Rana chensinensis ovum oil (RCOO) is an emerging source of unsaturated fatty acids (UFAs), but it is lacking in green and efficient extraction methods. In this work, using the response surface strategy, we developed a green and efficient CO₂ supercritical fluid extraction (CO₂-SFE) technology for RCOO. The response surface methodology (RSM), based on the Box–Behnken Design (BBD), was used to investigate the influence of four independent factors (pressure, flow, temperature, and time) on the yield of RCOO in the CO₂-SFE process, and UPLC-ESI-Q-TOP-MS and HPLC were used to identify and analyze the principal UFA components of RCOO. According to the BBD response surface model, the optimal CO₂-SFE condition of RCOO was pressure 29 MPa, flow 82 L/h, temperature 50 °C, and time 132 min, and the corresponding predicted optimal yield was 13.61%. The actual optimal yield obtained from the model verification was 13.29 ± 0.37%, and the average error with the predicted value was 0.38 ± 0.27%. The six principal UFAs identified in RCOO included eicosapentaenoic acid (EPA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA), and oleic acid (OA), which were important biologically active ingredients in RCOO. Pearson correlation analysis showed that the yield of these UFAs was closely related to the yield of RCOO (the correlation coefficients were greater than 0.9). Therefore, under optimal conditions, the yield of RCOO and principal UFAs always reached the optimal value at the same time. Based on the above results, this work realized the optimization of CO₂-SFE green extraction process and the confirmation of principal bioactive ingredients of the extract, which laid a foundation for the green production of RCOO.

This work demonstrated a method combining reversed-phase high-performance liquid chromatography (RP-HPLC) with chemometrics analysis to identify the authenticity of Ranae Oviductus. The fingerprint chromatograms of the Ranae Oviductus protein were established through an Agilent Zorbax 300SB-C8 column and diode array detection at 215 nm, using 0.085% TFA (v/v) in acetonitrile (A) and 0.1% TFA in ultrapure water (B) as mobile phase. The similarity was in the range of 0.779–0.980. The fingerprint chromatogram of Ranae Oviductus showed a significant difference with counterfeit products. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) successfully identified Ranae Oviductus from the samples. These results indicated that the method established in this work was reliable.

Natural products play an important role in drug discovery. This work employed a natural product 1-methylhydantoin as the lead compound to develop novel dual-active drugs. 1-Methylhydantoin was isolated from Oviductus Ranae, which is a traditional Chinese medicine that has been used for tussive and inflammation treatment for a long time. An in silico study screened the more active 1-methylhydantoin derivatives. Antitussive assessment indicated that the newly synthesized agent had similar bioactivity with the natural product. An anti-inflammatory model used xylene induced ear edema model. At the same dosage (100 mg/Kg), the newly prepared agent had an inhibition rate 53.18% which was much higher than that of the lead compound (22.69%). The results might be ascribed to the cyclooxygenases-1 (COX-1) and cyclooxygenases-2 (COX-2) selectivity, and the fitness of the compound, and the binding pocket. The anti-particulate matter (PM 2.5) acute pneumonia was evaluated through an in vivo model constructed by nasal instillation with PM 2.5 suspension. The results of the above models suggested that this novel agent had remarkable antitussive, anti-inflammatory, and anti-PM 2.5 acute pneumonia activities.

Oviductus Ranae is a nutritional product for both medicine and food. Its quality is uneven due to multiple factors. An efficient method was established to evaluate the quality of Oviductus Ranae using fingerprint techniques and chemometric methods based on the analysis of polyunsaturated fatty acids (PUFAs) in petroleum ether extract by high performance liquid chromatography (HPLC). During this process, 27 batches of Oviductus Ranae were analyzed by HPLC and two types of chromatographic fingerprints were established. The fingerprint that was obtained by matching six known peaks was used for the quantification of six PUFAs. Another fingerprint was obtained by matching sixteen peaks with a peak area ratio greater than 0.5% and it was used to classify the different qualities of Oviductus Ranae by further combining three different chemometric models. The 27 batches of Oviductus Ranae were divided into four categories, which was consistent with the analysis results of six PUFAs contents. The two high-quality samples with significantly higher contents were classified into one category, and samples with medium contents were divided into two categories, including eight and thirteen samples, respectively. The four inferior samples with lower contents were classified into one category. The results indicated that the newly developed method has potential application prospects for the quality evaluation of Oviductus Ranae.

In this study, 1-methylhydantoin cinnamic imides were synthesized from 1-methylhydantoin and trans-cinnamic acid, and their anti-inflammatory activity was investigated. The anti-inflammatory activity in vitro was evaluated by measuring the contents of NO, TNF-α and IL-1β in the supernatant of RAW264.7 cells stimulated by LPS. The cytotoxicity of 1-methylhydantoin cinnamoyl imides on RAW264.7 cells was detected using the CCK-8 method. The results showed that compounds 2 and 4 can significantly inhibit the release of NO and reduce the secretion of TNF-α and IL-1β. Compound 3 inhibited the production of TNF-α. The inhibition rate of COX was evaluated in vitro. The in vivo anti-inflammatory activities of the five compounds were evaluated by establishing an animal model of xylene ear swelling. The results showed that 1-methylhydantoin cinnamic imides could alleviate xylene-induced ear edema in mice in a dose-dependent manner. Among them, the effect of compound 5 was the most significant. Under the action of high dosage, its ear swelling inhibition rate was as high as 52.08%.