Protein-Based Fingerprint Analysis for the Identification of Ranae Oviductus Using RP-HPLC.

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.

[Application of rapid PCR to authenticate Ranae Oviductus].

Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.

[Identification of Ranae Oviductus original animal based on COI sequences].

Ranae Oviductus has a high economic and social value, but its adulterants are more numerous, which causes a great confusion to the market. Using DNA bar code technology based on COI sequence for PCR amplification and sequencing of the identified Rana dybowskii, R. chensinensis, R. huanrensis and R. amurensiss, the COI gene database of four species of Rana was established, and comparing the measured sequence with the sequence of GenBank, four kinds of Rana were identified. The MEGA (molecular evolutionary genetics analysis) 7 .0 software was used to calculate the genetic distance of K2P and construct the NJ (neighbor-joining) system cluster tree. The sequence of the four species of Rana measured were clustered into one group with the sequence of the four kinds of Rana downloaded from GenBank, but separated from the two outer groups downloaded from GenBank. The COI gene of the R. dybowskii was likely to have regional differences, however this technique failed to distinguish male and female Rana. The results showed that DNA bar code technology could accurately identify the base of original animal of R. oviductus. It indicates that DNA bar code COI provides a new method for the identification of R. oviductus.

Textual Research on Ranae Oviductus / 中国实验方剂学杂志

Ranae Oviductus has a good tonic effect and is commonly used for both medicine and food. The use of Ranae Oviductus was confused because the origin of Ranae Oviductus was roughly recorded in ancient herbal literatures. In order to clarify the confusing literatures and trace the origin of Ranae Oviductus，this paper conducted a textual research on the name，origin，distribution，harvesting and processing，efficacy of the Chinese medicine by consulting ancient herbal books，modern literatures and monographs of traditional Chinese medicine. The results of the textual research showed that Ranae Oviductus belongs to Manchu medicine，which was first applied by the Manchu people because of its tonic effect. The original animal of Ranae Oviductus has many names，which are all translated from Manchu language. By analyzing the descriptions of Ranidae in various herbal books，it is concluded that the earliest description of the original animals of Ranae Oviductus appeared in the Shengjing Tongzhi compiled by Agui in the Qing dynasty. After summarization of the taxonomic changes of some species of Rana，the original animals of Ranae Oviductus were preliminarily determined as Rana dybowskii，R. amurensis and R. huanrenensis. We excluded R. huanrenensis by its size and R. amurensis by its poor quality. Therefore，the original animal of Ranae Oviductus is R. dybowskii，the main production area is northeast China and the best capture time is in October. Ranae Oviductus is often eaten after being stewed. The study can provide the effective basis for the identification of the original animal of Ranae Oviductus，the distribution of production area and the utilization of resources.

Effect of Ranae Oviductus Protein Hydrolysate on Ethanol-induced L-02 Cell Injury / 中国实验方剂学杂志

Objective:To study the protective effect and mechanism of Ranae Oviductus protein hydrolysate （ROPH） on the expression of pathway-related proteins in ethanol-induced L-02 cell injury. Method:The ROPH was prepared by compound enzymatic hydrolysis. L-02 cell injury model was induced with 400 mmol·L^-1ethanol. Cell viability was detected by cell counting kit-8 （CCK-8） assay. Cell cycle and apoptosis were examined by flow cytometry. JC-1/Hochest staining was employed for qualitative investigation. The expression of related proteins in apoptosis， mitogen-activated protein kinase （MAPK） signaling pathway， and pyroptosis in L-02 cells was detected by Western blot. Result:The results of the CCK-8 assay showed that 400 mmol·L^-1ethanol could induce L-02 cell injury within 12 hours. Compared with the blank group， the model group showed decreased viability of L-02 cells （<italic>P</italic><0.01）， elevated percentage of the cell cycle in the G₀/G₁ phase （<italic>P</italic><0.01）， increased total cell apoptosis rate （<italic>P</italic><0.01）， reduced mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated expression of apoptosis-related proteins ［B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， Cytochrome C （Cyt C）， and cysteine-dependent aspartate specific protease-3 （Caspase-3）］ （<italic>P</italic><0.05， <italic>P</italic><0.01） and MAPK signaling pathway-related proteins ［C-Jun amino-terminal kinase （JNK） and p38 MAPK］ （<italic>P</italic><0.05， <italic>P</italic><0.01）， and potentiated expression of pyrolysis-related proteins Caspase-1 and interleukin-1<italic>β </italic>（IL-1<italic>β</italic>） （<italic>P</italic><0.05）. Compared with the model group， the ROPH treatment group exhibited improved cell cycle arrest （<italic>P</italic><0.05， <italic>P</italic><0.01）， diminished total cell apoptosis rate （<italic>P</italic><0.01）， elevated mitochondrial membrane potential in a dose-dependent manner， down-regulated expression of Bax， Cyt C， and Caspase-3 proteins （<italic>P</italic><0.05， <italic>P</italic><0.01）， up-regulated expression of Bcl-2 protein （<italic>P</italic><0.05， <italic>P</italic><0.01）， and a downward trend in expression of proteins related to MAPK signaling pathway and pyrolysis （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:ROPH could inhibit oxidative stress-triggered liver injury in ethanol-induced cells by improving mitochondrial membrane potential， reducing the expression of proteins in the mitochondria-mediated apoptosis pathway， and inhibiting the expression of proteins related to the MAPK signaling pathway and pyrolysis pathway to reduce the mitochondrial dysfunction and inflammatory response in ethanol-induced L-02 liver cells and inhibit oxidative stress， thereby exerting a therapeutic role in alcoholic liver injury.

Effect of Ranae Oviductus on Follicular Development Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of Ranae Oviductus (RO) on ovarian follicular development， phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway， and pregnancy function in rats, and the estrogen-like mechanism of OR. Method:Seventy female Wistar rats were randomly divided into a normal group， a progynova+ luteohormone group (1 mg·kg^-1+40 mg·kg^-1), a clomiphene group (10 mg·kg^-1), and high-dose(400 mg·kg^-1) and low-dose(200 mg·kg^-1) RO groups. Rats were administered correspondingly by gavage for eight weeks. After seven weeks of intragastric administration, the estrus cycle of all rats was measured. After eight weeks of intragastric administration， four rats from each group were selected to give birth. For other rats, blood was collected on the day of estrus， and the serum levels of estradiol (E₂)，progesterone (P)， testosterone (T)， follicle-stimulating hormone (FSH)， and luteotropic hormone (LH) were detected. Uterus and ovaries were extracted and weighed to calculate organ index. One ovary was made into pathological sections, and the follicles at different developmental stages and corpus luteum were counted. Real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）and Western blot were performed on the other ovary to detect mRNA and protein changes in the PI3K/Akt signaling pathway. Forty female Kunming mice were randomly divided into a normal group and RO groups (400 mg·kg^-1) with 14 days， 28 days， and 56 days of intervention. Mice in the RO groups were raised with male mice in cages after intragastric administration of OR for 14， 28， and 56 days， respectively. After 18 days, the number of intrauterine fetuses on both sides and the number of stunted fetuses were counted. Result:After eight weeks of intragastric administration of OR， the rats showed decreased uterine index (<italic>P</italic><0.05)， declining serum LH (<italic>P</italic><0.05)， reduced luteum (<italic>P</italic><0.01)， dwindled primary follicles (<italic>P</italic><0.05)， and increased rate of follicle atresia (<italic>P</italic><0.01). Additionally， more luteal or interstitial glands degenerated into interstitial structures in the ovarian cortex in a short time. The mRNA expression levels of PI3K and Akt in the ovary were elevated (<italic>P</italic><0.01)， while the mRNA expression levels of mTOR and PTEN were reduced (<italic>P</italic><0.01). The phosphorylation level of Akt protein showed a downward trend without significant difference. For the rats， the number of fetuses was decreased (<italic>P</italic><0.05). The pregnancy rate of mice was decreased to varying degrees after administration of RO for different durations， with the lowest in the 14 day RO group， as low as 30%. After 28 days of intragastric administration of RO， the difference in left and right uterine pregnancy increased (<italic>P</italic><0.05). Conclusion:Long-term administration of RO can lead to premature ovarian failure by over-stimulating the ovary， which is similar to clomiphene. Short-term administration can result in decreased pregnancy rate， excessive ovulation on one side， and inhibition of ovulation on the other side. The influence on follicles needs further exploration.

Application of rapid PCR to authenticate Ranae Oviductus / 中国中药杂志

Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.

Identification of Ranae Oviductus original animal based on COI sequences / 中国中药杂志

Ranae Oviductus has a high economic and social value, but its adulterants are more numerous, which causes a great confusion to the market. Using DNA bar code technology based on COI sequence for PCR amplification and sequencing of the identified Rana dybowskii, R. chensinensis, R. huanrensis and R. amurensiss, the COI gene database of four species of Rana was established, and comparing the measured sequence with the sequence of GenBank, four kinds of Rana were identified. The MEGA (molecular evolutionary genetics analysis) 7 .0 software was used to calculate the genetic distance of K2P and construct the NJ (neighbor-joining) system cluster tree. The sequence of the four species of Rana measured were clustered into one group with the sequence of the four kinds of Rana downloaded from GenBank, but separated from the two outer groups downloaded from GenBank. The COI gene of the R. dybowskii was likely to have regional differences, however this technique failed to distinguish male and female Rana. The results showed that DNA bar code technology could accurately identify the base of original animal of R. oviductus. It indicates that DNA bar code COI provides a new method for the identification of R. oviductus.

Antidepressive effects of petroleum ether extract from Ranae Oviductu and its possible mechanism / 中草药

Objective: To study the antidepressive effect of the petroleum ether extract from Ranae Oviductus (PERO) on chronic mild stress depressive rat model and its mechanism. Methods: The rats were divided into control, model, Fluoxetine (10 mg/kg, positive control), and different doses (30, 100, and 300 mg/kg) of PERO groups. The rats in PERO groups were administered once daily for 21 d, while the rats in the control and model groups were administered with equivalent normal saline. After 1 h for the rats in PERO and model groups and 0.5 h for the rats in Fluoxetine group of the last administration, the chronic unpredictable mild stress for 21 d was used to induce the depression in rats. The body weight was measured and the depressive-like behaviors were evaluated by the open-field test and sucrose preference test. Then the corticosterone level in the rat plasma was detected by enzyme-linked immunosorbent assay (ELISA) and the brain-derived neurotrophic factor (BDNF) protein levels in the hippocampus were measured by Western blotting analysis. Results: Compared with the control group, the body weight, sucrose preference, and motion distance in open-field test of rats were decreased, the corticosterone level in plasma was increased, and the BDNF level in hippocampus was decreased. Compared with the model group, PERO treatment alleviated the body weight decreasing, increased the sucrose preference and motion distance, reduced the corticosterone level in the plasma of rats, and increased the BDNF level in hippocampus of rats. Conclusion: The antidepressive effect of PERO is likely mediated by modulating the function of hypothalamic-pituitary-adrenal axis and increasing the expression of BDNF in brain tissues.