[Therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea in rats and its metabolomic analysis].

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics. Forty adult female rats were randomly divi-ded into a normal group, a model control group, ibuprofen(0.12 g·kg~(-1)) group, and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg~(-1)) groups, with eight rats in each group. The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin. Drugs were administered by gavage from the 4th day of modeling for 7 d. After the last administration, pitocin was injected intraperitoneally, and the writhing latency and writhing times within 30 min were recorded. The uterine and ovarian coefficients were determined. Estradiol(E_2), progesterone(Prog), oxytocin(OT), cyclooxyge-nase 2(COX-2), prostaglandin E_2(PGE_2), prostaglandin F_(2α)(PGF_(2α)), and Ca~(2+) levels in uterine tissues were measured by ELISA and biochemical kits. Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining. The protein expression of oxytocin receptor(OTR), prostaglandin E_2 receptor 3(EP3), and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry. The mRNA expression of OTR, PGE_2 receptors 1-4(EP1, EP2, EP3, and EP4), and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR. Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways. The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01), significantly reduce the uterine and ovarian coefficients(P<0.01), and improve their histomorphology. After treatment with Leonuri Herba, PGE_2 content was significantly increased(P<0.05), COX-2, PGF_(2α) and Ca~(2+) content, and PGF_(2α)/PGE_2 was significantly decreased(P<0.05 or P<0.01), and OT content was decreased, while E_2 and Prog content tended to further increase in uterine tissues of PD rats. Correspondingly, OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues. The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01), and the mRNA expression of EP1, EP3, and OTR showed a decreasing trend. The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment. The results indicate that Leonuri Herba is effective in the prevention and treatment of PD, and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.

The mechanism of Leonuri Herba in improving polycystic ovary syndrome was analyzed based on network pharmacology and molecular docking.

Background: Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder affecting women. Chinese herbs have been considered as an alternative treatment for PCOS, and Yi-mu-cao (Leonuri Herba) is one of the most commonly used herbs to treat PCOS, which can relieve symptoms of PCOS patients. But the mechanism of its treatment remains unclear. Method: The main active ingredients and potential targets of Leonuri Herba were obtained by TCMSP and Swiss Target Forecast, and the related targets of PCOS were obtained by searching DrugBank, GeneCard and DisGeNet databases. The Protein-Protein Interaction (PPI) network was constructed using STRING database. GO and KEGG were used to detect the enrichment pathways of key targets. Cytoscape software was used to construct the component-target-pathway network, analyze the PPI network core, and verify the reliability of target binding by molecular docking technology. Result: 8 components and 116 targets of Leonuri Herba on PCOS were screened. Common targets mainly involve the Lipid and atherosclerosis, Endocrine resistance, AGE-RAGE signaling in diabetic complications and other signaling pathways. It is suggested that it can form multi-target and multi-pathway regulatory network through quercetin, kaempferol and other active substances to regulate endocrine disorders and reduce inflammatory response, so as to systematically improve PCOS. Molecular docking experiments showed that the active constituents of Leonurus had good binding activity with potential targets of PCOS. Conclusion: In summary, this study elucidates the potential effect of Leonuri Herba on PCOS, which is helpful to provide reference for clinical practice. This is also conducive to the secondary development of motherwort and its monomer components, and precision medicine for PCOS.

Therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea in rats and its metabolomic analysis / 中国中药杂志

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics. Forty adult female rats were randomly divi-ded into a normal group, a model control group, ibuprofen(0.12 g·kg~(-1)) group, and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg~(-1)) groups, with eight rats in each group. The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin. Drugs were administered by gavage from the 4th day of modeling for 7 d. After the last administration, pitocin was injected intraperitoneally, and the writhing latency and writhing times within 30 min were recorded. The uterine and ovarian coefficients were determined. Estradiol(E_2), progesterone(Prog), oxytocin(OT), cyclooxyge-nase 2(COX-2), prostaglandin E_2(PGE_2), prostaglandin F_(2α)(PGF_(2α)), and Ca~(2+) levels in uterine tissues were measured by ELISA and biochemical kits. Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining. The protein expression of oxytocin receptor(OTR), prostaglandin E_2 receptor 3(EP3), and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry. The mRNA expression of OTR, PGE_2 receptors 1-4(EP1, EP2, EP3, and EP4), and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR. Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways. The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01), significantly reduce the uterine and ovarian coefficients(P<0.01), and improve their histomorphology. After treatment with Leonuri Herba, PGE_2 content was significantly increased(P<0.05), COX-2, PGF_(2α) and Ca~(2+) content, and PGF_(2α)/PGE_2 was significantly decreased(P<0.05 or P<0.01), and OT content was decreased, while E_2 and Prog content tended to further increase in uterine tissues of PD rats. Correspondingly, OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues. The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01), and the mRNA expression of EP1, EP3, and OTR showed a decreasing trend. The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment. The results indicate that Leonuri Herba is effective in the prevention and treatment of PD, and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.

Protection against acute cerebral ischemia/reperfusion injury by Leonuri Herba Total Alkali via modulation of BDNF-TrKB-PI3K/Akt signaling pathway in rats.

OBJECTIVE: To observe the brain protective effect of Leonuri Herba Total Alkali (LHA) on cerebral ischemia reperfusion injury in rats, so as to provide basis for clinical research. METHODS: Adult male SD rats were randomly assigned into sham group, middle cerebral artery occlusion/reperfusion (MCAO/R) group, and LHA + MCAO/R group (25 mg/kg, 50 mg/kg, and 100 mg/kg). Fourteen days before MCAO/R surgery, the rats in treatment groups were orally administered with LHA in ultrapure water once daily for 14 days, while rats in the sham and MCAO groups were given the same amount of saline in advance. After 1 h of administration on the 14th day, MCAO surgery was subjected. The neurological deficits, brain infarct volume, histopathology, immunofluorescence, inflammation indicators and the gene/protein expressions of BDNF-TrKB-PI3K/Akt signaling pathway in the rat brain tissue were evaluated 24 h after the MCAO/R-injury. RESULTS: It was found that rats in LHA pre-administration group showed significantly reduced neurological deficit scores, infarction volume, the serum levels of NSE and S100ß. Meanwhile, the content of Evans Blue (EB) in brain tissue from LHA group was decreased, as well as the levels of inflammatory cytokines and their gene levels. Moreover, LHA pre-administration inhibited the expression of CD44, GFAP, FOXO1 and promoted the expression of BDNF and NeuN. In addition, LHA pre-administration could up-regulate the protein expression of TrkB, p-PI3K, p-Akt, Bcl-2, and down-regulate the protein expression of Bax, and increase the level of Bcl-2/Bax. CONCLUSIONS: The study demonstrated that LHA pre-administration could regulate the PI3K/Akt pathway by increasing BDNF levels, and play a neuroprotective role in cerebral ischemia-reperfusion injury.

Comparison Between Traditional and Integrated Processing Technology of Leonuri Herba Based on Chemical Composition and Pharmacological Effect / 中国实验方剂学杂志

Objective: To elucidate the rationality of integrated processing technology of Leonuri Herba based on comparison of chemical constituents and pharmacological effect of Leonuri Herba between traditional and integrated processing technology. Method:The contents of stachydrine hydrochloride,leonurine hydrochloride,rutin,hyperoside and isoquercetin were used as indexes to compare the differences in the contents of chemical constituents between traditional and integration processing technology of Leonuri Herba.Effect of Leonuri Herba with different processing technology on auricular swelling induced by dimethylbenzene in mice were observed to compared the differences of their anti-inflammatory effect.And rat acute blood stasis model was used to compare the differences of Leonuri Herba with different processing technology on hemorheology and blood coagulation indexes. Result:Contents of stachydrine hydrochloride,leonurine hydrochloride,rutin,hyperoside and isoquercetin in products of integrated processing were 1.558%,0.168%,0.137%,0.113% and 0.078%,they were 1.482%,0.134%,0.125%,0.082% and 0.071% in products of traditional processing,respectively.Both of the processing methods could reduce the degree of swelling and the contents of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6 in mouse serum.And both of the processing methods could reduce the whole blood viscosity,prolong the thrombin time(TT),prothrombin time(PT),activated partial thromboplastin time(APTT) and reduce the concentration of plasma fibrinogen(FIB) in acute blood stasis model rats. Conclusion:Compared with the traditional processing technology,the integrated processing technology is better in guaranteeing the quality of Leonuri Herba decoction pieces and reducing the production cost,which indicates that the integrated processing of Leonuri Herba is reasonable.

[Standard decoction of Leonuri Herba].

To build an evaluation standard for quality of Leonuri Herba standard decoction. 13 batches of Leonuri Herba standard decoction with different quality were prepared. The contents of leonurine hydrochloride and stachydrine hydrochloride were determined; then the transfer rate and the extract rate were calculated and pH value was measured; and HPLC fingerprint method was established for analysis. The results of 13 batches of samples revealed that the transfer rate of leonurine hydrochloride and stachydrine hydrochloride was 30.0%-53.4% and 67.0%-82.6%, respectively; the extract rate was 12.1%-18.3% and the pH value was 5.87-6.22. Moreover, 12 common chromatographic peaks were determined based on fingerprint by using Similarity Evaluation System for Chromatographic fingerprint of Traditional Chinese Medicine (2012A). The similarity of 13 batches of samples was analyzed and compared, and the results showed that the similarity was higher than 0.9. In this study, the preparation method for Leonuri Herba decoction was standard, with high similarity in fingerprint, showing high precision, stability and repeatability in fingerprint analysis. Thus, this study can provide a reference for the quality control of Leonuri Herba dispensing granules.

The Effect of Leonuri Herba alkaloids on Senile BPH (male and female hormone induced) model rats.

Objective: To investigate the pharmacological effects of Leonuri Herba alkaloids (LHA) on prostate hyperplasia in older rats and the effect mechanism. Methods: Remove bilateral testes from BPH model rats, and conduct subcutaneous injection of testosterone and estradiol. At the same time, feed corresponding drugs to the rats by gastric perfusion for 30d. In the first 27d, conduct bladder fistula surgery. Three days after feeding, carry out the detection of the urine flow dynamics. Eyeball blood taking, determination of serum E2 levels, and quickly remove the prostate, thymus gland, spleen, kidney, lung, and bladder. 1/3 prostate homogenate, determine the level of PACP, T, DHT. 1/3 prostate was determined by mRNA expression in bFGF. The remaining 1/3 prostate was observed by light microscopy. Results: LHA could significantly decrease the animal prostate index, level of DHT, T, PACP, and elevate levels of E2 in the serum. It could also significantly reduce the maximum voiding pressure, intercontraction interval, and bladder resting pressure. Conclusion: LHA has good therapeutic effect on prostatic hyperplasia model rats induced by male and female hormone.

Effect of Total Alkali in Leonuri Herba on rat ear acne model of serum IL-6 level, Thymus and Spleen Tissue Morphology.

To investigate the effect of Total Alkali in Leonuri Herba (TALH) on rat ear acne model of serum IL-6 level, Thymus and Spleen Tissue Morphology. The rats were divided into the TALH group at high, medium and low doses (200 mg kg-1, 100 mg·kg-1, 50 mg·kg-1), the tanshinone group (360 mg·kg-1), model group and the blank group. All the rats were administrated once a day for 14 days. At the same time on the first day, the rats in the blank group were injected normal saline in the stomach and also had intradermal injection of sterile saline in the auricle. The rats in other groups (model group were lavaged with physiological saline) were injected intradermally auricle spare Staphylococcus epidermidis bacteria liquid. After the injection, the rat ear swelling rate was calculated for 5 consecutive days in terms of rat auricle thickness measurement. 1 h after the last administration, the eyeball blood was removed, centrifuged to separate serum, and measured to be at serum IL-6 level; The tissue sections of thymus and spleen were observed according to morphology criteria. Compared with the blank group, the level of IL-6 in serum of the rats in the model group was remarkably higher, and those in the thymus and spleen groups were remarkably lower, indicating the rat ear acne model is successful. Compared with the model group, the rats in the TALH group can remarkably make serum IL-6 level decrease, causing significant thymus cortical thickening and increased spleen sections, and remarkably increasing the lymphocytes of thymus and spleen. The effect of large dose in the TALH group is the best and increases with the increase of the dose and curative effect. The TALH treatment of acne may be related to the reduction of serum IL-6 levels and the resistance of the atrophy of the thymus and spleen on the rat ear acne model.

Standard decoction of Leonuri Herba / 中国中药杂志

To build an evaluation standard for quality of Leonuri Herba standard decoction. 13 batches of Leonuri Herba standard decoction with different quality were prepared. The contents of leonurine hydrochloride and stachydrine hydrochloride were determined; then the transfer rate and the extract rate were calculated and pH value was measured; and HPLC fingerprint method was established for analysis. The results of 13 batches of samples revealed that the transfer rate of leonurine hydrochloride and stachydrine hydrochloride was 30.0%-53.4% and 67.0%-82.6%, respectively; the extract rate was 12.1%-18.3% and the pH value was 5.87-6.22. Moreover, 12 common chromatographic peaks were determined based on fingerprint by using Similarity Evaluation System for Chromatographic fingerprint of Traditional Chinese Medicine (2012A). The similarity of 13 batches of samples was analyzed and compared, and the results showed that the similarity was higher than 0.9. In this study, the preparation method for Leonuri Herba decoction was standard, with high similarity in fingerprint, showing high precision, stability and repeatability in fingerprint analysis. Thus, this study can provide a reference for the quality control of Leonuri Herba dispensing granules.

Comparative analysis of the main bioactive components of Xin-Sheng-Hua granule and its single herbs by ultrahigh performance liquid chromatography with tandem mass spectrometry.

Xin-Sheng-Hua granule, a representative formula for postpartum hemorrhage, has been used clinically to treat postpartum diseases. Its main bioactive components comprise aromatic acids, phthalides, alkaloids, flavonoids, and gingerols among others. To investigate the changes in main bioactive constituents in its seven single herbs before and after compatibility, a rapid, simple, and sensitive method was developed for comparative analysis of 27 main bioactive components by using ultrahigh-performance liquid chromatography with triple quadrupole electrospray tandem mass spectrometry for the first time. The sufficient separation of 27 target constituents was achieved on a Thermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 µm) within 20 min under the optimized chromatographic conditions. Compared with the theoretical content, the observed content of each analyte showed remarkable differences in Xin-Sheng-Hua granule except thymine, p-coumaric acid, senkyunolide I, senkyunolide H, and ligustilide; the total contents of 27 components increased significantly, and the content variation degrees for the different components were gingerols > flavonoids > aromatic acids > alkaloids > phthalides. The results could provide a good reference for the quality control of Xin-Sheng-Hua granule and might be helpful to interpret the drug interactions based on variation of bioactive components in formulae.

[Determination of stachydrine hydrochloride and leonurine hydrochloride in Yimucao preparations by HPLC-MS].

To establish the quantitative method of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba. The contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba were determined by HPLC-MS. The chromatographic column was Waters XBridge Amide(4.6 mm×250 mm,5 µm). The mobile phase was acetonitrile-0.1% formic acid in gradient mode,at the flow rate of 1.0 mL• min⁻¹,with the split ratio of 1∶4. MS conditions for the ESI ion source,positive ion mode,selective ion scan(SIM) of stachydrine hydrochloride(m/z 144.0) and leonurine hydrochloride(m/z 312.0) was measured. The linear ranges of stachydrine hydrochloride was 0.562 8-281.4 µg•L-1（r=0.999 8）. The linear ranges of leonurine hydrochloride was 0.521 2-260.6 µg•L-1（r=0.999 8）. The method is accurate,simple,and reliable,and can be used to determine the contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba.

Determination of stachydrine hydrochloride and leonurine hydrochloride in Yimucao preparations by HPLC-MS / 中国中药杂志

To establish the quantitative method of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba. The contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba were determined by HPLC-MS. The chromatographic column was Waters XBridge Amide(4.6 mm×250 mm,5 μm). The mobile phase was acetonitrile-0.1% formic acid in gradient mode,at the flow rate of 1.0 mL• min⁻¹,with the split ratio of 1∶4. MS conditions for the ESI ion source,positive ion mode,selective ion scan(SIM) of stachydrine hydrochloride(m/z 144.0) and leonurine hydrochloride(m/z 312.0) was measured. The linear ranges of stachydrine hydrochloride was 0.562 8-281.4 μg•L-1（r=0.999 8）. The linear ranges of leonurine hydrochloride was 0.521 2-260.6 μg•L-1（r=0.999 8）. The method is accurate,simple,and reliable,and can be used to determine the contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba.

Effect of Leonuri Herba alkaloids on prostatic hyperplasia in older rats / 中草药

Objective: To investigate the pharmacological effects and mechanism of Leonuri Herba alkaloids (LHA) on prostate hyperplasia in older rats. Methods: The prostate hyperplasia model in older rats was induced by male and female hormone, the rats were divided into five groups: 50.0, 25.0, 12.5 mg/kg alkaloids dosage group, 30 mg/kg Longbishu Capsule suspension group, and model group given the same volume of saline. Otherwise aged rats and young rats were set as control groups administered with saline, and administered once daily for consecutive 30 d. The wet weight and prostate index in rat prostate were measured, and the expression of E2, DHT, T, bFGF, TGF-β1, EGF, and IGF-1 in serum and prostate tissue were detected after the experiment. The rat prostate tissue morphology changes and rat prostate cells ultrastructural changes were observed by light microscopy and transmission electron microscopy. Results: Compared with the model group, benign prostatic hyperplasia rat model was replicated successfully, low-dose LHA can significantly reduce the rat prostate wet weight and prostate index (P < 0.05), mid-dose LHA can significantly reduce the rat prostate index (P < 0.05). Each dose of LHA can significantly reduce the levels of T and DHT in prostate model rats (P < 0.01). Each dose of LHA can significantly reduce the expression of bFGF, EGF, and IGF-1 in prostate model rats (P < 0.01). Low-dose LHA can significantly increase the expression of TGF-β1 in prostate tissue. Each dose of LHA can significantly reduce the density of prostate model rats (P < 0.05, 0.01), and significantly increase the membrane surface and surface area values (P < 0.01). Each dose of LHA can significantly reduce intracytoplasmic mitochondria in prostate cells caused by model, and relieve the pathological changes of mitochondria cristae. Conclusion: LHA has good therapeutic effect to prostatic hyperplasia model rats induced by male and female hormone.

Toxicity assessment of Leonuri Herba aqueous extract orally administered to rats for 13 consecutive weeks.

ETHNOPHARMACOLOGICAL RELEVANCE: The Leonuri Herba has been traditionally used for women's disease in Asian countries. AIM OF THE STUDY: The objective of the present study was to evaluate the subchronic toxicity of Leonuri Herba aqueous extract in male and female F344 rats. MATERIAL AND METHODS: Leonuri Herba aqueous extract was administered orally once daily at dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg/day for 13 weeks. Toxicological assessment was performed including mortality, clinical signs, body and organ weights, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination. RESULTS: There were no treatment related differences in clinical signs, urinalysis, hematology and serum chemistry, except for a histopathologic examination. The squamous cell hyperplasia in the forestomach was observed in both sexes of rats given 2000 mg/kg/day of Leonuri Herba aqueous extract. CONCLUSION: In conclusion, the NOAEL (No Observed Adverse Effect Level) for Leonuri Herba aqueous extract was determined as 1000 mg/kg/day in both sexes of rats under the present experimental conditions. And the acceptable daily intake value for Leonuri Herba aqueous extract was calculated to be 10mg/kg body weight per day using a safety factor of 100 to the NOAEL.