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This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1β(IL-1β), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1β and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1β and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.
Subject(s)
Humans , Rats , Male , Animals , Interleukin-10 , Rats, Sprague-Dawley , Interleukin-4/metabolism , Bombyx , Interleukin-6/metabolism , Microglia/metabolism , Saline Solution/metabolism , Reperfusion Injury/metabolism , Brain Ischemia/metabolism , Cerebral Infarction , Reperfusion , NeuronsABSTRACT
Bombyx Batryticatus is a precious traditional Chinese animal drug commonly used in clinical practice in China, which has the effects of extinguishing wind, stopping convulsions, dispelling wind, relieving pain, resolving phlegm, and dissipating mass. The processing of Bombyx Batryticatus has a long history. As early as in the Liu Song period of the Southern and Northern Dynasties, there was a record of the processing of Bombyx Batryticatus with rice swill. In addition to the processing with bran, honey bran, and ginger juice, which are still used today, there are also processing methods such as rendering, flour processing, wine processing, salt processing, oil processing, charcoal, and red dates processing in ancient times. After processing, the fishy smell of Bombyx Batryticatus can be removed, and avoid nausea and vomiting caused by the direct taking. Furthermore, processing can also facilitate the removal of surface hairs and toxicity reduction, making the medicinal material crispy and easy to crush. Previous studies have shown that the main chemical constituents of Bombyx Batryticatus include protein polypeptides, sterols, and flavonoids, with anticonvulsant, anticoagulation, antithrombotic, anti-cancer, hypnotic, hypoglycemic, and other pharmacological effects. This paper reviewed the processing historical evolution, chemical constituents, and pharmacological effects of Bombyx Batryticatus to lay a foundation for the research on the processing mechanism, quality control, and active core substances of Bombyx Batryticatus.
Subject(s)
Animals , Bombyx , China , Evolution, Chemical , Flavonoids , FruitABSTRACT
OBJECTIVE:To evaluate the quality of Bombyx batryticatus medicinal materials in the market by multi-index,and provide reference for clinical rational drug use. METHODS:Referring to the Chinese Pharmacopoeia (2015 edition,part 1),the appearance characters of samples were observed,moisture and the total ash were detected,Fourier transform infrared spectroscopy was adopted to identify the medicinal materials,UV-Visible spectrophotometry and potassium permanganate titration were respec-tively used to determine the contents of active ingredient protein and ammonium oxalate. RESULTS:The moisture ranged in 2.84%-4.07%(RSD<5%,n=3),total ash in 7.79%-34.32%(RSD<5%,n=3),UV and FTIR spectra were basically similar, and both can reflect the characteristic peaks of protein peptides. Protein content ranged in 29.87%-57.19%(RSD<0.5%,n=3), ammonium oxalate content in 0.27%-4.77%(RSD<5%,n=3). CONCLUSIONS:The 10 batches of samples are authentic and processed products,moisture fits the requirement in pharmacopoeia while total ash does not;and the main ingredient is protein pep-tides. However,effective protein polypeptide content shows great differences as well as ammonium oxalate.
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To identify the commercial medicinal materials of Bombyx Batryticatus, two-dimensional DNA barcode was used to construct the "Internet Plus" identification system for Chinese medicine, which should benefit the cross-platform communication of DNA barcode information. Bombyx Batryticatus contained Bombyx mori Linnaeus and Beauveria bassiana (Bals.) Vuillant. Both COI and ITS sequences were obtained via PCR amplification for total genomic DNA extracted from raw materials using the animal genomic DNA kit, while only ITS but no COI sequences was obtained when using the plant genomic DNA kit. The ITS sequences obtained using the animal genomic DNA kit were consistent with those using plant genomic DNA kit. The medicinal materials yielded COI sequences and identified as B. mori. According to analysis of ITS sequences, the main species of the medicinal materials were identified as B. bassiana and few were identified as other fungi. NJ trees analysis based on ITS sequences suggests that it can be easily distinguished from other fungi. Our results showed that total genomic DNA of B. mori and B. bassiana was extracted simultaneously using the animal genomic DNA kit, which could effectively solve the problem in species identification of animal and fungi mixture materials. COI and ITS regions as DNA barcodes can stably and accurately identify Bombyx Batryticatus. The "Internet Plus" two-dimensional DNA barcode system will promote the standardization and normalization of Chinese medicinal materials market.
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Objective: To study the chemical constituents of Bombyx batryticatus. Methods: The chemical constituents of B. batryticatus were isolated by different column chromatographic techniques, including silica gel and Sephadex LH-20 columns. The structures of these compounds were identified by a comprehensive analysis of the spectroscopic data. Results: Eleven compounds were isolated from the EtOAc extract of B. batryticatus. The compounds were identified as cyclo (D)-Pro-(D)-Val (1), cyclo (S)-Pro-(R)-Leu (2), cyclo (D)-Pro-(D)-Ile (3), cyclo (D)-Pro-(D)-Phe (4), aurantiamid (5), (+)-pinoresinol (6), isololiolide (7), butyl-2-pyrrolidone-5-carboxylate (8), (-)-medioresinol (9), (-)-lariciresinol (10), and methylphydroxybenzeneacetate (11). Conclusion: All the compounds are isolated from B. batryticatus for the first time.
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Objective: To investigate the chemical constitutes of Bombyx Batryticatus. Methods: The compounds were isolated by means of solvent extraction, repeated silica gel column chromatography, ODS, and preparative RP-HPLC. The structures were identified by spectral analysis and chemical evidence. Results: Four compounds of steroids were isolated from the Bombyx Batryticatus and were identified as (3α,6β)-3-benzyl-6-isopropyl-4-methyl-2,5-morpholinedione (1), (3α,6α)-3-benzyl-6-isopropyl- 4-methyl-2,5-morpholinedione (2), 5α,8α-epidioxyergosta-6,22-dien-3β-ol (3), 22E-3β-hydroxy-5α,6α-epoxyergosta-22-en-7-one (4), 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3β,7α-diol (5), 7α-methoxy-(22E,24R)-5α,6α-epoxyergosta-8(14),22-diene-3β-ol (6), (22E,24R)-ergosta-5,7,22-trien-3β-ol (7), stigmasta-7,22-diene-3β,5α,6α-triol (8), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-diene-6-one (9), (22E,24S)-5α,8α-epidioxy-24-methylcholesta-6,9(11),22-trien-3β-ol (10), homoarginine (11), and β-sitosterol (12). Conclusion: Compounds 1-11 are isolated from Bombyx Batryticatus for the first time.
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Objective To explore the anticoagulating active components in water extraction, alcohol sedimentation and fraction separated by gelfiltration chromatography of Bombyx Batryticatus. Methods Anticoagulating active fractions were prepared by the methods of water extraction, 70% alcohol sedimentation and gelfiltration chromatography. Components (such as porteins or polypeptide, amino acids, oxalic acid ammonium) in each fraction were determined by chemical reaction indentification, Kjeldahl method, conductance, UV and HPLC. Results Polypeptide, amino acids and oxalic acid ammonium were the main components in anticoagulating active fractions, content of which reached more than 80%. The content of oxalic acid ammonium decreased about 52% processed by alcohol sedimentation, while the content of polypeptide and amino acids only decreased about 8%. The content of peptides and amino acids increased about 28% purified by gelfiltration chromatography compared with the one in water extraction and about 39% compared with the one in alcohol sedimentation. The content of oxalic acid ammonium was the same as the one in alcohol sedimentation, but decreased about 1 time compared with the one in water extraction. The content of 15 kinds of amino acids were 40%～50% in the solids of each fractions, which maked up to 60%～76% of the content of proteins determined by Kjeldahl method. Molecular weight range of peptides is 1.0～4.4 kDa. Conclusion This study provides a experimental basis for further separation and purification of anticoagulating active components from Bombyx Batryticatus.
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Object To study and identify the chemical constituents of Bombyx Batryticatus. Methods The constituents were isolated from the above materials by column chromatography using silica gel and Sephadex LH-20, purified by crystallization, and identified by spectroscopic methods. Results Eight compounds were isolated and identified. They are 6-methoxy-7-O-?-D-(4′-methoxy) glucopyranosyl coumarin (Ⅰ), ergost-6, 22-dien-3?, 5?, 8?-triol (Ⅱ), palmitic acid (Ⅲ), meso-erythritol (Ⅳ), D-mannitol (Ⅴ), uracil (Ⅵ), ?-sitosterol (Ⅶ), and daucosterol (Ⅷ). Conclusion Coumpound Ⅰ is a new coumarin glycoside.
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Objective To study the mechanism of herbal medicines with the actions of tonifying Qi,calming wind and eliminating phlegm in treating epilepsy. Methods Rat models of chronic epilepsy were induced by penicillin. Changes of electrocorticogram (ECOG) and the contents of cerebral gamma-aminobutyric acid (GABA) and glutamate (GLU) in rats were observed. Results Radix Astragali increased the content of GABA and the effect was stronger with Borneol Syntheticum added but had less effect in decreasing GLU. Both Bombyx Batryticatus and Arisaema cum Bile could decrease GLU content and Arisaema cum Bile in particular. The effect of Radix Astragali was stronger in decrease of ECOG frequency but weaker than that in modifying the amplitude of ECOG,while Bombyx Batryticatus and Arisaema cum Bile were in the contrary. The effect of Radix Astragali in improving ECOG did not differ from that with Borneolm Syntheticum added. Conclusion Qi-tonifying,wind-calming and phlegm-eliminating herbal medicines are effective in treating epilepsy and their mechanism may be related to the regulation of electrocorticogram and the content of cerebral GABA and GLU.