción de nutrientes y sirven como puerta de entrada a patógenos. Debido a esto, se requieren manejos que permitan la regulación de las poblaciones mediante estrategias que no atenten contra el equilibrio del ecosistema y que presenten una ventaja competitiva frente a los tratamientos tradicionales. En este estudio se evaluó la respuesta poblacional de fitonematodos del plátano Dominico Hartón, la presencia de hongos micorrícicoarbusculares (HMA) y la actividad de lombrices a la inoculación con HMA, lixiviado de compost de raquis de plátano y lombricompost. Los resultados obtenidos en la investigación sugieren que la respuesta de la población de fitonematodos a los tratamientos evaluados, mostró gran dependencia de las características parasíticas de cada género y que la aplicación de lombricompost, HMA y lixiviado de raquis de plátano pueden tener potencial en la regulación de las poblaciones de fitonematodos en el cultivo.

Plantain cultivation in Colombia occupies one of the main areas of the economy: plantain is part of the shopping basket and is a source of employment in the areas where it is cultivated. The production of this fruit is threatened by the attack of phytoparasitic nematodes affecting the root system, reducing the absorption of nutrients and serving as a gateway to pathogens. Because of this, maneuvers which allow the regulation of populations through strategies that do not threaten the balance of the ecosystem and that show a competitive advantage over traditional treatments. The phytonematodes population response of Dominico-Harton plantain, the presence of arbuscular mycorrhizal fungi (AMF) and earthworm activity to HMA inoculation, leachate from plantain compost rachis and earthworm compost were evaluated in this study. The results obtained from this research suggest that the phytonematodes population response to the treatments evaluated, showed strong dependence on parasitic characteristics of each gender and than the application of earthworm compost, AMF and leachate from plantain compost rachis may have a potential in the regulation of phytonematodes populations in cultivation.

Determination of Hexabromocyclododecanes Enantiomers in Earthworm and Soil by Chiral High Performance Liquid Chromatography-Isotopic Dilution-Tandem Mass Spectrometry / 分析化学

An enantioselective method was developed for the separation and determination of three chiral hexabromocyclododecanes ( HBCDs ) including α-HBCD, β-HBCD, γ-HBCD in soil and earthworm by HPLC-ID-MS/MS. d18-HBCDs used as internal standards were added to the samples before extraction. HBCDs enantiomers were extracted from soil by accelerated solvent extraction ( ASE ) with n-hexane/DCM (1:1,V/V) at 100℃ and 10 MPa for 5 min, and further cleaned up using silica column. HBCDs enantiomers were extracted from earthworm by vortex turbulence with ethyl acetate. The extracts were orderly sulphonated by sulfuric acid, and purified by silica column. For all HBCDs enantiomers, good linearities were obtained in the concentration range of 0. 25-50 ng/mL. Limits of detection ( LOD) and limits of quantification ( LOQ) were 0. 00544-0. 00766 ng/g and 0. 0173-0. 0244 ng/g, respectively in soil. The recoveries of spiked samples at 0. 05 and 2. 5 ng/g levels were 80. 0%-95. 9% with relative standard deviations ( RSD, %) of 5. 7%-11. 9% in soil. Limits of detection (LOD) and limits of quantification (LOQ) were 0. 0103-0. 0148 ng/g and 0. 0328-0. 0471 ng/g, respectively in earthworm. The recoveries of spiked samples at 0. 1 and 5 ng/g levels were 78. 0% -94. 4% with relative standard deviations ( RSD, %) of 6. 1% -12. 2% in earthworm. This method can meet the requirements of determination of trace HBCDs in soil and earthworm.

<b>Efficacy of a Supplement Containing Freeze-dried Earthworm Powder for Atherosclerosis</b> / 日本東洋医学雑誌

We studied the effects of a supplement containing freeze-dried earthworm powder : “Protein Related compound Derived from Redworm (PR-DR)” for atherosclerosis in 19 patients (10 females, average age : 61.5 ± 6.9), whose vascular ages were estimated to be older than their actual ages based on the index of atherosclerosis ; Intima-Media Thickness (IMT) by carotid ultrasonography and Cardio-Ankle Vascular Index (CAVI). After we obtained informed consent, they took daily dose of 6 PR-DR capsules for 6 months. Each capsule contains 40 mg of powdered extract of <i>Lumbricus rubellus</i>, which is known to contain fibrin-degrading an enzyme and to increase the effect of t-PA.<br>Before and 6 months after intake, we found significant decrease in 3 indicators of atherosclerosis. The scores of difference between 2 time points (6 month-pre) were IMT-Cmax : -0.15 ± 0.16 mm, maxIMT : -0.22 ± 0.17 mm, plaque score : -1.57 ± 1.12 (all p < 0.01 by Wilcoxon signed-rank test). As statins and EPA also decrease atherosclerosis, we excluded 6 patients who were taking these medications. Even in the remaining 13 patients (7 females, average age : 61.2 ± 7.9), 3 indicators were still significantly lower (IMT-Cmax : -0.07 ± 0.07 mm, maxIMT : -0.19 ± 0.17 mm, plaque score : -1.39 ± 1.04 ; all p < 0.01) after 6 months of intake. Thus, the results showed that PR-DR has anti-atherosclerosis effect and can be used clinically for the prevention of atherosclerosis and diseases associated with it.

Microbial diversity and digestive enzyme activities in the gut of earthworms found in sawmill industries in Abeokuta, Nigeria / Diversidad microbiana y actividad de enzimas digestivas en el intestino de las lombrices de tierra en los aserraderos de Abeokuta, Nigeria

The growing demand for wood has resulted in large volumes of wood wastes that are daily released to the soil from the activities of sawmills in South-Western Nigeria. In an attempt to setup a bioremediation model for sawdust, this study therefore aimed at evaluating microbial diversity, and the level of digestive enzymes in the gut of earthworms (Eudrilus eugeniae, Libyodrilus violaceous and Hyperiodrilus africanus) of sawmill origin. Four major sawmills located in Abeokuta (7o9’12” N - 3o19’35” E), namely Lafenwa, Sapon, Isale-Ake and Kotopo sawmills were used for this study. The arboretum of the Federal University of Agriculture, Abeokuta was used as control. Gut microbial analysis was carried out using the pour-plate method while digestive enzyme activities in the earthworm guts were done by the spectrophotometric method. Higher microbial counts (28.5±0.1x10³-97.0±0.1x10³cfu for bacteria and 7.0±0.1x10³-96.0±0.1x10³cfu for fungi) and microbial diversity were recorded in the gut of earthworms of the sawmill locations than those of the control site (17.5±0.1x10³cfu for bacteria and 4.5±0.1x10³cfu for fungi). Streptococcus mutans and Proteus spp.were common in the gut of E. eugeniae, and L. violaceous from the study sawmills, while Streptococcus mutans were also identified in H. africanus, but absent in the gut of E. eugeniae from the control site. Cellulase (48.67±0.02mg/g) and lipase (1.81±0.01mg/g) activities were significantly higher (p<0.05) in the gut of earthworms from the control site than those of the study sawmills. Furthermore, amylase (α and β) activity was highest in the gut of earthworms from the sawmills. Variations observed in the gut microbial and digestive enzyme activities of earthworms from the study sawmills as compared to the control site suggests that earthworms, especially E. eugeniae, could be a better organism for use as bioremediator of wood wastes. Rev. Biol. Trop. 62 (3): 1241-1249. Epub 2014 September 01.

La creciente demanda de madera ha resultado en grandes volúmenes de residuos de madera que se lanzan diariamente a la tierra, procedentes de las actividades de los aserraderos en el suroeste de Nigeria. Se realizó este estudio en un intento de configurar un modelo de biorremediación de aserrín, con el objetivo de evaluar la diversidad microbiana y el nivel de las enzimas digestivas en el intestino de las lombrices de tierra (Eudrilus eugeniae, Libyodrilus violáceo y Hyperiodrilus africanus) de origen aserradero. Para este estudio se utilizaron cuatro grandes aserraderos ubicados en Abeokuta (7°9’12 ‘’ N - 3°19’35 ‘’ E), a saber: Lafenwa, Sapon, Isale-Ake y Aserraderos Kotopo. El arboreto de la Universidad Federal de Agricultura, Abeokuta se utilizó como control. El análisis microbiano se llevó a cabo utilizando el método de vertido de placa, mientras que las actividades de enzimas digestivas en los intestinos de las lombrices se realizaron por el método espectrofotométrico. Los recuentos microbianos más altos fueron 28.5±97.0±0.1x10³-0.1x10³cfu para las bacterias y 7.0±0.1x10³-96.0±0.1x10³cfu para los hongos y la diversidad microbiana registrada en el intestino de las lombrices de tierra de los lugares de aserraderos y las del sitio de control fueron: 17.5±0.1x10³cfu para bacterias y 4.5±0.1x10³cfu para hongos. Streptococcus mutans y Proteus spp. eran comunes en el intestino de E. eugeniae, y L. violaceous de los aserraderos de estudio, mientras que también se identificaron Streptococcus mutans en H. africanus, que estuvieron ausentes en el intestino de E. eugeniae en el sitio de control. Actividades de celulasa (48.67±0.02mg/g) y lipasa (1.81±0.01mg/g) fueron significativamente mayores (p<0.05) en el intestino de las lombrices de tierra en el sitio de control que las de los aserraderos de estudio. Por otra parte, la actividad de la amilasa (α y β) fue mayor en el intestino de las lombrices de tierra de los aserraderos. Las variaciones observadas en la microbiota intestinal y la actividad de las enzimas digestivas de las lombrices de tierra de los aserraderos de estudio, en comparación con el sitio de control, sugieren que las lombrices de tierra, especialmente E. eugeniae, podrían ser un mejor organismo para su uso como biorremediador de residuos de madera.

From Earthworm Research to See How to Make Progress in Dilemma of Traditional Chinese Medicine Resources and Quality / 世界科学技术-中医药现代化

Resource of traditional Chinese medicine (TCM) is one of the greatest treasures of China. It made magnif-icent contribution in Chinese lives and breeds. Quality is the foundation of safety, effective and quality control of TCM. With the social development, problem with raising need and falling resource and quality had became more and more serious. It slowed down the speed of TCM modernization and internationalization. This study used Shanghai earthworm, the authentic TCM of Shanghai, as an example. Focus on resource and quality problems, such as species confusion, resource shortage, rough machining, low quality and no standards, this study tried to work out the reason-able and feasible solution. This effort funded a way out of a stalemate of TCM resources and quality. It is important to TCM modernization and internationalization.