A Strategy for Rapid Discovery of Marker Peptides Associated with Fibrinolytic Efficacy of Pheretima aspergillum Based on Bioinformatics Combined with Parallel Reaction Monitoring.

Quality control of animal-derived traditional Chinese medicines has improved dramatically as proteomics research advanced in the past few decades. However, it remains challenging to identify quality attributes with routine proteomics approaches since protein with fibrinolytic activity is rarely reported in pheretima, a typical animal-derived traditional medicine. A novel strategy based on bioinformatics combined with parallel reaction monitoring (PRM) was developed here to rapidly discover the marker peptides associated with a fibrinolytic effect. Potential marker peptides were found by lumbrokinase sequences' alignment and in silico digestion. The fibrinogen zymography was used to visually identify fibrinolytic proteins in pheretima. As a result, it was found that the fibrinolytic activity varied among different portions of pheretima. Fibrinolytic proteins were distributed regionally in the anterior and anterior-mid portion and there was no significant fibrinogenolytic activity observed in the mid-posterior and posterior portion. Finally, PRM experiments were deployed to validate and quantify selected marker peptides and a total of 11 peptides were identified as marker peptides, which could be potentially used in quality control of pheretima. This strategy provides a robust workflow to benefit the quality control of other animal-derived traditional medicines.

Effect of Pheretima aspergillum on reducing fibrosis: A systematic review and meta-analysis.

Background: Pheretima aspergillum (common name: Earthworm, Chinese name: dilong) has been used in traditional Chinese medicine for thousands of years. Recently, a few scientific studies have investigated the antifibrotic effects of Dilong extract (DE) and produced controversial results. We conducted a meta-analysis to make an informed decision on the antifibrotic effects of Dilong extract. Methods: The studies on antifibrotic effects of Dilong extract published until July 2022 in the scientific databases [PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), VIP database for Chinese Technical Periodicals, SinoMed and WanFang database] were reviewed. The RevMan 5.4.1 software was used for standardized mean difference (SMD) analysis. Two researchers independently reviewed all the studies, and their quality was assessed using the Cochrane risk of bias tool. Results: A total of 325 studies were found in the scientific databases; however, only 13 studies met the criteria for analysis. Dilong extract treatment was associated with antifibrotic effects via inhibiting the transforming growth factor beta 1 (TGF-ß1, SMD = -3.16, 95% CI: -4.18, -2.14, p < .00001) and alpha-smooth muscle actin (α-SMA: SMD = -2.57, 95% CI: -3.47, -1.66, p < .00001). Conclusion: Dilong extract effectively reduces tissue fibrosis; thus, further scientific studies should be conducted to investigate and develop it for clinical use. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42022357141.

Investigation of the chemical compounds in Pheretima aspergillum (E. Perrier) using a combination of mass spectral molecular networking and unsupervised substructure annotation topic modeling together with in silico fragmentation prediction.

Untargeted mass spectrometry analysis is one of the most challenging and meaningful steps in the rapid structural elucidation of the highly complex and diverse constituents of traditional Chinese medicine. Specifically, it is a laborious and time-consuming way to identify unknown compounds. Herein, a workflow was proposed to expedite the annotations of the chemical structures in Pheretima aspergillum (E. Perrier) (Di-Long, DL). First, ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) was performed to obtain the untargeted mass spectral data. Then, the spectral data were uploaded to the Global Natural Products Social Molecular Networking (GNPS) platform to create a network and extract the Mass2Motifs (co-occurring fragments and neutral losses) using unsupervised substructure annotation topic modeling (MS2LDA). Finally, a structural analysis was performed using the proposed workflow of MS2LDA in combination with mass spectral molecular networking and in silico fragmentation prediction. As a result, a total of 124 compounds from DL were effectively characterized, of which 89 (7 furan sulfonic acids, 57 phospholipids and 25 carboxamides) were identified as potentially new compounds from DL. The results presented in this article significantly improve the understanding of the chemical composition of DL and provide a solid scientific basis for the future study of the quality control, underlying pharmacology and mechanism of DL. Moreover, the proposed workflow was used for the first time to accelerate the annotations of unknown molecules from TCM. Furthermore, this workflow will increase the efficiency of characterizing the 'unknown knowns' and elucidation of the 'unknown unknowns' from TCM, which are crucial steps of discovering the natural product drugs in TCM.

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.

Anti-Asthma Effect of an Active Components Group from Decoction of Pheretima Aspergillum and Its Chemical Composition Characterized by Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry.

The decoction of Pheretima aspergillum is used as a traditional medicine for treatment of asthma in China with a long clinical history. The aim of the study was to investigate the anti-asthma activities of an active components group obtained from the decoction of Pheretima aspergillum in histamine and ovalubumin induced asthma guinea pigs. The experimental asthma model was established with spray of histamine and ovalbumin (OVA) followed by intragastric administration of Pheretima aspergillum extract and fractions. The incubation period of asthma, serum IFN-γ, IL-4, and LTB4 levels were tested and determined. In addition, liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) was used to identify the bioactive components in active fraction. The significant increase of asthma incubation period, serum IFN-γ level were observed in the asthma guinea pigs treated with the active components group. The serum IL-4 and LTB4 levels were obviously decreased compared to that of model controls. In addition, twenty oligopeptides were first identified or characterized in the active fraction from the decoction of Pheretima aspergillum. The results indicated that Pheretima aspergillum can inhibit infiltration and diffusion of inflammatory cells in asthma model guinea pigs, and regulate IFN-γ, IL-4, and LTB4 secretions. It is reasonable to assume that the anti-asthma activities of Pheretima aspergillum mainly resulted from these oligopeptides. Also, synergistic effects among their compounds may exist in the anti-asthma activity of Pheretima aspergillum.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification.

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/µl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

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.

Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.

BACKGROUND: Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells. METHOD: RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot. RESULTS: GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 µg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE2, TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1ß and IL-6. CONCLUSION: These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.

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.

Specific Polymerase Chain Reaction Molecular Identification of Pheretima aspergillum (E. Perrier) / 广州中医药大学学报

Objective To screen out the polymerase chain reaction ( PCR) primers for specifically identifying Pheretima aspergillum (E. Perrier) , so as to establish a rapid and accurate method to identify the origin of Pheretima aspergillum. Methods Four kinds of earthworms recorded in pharmacopoeia and six kinds of their adulterants commonly seen in the market were collected. The 12SrRNA sequences related to earthworm were downloaded from GenBank database. PCR amplification for ten kinds of samples was performed using the universal primers, and then the products were sequenced. According to the differences between the above sequences, three pairs of specific primers in the non-conservative district were designed for identification of Pheretima aspergillum. Results High-specificity PCR amplification for Pheretima aspergillum with 12St/12Stf primer occurred when the annealing temperature was increased to 64℃, and only Pheretima aspergillum had single amplification strip, while no strips were found in the other adulterants under the same condition. The achieved specific primers could be well verified in 15 kinds of medicinal materials of earthworm purchased in the market, which had the same morphological features showed by the traditional identifying method. Conclusion With 12St/12Stf primer as a specific marker, the identifica tion of Pheretima aspergillum is rapid and accurate in related species of Pheretima aspergillum, and avoids the effect of some factors such as integrity of experimental materials and drying process.

HPLC in determination of hypoxanthine, xanthine, uridine and uracil in Shanghai Pheretima and Pheretima aspergillum (E. Perrier) / 第二军医大学学报

Objective To establish an effective method for the determining hypoxanthine, xanthine, uridine and uracil in earthworm in Shanghai Pheretima and Pheretima aspergillum (E. Perrier), contributing to quality control of the medicinal material. Methods Hypoxanthine, xanthine, uridine and uracil were extracted from the earthworms with 0.9% NaCl by ultrasonic and determined by HPLC. The chromatographic conditions were: SORBAX SB-Aq column (250 mm×4.6 mm, 5 μm, Aglient Co.,Ltd), 5 mmol/L KH2PO4 (pH 2.9) as the mobile phase with a flow rate of 1 mL/min,the detection wavelength was 254nm, the column temperature was set at 30℃, and the injection volume was 10 μL. Hypoxanthine, xanthine, uridine and uracil were determined by HPLC at the same time. Results The linear range was 0.5-100 μg (r=0.999 9) for hypoxanthine, with the average recovery being 99.37%, RSD=1.36% (n=6). The linear range was 0.5-100 μg (r=0.993 1) for xanthine, with the average recovery being 91.57%, RSD=1.40% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uridine, with the average recovery being 95.31%, RSD=1.64% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uracil, with the average recovery being 100.21%, RSD=1.98% (n=6). Conclusion The current method is reproducible and has satisfactory recovery, and it can be used to determine hypoxanthine, xanthine, uridine and uracil in earthworm medicinal material.

Effect of oral administration of Pheretima aspergillum (earthworm) in rats with cerebral infarction induced by middle-cerebral artery occlusion.

We investigated the curative effect of Pheretima aspergillum (earthworm, PA) on rats with middle cerebral artery occlusion (MCAo). The MCAo-induced cerebral infarction was established and its underlying mechanisms by counting the infarction areas and evaluating the rats' neurological status. Immunostaining was used to test the expression of NeuN, and glial fibrillary acidic (GFAP), S100B, and brain-derived neurotrophic factor (BDNF) proteins. Our results showed that oral administration of PA for two weeks to rats with MCAo successfully reduced cerebral infarction areas in the cortex and striatum, and also reduced scores of neurological deficit. The PA-treated MCAo rats showed greatly decreased neuronal death, glial proliferation, and S100B proteins in the penumbra area of the cortex and in the ischemic core area of the cortex, but BDNF did not changed. These results demonstrated novel and detailed cellular mechanisms underlying the neuroprotective effects of PA in MCAo rats.