Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium.

ETHNOPHARMACOLOGICAL RELEVANCE: Di-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. AIM OF THE STUDY: Considering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. MATERIALS AND METHODS: Transcriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. RESULTS: We confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. CONCLUSIONS: Our work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

[Enhancing effects of chemical permeation enhancer propylene glycol and ultrasonic technology on transdermal permeation of Baimai Ointment].

This study aimed to improve the transdermal permeation quantity of Baimai Ointment by investigating the enhancing effects of physical and chemical permeation promoting methods on transdermal permeation of Baimai Ointment. The improved Franz diffusion cell method was used for in vitro transdermal experiment. The abdominal skin of mice was used, and the skin was treated with 3% propylene glycol in the chemical enhancement group. Ultrasonic technology was introduced in the physical enhancement group. The conditions of ultrasonic technology were optimized by single factor trial. Taking Q_(EF) and ER as the indexes of penetration promotion performance, the enhancing effects of the two methods were compared. The results showed that the promotion performance of 3% propylene glycol for ammonium glycyrrhizinate, nardosinone and curcumin of the chemical enhancement group were 1.74, 1.60, and 3.73 times higher than those of the blank group, respectively. The overall permeation efficiency of the Baimai Ointment was significantly improved. The comprehensive promoting effect on each component was curcumin>ammonium glycyrrhizinate>nardosinone. In the physical enhancement group, the penetration promoting effect of ultrasonic power 1.0 W was better than that of 2.0 W and 0.5 W, ultrasonic time 5 min was better than 3 min and 8 min, and the ultrasonic frequency 1 MHz was better than 3 MHz. Therefore, the optimal ultrasonic condition was 1.0 W-5 min-1 MHz. Under this condition, in terms of the transdermal permeation for ammonium glycyrrhizinate, the Q_(EF) and ER of the ultrasonic technology were better than those of 3% propylene glycol. In terms of the transdermal permeation for nardosinone and curcumin, the QEF and ER of 3% propylene glycol were better than those of the ultrasonic technology. Therefore, 3% propylene glycol combined with ultrasonic technology can be used to promote permeation of Baimai Ointment that contains both water-soluble and fat-soluble components in the clinical application. This study provides a theoretical basis for the clinical application of Baimai Ointment and other transdermal preparations.

[Analysis on quality value transfer of substance benchmarks of Taohong Siwu Decoction].

By preparing 15 batches of substance benchmarks of Taohong Siwu Decoction, the methodology of the characteristic spectrums of substance benchmarks was established. The paste-forming rate range, the contents and the transfer rate range of the index components, hydroxy safflower yellow A, ferulic acid and paeoniflorin, the characteristic peaks and the similarity range of the characteristic spectrums of Taohong Siwu Decoction were determined to define key quality attributes of substance benchmarks of Taohong Siwu Decoction.In the 15 batches of substance benchmarks of Taohong Siwu Decoction, the similarity of characteristic spectrums was higher than 0.9. Furthermore, based on summarization of the characteristic peak information, there were 13 characteristic peaks in the whole decoction. Baishao had three characteristic peaks, Honghua had seven characteristic peaks, and Chuanxiong and Danggui had three characteristic peaks. The paste-forming rate of the 15 batches of substance benchmarks was controlled at 33.11%-40.62%. The content of hydroxy safflower yellow A was 0.129%-0.203%, with the average transfer rate of 16.596%±0.669%.The content of ferulic acid was 0.043%-0.055%, with the average transfer rate of 20.489%±1.772%.The content of paeoniflorin was 0.676%-0.943%, with the average transfer rate of 29.112%±3.273%.The quality value transfer of substance benchmarks of classical prescription Taohong Siwu Decoction was analyzed by the combination of characteristic spectrums, paste-forming rate and the content of index components. The established substance benchmark quality evaluation method was stable and feasible, and could provide a basis for quality control and subsequent development of relevant preparations of Taohong Siwu Decoction.

[Characteristic spectrum and decoction process of classical prescription Qingwei San].

In this experiment, by determination of the HPLC characteristic spectrum of the classical prescription Qingwei San decoction, the contents of isoferulic acid, palmatine and paeonol in Qingwei San decoction and the extraction rate were investigated. The factors such as the crushing degree of decoction pieces, the amount of decocting water, the decocting time, the filter material and the decocting container involved in Qingwei San decoction process were examined to make a detailed comparison of Qingwei San's decoction processes during the development.HPLC characteristic spectrum method of Qingwei San was established, and then the decoction process parameters of Qingwei San were optimized, with the similarity of characteristic spectrum, the concentration of the index components and the extraction rate as indexes. The decoction process of Qingwei San was determined as follows: Qingwei San decoction pieces were weighed according to the prescription amount and pulverized into the most coarse powder; the powder was put in a ceramic pot, added with 225 mL water, heated to boiling, cooked for 50 minutes with gentle heat(100 W), and filtered with a layer of 300 mesh nylon cloth.The similarity of Qingwei San's characteristics pectrum of different decoction methods was all above 0.9, and the concentration of isoferulic acid, palmatine and paeonol in Qingwei San under determined decoction process was 40.74, 26.73, 65.73 µg·mL~(-1), respectively, with an extraction rate of 33.80%.The characteristic spectrum determined in this experiment can better express the information and index components of Qingwei San, and if combined with the extraction rate information, it can provide the general information, index component content and extraction information. The decoction process after detailed investigation can better reflect the quality of Qingwei San decoction, with easier control and operation. It can provide a basis for the subsequent research and development of Qingwei San decoction standard, and can also provide experimental basis and reference for the decoction process research of other classical prescriptions.