[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Herbal Textual Research on Scorpio in Famous Classical Formulas and Its Resource Status / 中国实验方剂学杂志

By reviewing the materia medica， medical books and scripture history， combining modern and contemporary literature and field investigation， this paper systematically reviewed the name， origin， scientific name， producing area， quality， harvesting and processing of Scorpio in famous classical formulas to clarify the relationship between ancient and modern times and provide a reference basis for the development of related famous classical formulas. After the textual research， it can be seen that there are many names of Scorpio， and most of the materia medica use Xie or Quanxie as the official name. The origin of Scorpio used in the past dynasties is Buthus martensii， which is the same in ancient and modern times. B. martensii is mainly distributed in the central and eastern parts of China， as well as Mongolia， Korea and other places， located in East Asia. Therefore， pharmaceutical workers in China mostly continue to use the early Chinese name， that is， Dongya Qianxie， while modern Scorpiones taxonomists set its Chinese name as Mashi Zhengqianxie. In order to maintain the stability and continuity of the origin of Scorpio， the previous editions of Chinese Pharmacopoeia have always used the name of B. martensii. The geo-authentic producing area of Scorpio， which has been respected in the past dynasties， was Qingzhou， Shandong. Until the Republic of China， due to the different processing methods in the production area， the geo-authentic producing area expanded to Yu county， Henan， with Yu county as the distribution center， the best quality of Scorpio is produced by boiling in clear water and drying after boiling. The origin processing of Scorpio is mainly divided into clean water and salt water boiling and then drying， where the method of boiling with salt water was first described in Bencao Yuanshi， the purpose is antiseptic and suitable for storage， and the salt should be washed away when used clinically. There are few processing methods of Scorpio， in ancient times， it was used for roasting or frying after removing the feet， in modern times， it is mostly to remove impurities， wash and dry. Scorpio is not only used for medicinal purposes， but also has the habit of eating in many areas， so the consumption of resources is relatively large， and it is still mainly harvested from the wild， resulting in the decrease of wild resources year by year. Based on the research conclusion， it is recommended that B. martensii， which is produced in Qingzhou， Luyi or Yuzhou， should be used in the the development of famous classical formulas containing Scorpio， and the origin processing is preferable to be dried after boiling with water， and the processing specification should be selected as raw products. And B. martensii in geo-authentic producing areas was used as the seed source to establish a standardized breeding base to ensure the sustainable development of the resources of Scorpio.

Makatoxin-3, a thermostable Nav1.7 agonist from Buthus martensii Karsch (BmK) scorpion elicits non-narcotic analgesia in inflammatory pain models.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic pain management represents a serious healthcare problem worldwide. The use of opioid analgesics for pain has always been hampered by their side effects; in particular, the addictive liability associated with chronic use. Finding a morphine replacement has been a long-standing goal in the field of analgesia. In traditional Chinese medicine, processed Buthus martensii Karsch (BmK) scorpion has been used as a painkiller to treat chronic inflammatory arthritis and spondylitis, so called "Scorpio-analgesia". However, the molecular basis and the underline mechanism for the Scorpio-analgesia are still unclear. AIM OF THE STUDY: The study aims to investigate the molecular basis of "Scorpio analgesia" and identify novel analgesics from BmK scorpion. MATERIALS AND METHODS: In this study, the analgesic abilities were determined using formalin-, acetic acid- and complete Freund's adjuvant-induced pain models. The effect of BmK venom and processed BmK venom on Nav1.7 were detected by whole-cell voltage-clamp recordings on HEK293-hNav1.7 stable cell line. Action potentials in Dorsal root ganglion (DRG) neurons induced by Makatoxin-3-R58A were recorded in current-clamp mode. The content of Makatoxin-3 was detected using competitive enzyme-linked immunosorbent assay based on the Makatoxin-3 antibody. High performance liquid chromatography, western blot and circular dichroism spectroscopy were used to analysis the stability of Makatoxin-3. RESULTS: Here we demonstrate that Makatoxin-3, an α-like toxin in BmK scorpion venom targeting Nav1.7 is the critical component in Scorpio-analgesia. The analgesic effect of Makatoxin-3 could not be reversed by naloxone and is more potent than Nav1.7-selective inhibitors and non-steroidal anti-inflammatory drugs in inflammatory models. Moreover, a R58A mutant of Makatoxin-3 is capable of eliciting analgesia effect without inducing pain response. CONCLUSIONS: This study advances ion channel biology and proposes Nav1.7 agonists, rather than the presumed Nav1.7-only blockers, for non-narcotic relief of chronic pain.

Isolation, identification and electrophysiological activity of BmK M2 from Buthus martensii Karsch venom / 中国药科大学学报

@#This study aimed to isolate and identify novel toxin peptides targeting voltage-gated sodium channels (VGSGs) from the venom of the Buthus martensii Karsch (BmK) scorpion. Using G50-gel filtration, HPLC, peptide fingerprinting and amino acid sequencing, a novel sodium channel modulator, BmK M2, was identified from BMK scorpion. BmK M2 is a relatively abundant long chain polypeptide toxin in BmK scorpion venom with a molecular weight of 7 235.59, consisting of 64 amino acids and 4 pairs of disulfide bonds.Sequence alignment showed that the amino acid sequence of BmK M2 had high sequence and structural similarity to that of the discovered sodium channel toxins of BmK M1, BmK M3 and BmK M9, etc.BmK M2 is a potential new sodium channel modulator.Electrophysiological results revealed that BmK M2 can significantly enhance the activation, delay the steady-state inactivation and closed-state inactivation of Nav1.7, but has no activity on Nav1.8.BmK M2 can be used as a novel peptide probe for the study of the structure and function of Nav1.7 and the development of drugs targeting Nav1.7.

Discovery of Guanidine Derivatives from Buthus martensii Karsch with Metal-Binding and Cholinesterase Inhibition Properties.

Two rare guanidine-type alkaloids, Buthutin A (1) and Buthutin B (2), along with two other compounds (3, 4), were isolated from Buthus martensii Karsch, and determined using extensive spectroscopic data analysis and high resolution-mass spectrometry. Compound 1 showed the most potent inhibition on AChE and BChE with IC50 values of 7.83 ± 0.06 and 47.44 ± 0.95 µM, respectively. Kinetic characterization of compound 1 confirmed a mixed-type of AChE inhibition mechanism in accordance with the docking results, which shows its interaction with both catalytic active (CAS) and peripheral anionic (PAS) sites. The specific binding of compound 1 to PAS domain of AChE was also confirmed experimentally. Moreover, compounds 1 and 3 exhibited satisfactory biometal binding abilities toward Cu2+, Fe2+, Zn2+ and Al3+ ions. These results provide a new evidence for further development and utilization of B. martensii in health and pharmaceutical products.

Study of Anti-Inflammatory and Analgesic Activity of Scorpion Toxins DKK-SP1/2 from Scorpion Buthus martensii Karsch (BmK).

Buthus martensii Karsch (BmK), is a kind of traditional Chinese medicine, which has been used for a long history for the treatment of many diseases, such as inflammation, pain and cancer. In this study, DKK-SP1/2/3 genes were screened and extracted from the cDNA library of BmK. The DKK-SP1/2/3 were expressed by using plasmid pSYPU-1b in E. coli BL21, and recombinant proteins were obtained by column chromatography. In the xylene-induced mouse ear swelling and carrageenan-induced rat paw swelling model, DKK-SP1 exerted a significant anti-inflammatory effect by inhibiting the expression of Nav1.8 channel. Meanwhile, the release of pro-inflammatory cytokines (COX-2, IL-6) was decreased significantly and the release of anti-inflammatory cytokines (IL-10) were elevated significantly. Moreover, DKK-SP1 could significantly decrease the Nav1.8 current in acutely isolated rat DRG neurons. In the acetic acid-writhing and ION-CCI model, DKK-SP2 displayed significant analgesic activity by inhibiting the expression of the Nav1.7 channel. Moreover, DKK-SP2 could significantly inhibit the Nav1.7 current in the hNav1.7-CHO cells.

Scorpion Venom peptide, AGAP inhibits TRPV1 and potentiates the analgesic effect of lidocaine.

The current study was designed to test the hypothesis that BmK AGAP (AGAP) potentiates the analgesic effect of lidocaine. The chronic constrictive injury was performed on 72 rats to induce a rapid onset and long-lasting pain. The rats were randomly assigned to one of six groups; Group A (n = 12) received an intrathecal administration of saline, Group B (n = 12) received an intrathecal injection of lidocaine, Group C (n = 12) received an intrathecal administration of AGAP, Group D, E, and F (n = 12 each) received an intrathecal administration of lidocaine 0.005 mg/ml + AGAP 25, 50, 100 µg/kg respectively. The von Frey filaments were used to assess mechanical allodynia. Nav1.7 and TRPV1 currents were recorded by the whole-cell aspiration patch-clamp technique, and KCNQ2/3 currents were recorded by the whole-cell drilling patch-clamp technique. The whole-cell aspiration patch-clamp technique showed that AGAP inhibited TRPV1and KCNQ2/3 currents and increased the analgesic effect of lidocaine. AGAP may have a synergistic effect with lidocaine which demonstrates a potential therapeutic approach for optimizing post-operative analgesia.

Optimization of Scorpion Protein Extraction and Characterization of the Proteins' Functional Properties.

Scorpion has long been used in traditional Chinese medicine, because whole scorpion body extract has anti-cancer, analgesic, anti-thrombotic blood anti-coagulation, immune modulating, anti-epileptic, and other functions. The purpose of this study was to find an efficient extraction method and investigate some of physical and chemical parameters, like water solubility, emulsification, foaming properties, and oil-holding capacity of obtained scorpion proteins. Response surface methodology (RSM) was used for the determination of optimal parameters of ultrasonic extraction (UE). Based on single factor experiments, three factors (ultrasonic power (w), liquid/solid (mL/g) ratio, and extraction time (min)) were used for the determination of scorpion proteins (SPs). The order of the effects of the three factors on the protein content and yield were ultrasonic power > extraction time > liquid/solid ratio, and the optimum conditions of extraction proteins were as follows: extraction time = 50.00 min, ultrasonic power = 400.00 w, and liquid/solid ratio = 18.00 mL/g. For the optimal conditions, the protein content of the ultrasonic extraction and yield were 78.94% and 24.80%, respectively. The solubility, emulsification and foaming properties, and water and oil holding capacity of scorpion proteins were investigated. The results of this study suggest that scorpion proteins can be considered as an important ingredient and raw material for the creation of water-soluble supramolecular complexes for drugs.

Monitoring the Early Antiproliferative Effect of the Analgesic-Antitumor Peptide, BmK AGAP on Breast Cancer Using Intravoxel Incoherent Motion With a Reduced Distribution of Four b-Values.

Background: The present study aimed to investigate the possibility of using intravoxel incoherent motion (IVIM) diffusion magnetic resonance imaging (MRI) to quantitatively assess the early therapeutic effect of the analgesic-antitumor peptide BmK AGAP on breast cancer and also evaluate the medical value of a reduced distribution of four b-values. Methods: IVIM diffusion MRI using 10 b-values and 4 b-values (0-1,000 s/mm2) was performed at five different time points on BALB/c mice bearing xenograft breast tumors treated with BmK AGAP. Variability in Dslow, Dfast, PF, and ADC derived from the set of 10 b-values and 4 b-values was assessed to evaluate the antitumor effect of BmK AGAP on breast tumor. Results: The data showed that PF values significantly decreased in rBmK AGAP-treated mice on day 12 (P = 0.044). PF displayed the greatest AUC but with a poor medical value (AUC = 0.65). The data showed no significant difference between IVIM measurements acquired from the two sets of b-values at different time points except in the PF on the day 3. The within-subject coefficients of variation were relatively higher in Dfast and PF. However, except for a case noticed on day 0 in PF measurements, the results indicated no statistically significant difference at various time points in the rBmK AGAP-treated or the untreated group (P < 0.05). Conclusion: IVIM showed poor medical value in the early evaluation of the antiproliferative effect of rBmK AGAP in breast cancer, suggesting sensitivity in PF. A reduced distribution of four b-values may provide remarkable measurements but with a potential loss of accuracy in the perfusion-related parameter PF.

Thermostable potassium channel-inhibiting neurotoxins in processed scorpion medicinal material revealed by proteomic analysis: Implications of its pharmaceutical basis in traditional Chinese medicine.

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".

Peptides with therapeutic potential in the venom of the scorpion Buthus martensii Karsch.

The scorpion Buthus martensii Karsch (BmK) has generated significant interest due to the presence of biologically active peptides in its venom. In the past decade, dozens of different peptides from BmK have been identified. Most of the peptides are neurotoxins and are responsible for the toxicity of BmK venom. Other peptides, including neurotoxins and non-disulfide-bridged peptides, show potential anticancer, antimicrobial, analgesic, and anti-epileptic therapeutic effects. These peptides are attractive candidates for drug development, and peptide derivatives have also been designed to enhance their therapeutic potential, such as ADWX-1 and Kn2-7. In this review, we provide an overview of the most promising peptides found in BmK venom and of modified peptide derivatives showing therapeutic potential.

Neo-5,22E-Cholestadienol Derivatives from Buthus martensii Karsch and Targeted Bactericidal Action Mechanisms.

The discovery and search for new antimicrobial molecules from insects and animals that live in polluted environments is a very important step in the scientific search for solutions to the current problem of antibiotic resistance. Previously, we have reported that the secondary metabolite with the antibacterial action discovered in scorpion. The current study further isolated three new compounds from Buthus martensii karsch, while compounds 1 and 2 possessed 5,22E-cholestadienol derivatives whose structure demonstrated broad spectrum bactericide activities. To explore the antibacterial properties of these new compounds, the result shows that compound 2 inhibited bacterial growth of both S. aureus and P. aeruginosa in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio ≤ 2). Similarly, with compound 1, a ratio of MBC/MIC ≤ 2 indicates bactericidal activity inhibited bacterial growth of P. aeruginosa. Remarkably, this suggests that two compounds can be classified as bactericidal agents against broad spectrum bactericide activities for 5,22E-cholestadienol derivatives from Buthus martensii karsch. The structures of compounds 1⁻3 were established by comprehensive spectra analysis including two-dimensional nuclear magnetic resonance (2D-NMR) and high-resolution electrospray ionization-mass spectrometry (HRESI-MS) spectra. The antibacterial mechanism is the specific binding (various of bonding forces between molecules) using compound 1 or 2 as a ligand based on the different receptor proteins'-2XRL or 1Q23-active sites from bacterial ribosome unit A, and thus prevent the synthesis of bacterial proteins. This unique mechanism avoids the cross-resistance issues of other antibacterial drugs.

Fluorescence Coupled with Macro and Microscopic Examinations of Morphological Phenotype Give Key Characteristics for Identification of Crude Drugs Derived from Scorpions.

Microscopic examination of crude drug components has been the traditional method to identify the origin of biological materials. For the identification of components in a given mixture via microscopy, standard reference photographs of fragments derived from different organs and tissues of individual species are required. In addition to these reference photographs, a highly observant eye is needed to compare the morphological characteristics observed under the microscope with those of the references and to then identify the origin of the materials. Therefore, if other indexes are available to be coupled with microscope examination, the accuracy of identification would be significantly improved. Here, we prepared standard reference photographs for microscopic examination to identify powdered and fragmented materials in the crude drug "Quanxie" derived from individual organs of dried scorpion (Buthus martensii KARSCH). Since a remarkable characteristic of scorpion bodies is that they fluoresce under UV light, two methods to identify "Quanxie" were established, including fluorescence fingerprint analysis and microscopic fluorescent luminance imaging analysis. In the former, at least 0.1 g of powered materials was used, which could be recovered after the measurement, and in the latter, only small amounts of powders were used for microscopic examinations. Both methods could distinguish powders of "Quanxie" from those of other micro-morphologically similar crude drugs, namely, "Chantui," "Sangpiaoxiao," and "Jiangcan." The combination of these methods should improve the swiftness and accuracy of "Quanxie" identification.

BmK CT enhances the sensitivity of temozolomide-induced apoptosis of malignant glioma U251 cells in vitro through blocking the AKT signaling pathway.

Temozolomide (TMZ) is a drug that has been demonstrated to improve the survival time of patients with glioblastoma multiforme (GBM) when administered with concomitant radiotherapy. However, chemoresistance is one of the major obstacles in the treatment of GBM. In the present study, an MTT assay and flow cytometry were used to demonstrate that chlorotoxin-like toxin in the venom of the scorpion Buthus martensii Kirsch (BmK CT) markedly inhibited cell proliferation and induced apoptosis in U251 cells when combined with TMZ. In combination with TMZ, BmK CT exhibited a significant and synergistic anti-tumor effect by inhibiting protein kinase B (AKT) independently and triggering the apoptosis signaling cascade in vitro. Furthermore, BmK CT increased the expression of phosphatase and tensin homolog at the transcriptional level, which is a key negative regulator of the AKT signaling pathway. The results of the present study demonstrated that BmK CT enhanced the sensitivity of TMZ-induced U251 cell apoptosis through the downregulation of phosphorylated AKT levels, suggesting that BmK CT and TMZ combination therapy may be a novel approach for glioma therapy.

Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels.

Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain α-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7±6.6nM for rNav1.4, 454.2±50.1nM for hNav1.5 and 30.9±10.3µM for hNav1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNav1.4 and hNav1.5 channels toward a more negative direction and present the typical features of a ß-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNav1.4 and hNav1.5 channels. The V1/2 values of the steady-state inactivation were altered to be more positive for rNav1.4 and more negative for hNav1.5. Moreover, the recovery of the hNav1.5 channel from inactivation was more significantly delayed than that of the rNav1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both α- and ß-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.

Research progress on pharmacological activity of peptides from Buthus martensii / 中草药

Buthus martensii is a precious Chinese materia medica, which has been extensively studied due to its various pharmacological effects such as anti-microbial activity, anti-tumor activity, analgesic activity, etc. This paper reviews domestic and foreign researches about the pharmacological activities of peptides from B. martensii, which will provide references for the utilization of these active medicinal peptides in B. martensii.

Investigation of Binding Modes and Functional Surface of Scorpion Toxins ANEP to Sodium Channels 1.7.

The depressant ß toxin anti-neuroexcitation peptide (ANEP) from the Chinese scorpion Buthus martensii Karsch has analgesic activity by interacting with receptor site 4 of the voltage-gated sodium channels (VGSCs). Here, with molecular dynamics simulations, we examined the binding modes between ANEP and the site 4 of mice sodium channel 1.7 (mNav1.7), a subtype of VGSCs related to peripheral pain. Homology modeling, molecular mechanics, and molecular dynamics in the biomembrane environment were adopted. The results suggested that ANEP bound to the resting site 4 mainly by amino acid residues in the ß2-ß3 loop and the 'NC' domains, and the activate site 4 mainly by amino acid residues in the hydrophobic domain of N-groove and residues in the 'pharmacophore'. Effects analysis of 14 mutants in the predicted functional domains of ANEP on mouse twisting models showed that the analgesic activity of mutants L15 and E24 of the 'pharmacophore', W36, T37, W38, and T39 forming the loop between the ß2- and ß3-strands and N8, V12, C60, and K64 in the NC domain increased distinctly after these residues were substituted for Ala, respectively. The binding modes and the active sites predicted were consistent with available mutagenesis data, and which is meaningful to understand the related mechanisms of ANEP for Nav1.7.

Transcriptome analysis of Spodoptera frugiperda 9 (Sf9) cells infected with baculovirus, AcMNPV or AcMNPV-BmK IT.

OBJECTIVES: To analyze the transcriptome of Spodoptera frugiperda 9 (Sf9) cells infected with AcMNPV or AcMNPV-BmK IT. RESULTS: A comprehensive transcriptome profile for Sf9 cells infected with AcMNPV or AcMNPV-BmK IT is shown. 43127572, 46529744 and 47235310 RNA-Seq profiles permitted the quantification of expression levels for control (C), AcMNPV-BmK IT treatment (ABT) and AcMNPV treatment (AT) groups. There were 997 up-regulated or down-regulated candidate genes with significant different expression level in these treatment groups. CONCLUSION: These results provide a broad spectrum of candidate genes that are critically involved in the molecular regulation mechanism of Sf9 cells infected with AcMNPV-BmK IT.

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli.

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purified to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.

Chemical constituents from Buthus martensii / 中成药

AIM To study the chemical constituents from Buthus martensii Karsch.METHODS The dichloromethane and 70％ methanol fractions of B.martensii extract were isolated and purified by silica,ODS,Sephadex LH-20 and mid-pressure preparative column,then the structures of obtained compounds were identified by physiochemical properties and spectral data.RESULTS Nine compounds were isolated and identified as cholest4-en-3-one (1),cholesterol (2),uracil (3),1-stearyl-2-lyso-sn-glycero-3-phosphatidylcholine (4),glycerol (5),oleic acid (6),proline (7),alanine (8),leucine (9).CONCLUSION Compounds 1,4,5 are isolated from B.martensii for the first time.