Angelicin: A leading culprit involved in fructus Psoraleae liver injury via inhibition of VKORC1.

ETHNOPHARMACOLOGICAL RELEVANCE: The adverse effects of Fructus Psoraleae (FP), especially liver injury, have attracted wide attention in recent years. AIM OF THE STUDY: To establish a system to explore potential hepatotoxic targets and the chief culprit of liver injury based on clinical experience, network pharmacological method, molecular docking, and in vitro and in vivo experiments. MATERIALS AND METHODS: Clinical applications and adverse reactions to FP were obtained from public literatures. Components absorbed in the blood were selected as candidates to search for potential active targets (PATs) of FP. Subsequently, potential pharmacological core targets (PPCTs) were screened through the "drug targets-disease targets" network. Non-drug active targets (NPATs) were obtained by subtracting the PPCTs from the PATs. The potential hepatotoxic targets (PHTs) of FP were the intersection targets obtained from Venn analysis using NPATs, hepatotoxic targets, and adverse drug reaction (ADR) targets provided by the databases. Then, potential hepatotoxic components and targets were obtained using the "NPATS-component" network relationship. Molecular docking and in vitro and in vivo hepatotoxicity experiments were performed to verify the targets and related components. RESULTS: Overall, 234 NPATs were acquired from our analysis, and 6 targets were identified as PHTs. Results from molecular docking and in vitro and in vivo experiments showed that angelicin is the leading cause of liver injury in FP, and VKORC1 plays an important role. CONCLUSION: The results indicate that six targets, especially VKORC1, are associated with the PHTs of FP, and angelicin is the leading culprit involved in FP liver injury via inhibition of VKORC1.

[A novel mouse allergy tested model and its application for traditional Chinese medicine injections's allergy evaluation].

When the drug induces the organism to produce a type Ⅰ allergic reaction, the combination of IgE and mast cells results in the degranulation of the mast cells. Release of vasoactive substances, increase in vascular permeability, and exudation of intravascular substances outside the blood vessels. Based on this pathophysiological mechanism, a mouse model that can objectively and quantitatively assess the allergic response to the injection has been established. ICR mice were sensitised by intraperitoneal injection of different doses of OVA once every two days for three times. 14 days after the last sensitization, a combination OVA solution of 4 times the sensitizing dose and Evans blue were injected intravenously into mice for the challenge. Compared with the normal group, OVA 0.625/2.5, 1.25/5, 2.5/10, 5/20 mg·kg~(-1) sensitized and challenged can induce allergic reactions mainly manifested by blue staining of the auricle in mice. Direct injection of OVA intravenously did not cause an auricular blue colouration reaction in mice. The passive cutaneous anaphylaxis reaction in mice was conducted with the aforementioned OVA-sensitized mouse serum, and there were obvious blue spots on the mouse's back. In addition, the content of anti-OVA-IgE in 5 mg·kg~(-1) OVA-sensitized mice was significantly increased. Ears and lungs of mice sensitized to OVA showed evident exudation inflammation. Significantly elevated inflammatory factors(VEGF and IL-10) were also detected in the serum of OVA-sensitized mice. The equivalent dose of OVA caused obvious allergic reactions in both guinea pigs and mice. Compared with nude mice, ICR and BALB/c mice are more sensitive to OVA sensitization. Injections of selected TCMI did not induce type Ⅰ allergic reactions in mice and guinea pigs, but there was a risk of inducing pseu-doallergic reactions in mice. The model is problematic and may well reflect the sensitization effect of allergens. It obtains the benefits of simple operation, accuracy, low cost, easy extension, and high repeatability. It is suitable for predicting and researching for IgE-dependent type Ⅰ allergic reactions.

[Research progress of intervention of Chinese herbal medicine and its active components on human coronavirus].

The outbreak caused by 2019 novel coronavirus(2019-nCoV) is still spreading, posing a great threat to the safety and health of general population. However, there have not been any effective drugs for treatment, with symptomatic treatment and prevention prevailing. The treatment plans of severe acute respiratory syndrome(SARS) and Middle East respiratory syndrome(MERS) are often used for reference in clinic. The advantages of traditional Chinese medicine(TCM) in treating SARS and MERS are that it can intervene and block the progression of disease in early stage, significantly reduce symptoms, shorten the treatment duration of patients, reduce complications and side effects caused by hormone therapy. The coronavirus disease 2019(COVID-19) belongs to the category of TCM epidemic diseases. Chinese patent medicines and prescriptions in medical observation and clinical treatment were recommended in the "pneumonia diagnosis and treatment plan for new coronavirus infection"(trial version fifth) of the National Health Commission of the People's Republic of China. Qingfei Paidu Decotion was recommended for the treatment of COVID-19 by the National Health Commission of the People's Republic of China and National Administration of Traditional Chinese Medicine. TCM shows good clinical efficacy and great potential in the treatment of COVID-19. Previous studies of TCM have shown broad-spectrum antiviral activity, providing a variety of sources for the discovery of new antiviral drugs. In this paper, we reviewed traditional Chinese medicines and its active ingredients in the hope of bringing novel inspirations to the drug screening and clinical treatment for COVID-19.

[Safety of animal traditional Chinese medicine (TCM) injections].

Animal medicine injection is an important part of traditional Chinese medicine (TCM) injections. All or part of animals with a significant curative effect and little side reactions as raw materials as well as modern technology are used to produce traditional Chinese medicine injections with a reliable and rapid drug efficacy and high bioavailability. Due to the complex composition of traditional Chinese medicine injections, imperfect quality standards, and unreasonable clinical use, the incidence of adverse reactions of traditional Chinese medicine injections has been significantly higher than that of traditional Chinese medicine for oral use. Animal medicine injections contain rich protein and fat, and heteroproteins are the main sensitization source in animal medicine injections. At present, the adverse reactions of animal medicine injections are mainly manifested in the anaphylaxis-like reactions at skin, mucous membranes and organ systems. The adverse reactions that occur during the first medication are more common. Specific causes for allergic-like adverse reactions in animal injections and related substances in traditional Chinese medicine injections made of animals that induce allergies or anaphylactoid reactions are currently not specifically reported. This article reviews the current adverse reactions of animal TCM injections, allergies and pseudoallergic reactions of animal TCM injections, the pharmacokinetics of animal TCM injections, and the combined use of drugs, in order to improve the quality standards of Chinese medicine injections for animals and provide reference for further safety related research.

[Preclinical study on adverse reactions of Xingnaojing injection].

In this study, different batches of Xingnaojing injection products were first selected for pseudoallergic mice test, and the results showed that after injection of 6.6-fold clinical dose Xingnaojing injection, the mice showed a slight pseudoallergic reaction, while other mice injected with other batches of injections showed no obvious pseudoallergic reaction. Therefore, it is preliminarily believed that this mice model can effectively indicate the risk of pseudoallergic reactions in the clinical application of Xingnaojing injections. In addition, by changing some of the processes, a high concentration of Xingnaojing injection was prepared for mice pseudoallergic test and guinea pig systemic allergy test. The results showed no significant type Ⅰ allergic reaction in guinea pigs. Mild pseudoallergic reactions occurred in mice after a 6.6-fold clinical dose injection. Therefore, it is considered that for sensitive or idiosyncratic people, the concentration of certain chemical components in Xingnaojing injection will increase after entering the body, which may increase the risk of pseudoallergic reaction. However, due to the limitations of test models, the risk of Xingnaojing injection to induce allergic reactions cannot be ruled out. Finally, by increasing the content of borneol and Tween and (or) sodium chloride in Xingnnaojing Injection and testing its pseudoallergic reactions, the results showed that the combination of these three ingredients may produce new trace sensitization substance and induce pseudoallergic reactions.

[Mechanism of nephrotoxicity of rhubarb in rats].

The aim of this study was to investigate the renal toxicity of rhubarb and its mechanism. The SD rats were randomly divided into three groups: normal group and two rhubarb extract groups (16, 2 g·kg⁻¹). According to the dose conversion method between human and animal, rhubarb 16 g·kg⁻¹ and 2 g·kg⁻¹ were equivalent to 10 times and 1.25 times of human clinical dose respectively. Rhubarb extract was administered by a gastric gavage to rats once daily for 30 days. Serum urea nitrogen （BUN）, creatinine （CRE） and urine KIM-1, NGAL and renal morphology were analyzed. The expressions of OAT1, OAT3 and clusterin mRNA in kidney were measured. The results showed that the low dose of rhubarb had no obvious renal toxicity. The high dose group showed mild and moderate renal injury and a down-regulation of clusterin mRNA expression in the kidney tissue. The renal toxicity in male animals was heavier than that in female animals. There was no significant change in blood BUN and CRE in the high dose group. But urine NGAL level of the high dose group increased by 51.53% compared with normal group, of which male animals increased more significantly (P<0.05, compared with the normal group). The expressions of renal OAT1 and OAT3 mRNA in the low dose group were obviously higher than that in the normal group. The results indicated that the high dose of rhubarb could cause the renal toxicity. The dosage should be controlled reasonably in the clinical use. OAT1 and OAT3 mRNA related to anionic transport in kidney tissue played a compensatory protective role in rhubarb-induced renal injury. But the compensatory effect is relatively weak at the high dose level. In addition, routine renal function indicators BUN and CRE had limitation for monitoring the kidney toxicity of rhubarb. It is suggested that urine NGAL detection might be helpful for monitoring the renal toxicity of rhubarb.

[Pseudoallergic reaction characteristics of Qingkailing injection and preliminary screening of allergic substances].

This study aimed to explore the characteristics and the influencing factors of Qingkailing injection (QKLI) pseudoallergic reaction, and screen out the possible pseudoallergenic substances. The results showed that ICR and Kunming mice had stronger pseudoallergic reactions than BALB/c and C57 mice after being injected with the same dose of QKLI. The pseudoallergic reaction induced by QKLI that was prepared with 0.9% saline was stronger than that prepared with 5% glucose. When the dose was twice of the clinical dose, some batches of QKLI could cause significant or suspected pseudoallergic reactions; when the dose dropped to clinically equal times, all of the batches did not induce pseudoallergic reactions in mice. Different batches of QKLI induced different pseudoallergic reactions in mice. Therefore, QKLI's pseudoallergic reactions might have a certain relationship with different body constitutions. Different solvents might affect the safety of QKLI. QKIL-induced pseudoallergic reactions had the different characteristics between batches, and the dosage should be strictly controlled in clinical use. After the comparison of pseudoallergic reactions induced by different components and different intermediates of QKLI in mice, it was preliminary believed that pseudoallergenic substances might exist in intermediate Isatidis Radix extracts and Gardenia extracts, but specific pseudoallergens shall be furthered studied in subsequent experiences.

[Pharmacokinetic characteristics of fourteen main components of Gegen Qinlian Decoction in rats].

Animal traditional Chinese medicine has a long history of application in China, and its clinical application is very extensive. Due to the complex chemical composition in animal traditional Chinese medicine, the basis of chemical research is relatively weak, which leads to the unclear composition and toxic components of many animal Chinese medicines. The relationship between the medicinal and toxic components of animal Chinese medicine has not yet been elucidated. The non-clinical safety evaluation of animal traditional Chinese medicine mainly includes acute toxicity, long-term toxicity, safety pharmacology, reproductive toxicity, genotoxicity experiments, and experimental studies such as carcinogenicity are needed when necessary. The current preclinical safety research on animal traditional Chinese medicine is mainly based on the study for toxic animal traditional Chinese medicines. Most animal Chinese medicines have not carried out systematic preclinical and clinical safety studies. The research method is mainly focused on acute toxicity test. It is necessary to carry out systematic preclinical safety studies on animal traditional Chinese medicines, to clarify the possible side effects and its characteristics, its toxic target organs, toxic doses and poisoning mechanisms induced by different animal traditional Chinese medicines. Finally, this paper suggests that in the preclinical safety study of animal traditional Chinese medicine, in-depth research and comparison should be carried out in combination with chemical substance foundation, origin, and collection season, and the safety of "non-toxic" animal traditional Chinese medicine should be carried out when necessary. In addition, it is necessary to rationally use the cutting-edge technologies and methods of toxicology research to fully clarify the preclinical safety information of animal Chinese medicines.

[Adverse reaction and screening of sensitizing substance of Shuxuening injection].

In this study, by the means of the active systemic allergy test in guinea pigs, passive skin allergy test in rats and pseudoallergic test in mice, it was determined that the "allergic reaction" of Shuxuening injection(SXNI) may not be a true IgE-mediated allergic reactions, but mainly of pseudoallergic reaction. Further pseudoallergic test proved that the pseudoallergic reactions of SXNI had difference between batches and showed dose dependence, so it was recommended to establish SXNI pseudoallergic reaction detection method for timely detecting and controlling the product risk of each batch products. In addition, as the pseudoallergic reactions of SXNI were dose-dependent, the dose and concentration of SXNI should be strictly controlled in clinical use. Then the main pseudoallergenic reaction test was conducted for the main monomer components in SXNI and the different fractions of Ginkgo biloba extract in mice, and the results showed that the sensitizing substances may mainly exist in YXY-3 fractions containing flavonol glycosides. By further chemically separating YXY-3, we got four chemical components. Among these four components, YXY-3-1 and YXY-3-2 were testified as the main allergenic components in SXNI through pseudoallergic test in mice. To make sure the specific chemical constituent that is responsible for the pseudoallergic reaction, in-depth study in follow-up experiments should be needed.

[Review on requirements of drug allergy or pseudoallergic reactions in pre-clinical evaluation].

Drug allergy and pseudoallergic reactions are main adverse drug reactions. Allergy is mainly induced by the immunogenicity of drug, drug metabolic products or drug additive. Pseudoallergic reactions may result from the irritation or activation of inflammatory material release. Pre-clinical evaluation of drug allergy and pseudoallergic reactions is included in immunotoxicity evaluation. Now there is no in vivo or in vitro method that could predict all kinds of allergy or pseudoallergic reactions due to the different mechanisms. In the past few years, FDA, SFDA OECD, ICH and WHO have published several guidelines on per-clinical immunotoxicity evaluation, however, no agreement has been reached on allergy and pseudoallergic reactions evaluation. This article reviews the requirements of allergy and pseudoallergic reactions in pre-clinical evaluation.

[Genotoxicity research thought and method on traditional Chinese medicine].

Genotoxicity research takes an important place in traditional Chinese medicine safety evaluation. Genotoxicity test on traditional Chinese medicine has been paid great attention since 1970s. Currently, the most developed genotoxicity test methods included: bacterial reverse mutation test and mouse lymphoma assay which are used to detect relevant genetic changes, micronucleus test and chromosomal analysis which are used to measure chromosomal aberration, and single cell electrophoresis assay which is used to test DNA damage. This article reviews research progress on genotoxicity of traditional Chinese medicine, evaluation methods of genotoxicity, the problems and solutions on genotoxicity evaluation of traditional Chinese medicine, and new technique used in genotoxicity test.

[Evaluation and study of pseudo-allergic reactions of three kinds of traditional Chinese medicine injections and its mechanisms].

To evaluated the pseudo-allergic reactions of cordate houttuynia, pulse-activating and Qingkailing injection in mice, the ICR mouse were divided into different test groups, then were intravenously injected with three traditional Chinese medicine injections, positive control compound 48/80 and physiological saline as normal control. All test substances were mixed with 0.4% Evans blue. The reaction and vascular permeability of the ears were observed and measured 30 min after injection. At the same time, the mechanisms of the traditional Chinese injections' pseudo-allergic reactions was studyed. ICR mice were injected with the test substances as above without EB, blood in a part of mice were collected after 5 min, and the level of histamine in the plasma were measured. Blood in the other part of mice were collected after 30 min, and the level of VEGF, TNF-α and IL-10 in the serum were measured. The reasults showed that except the cordate houttuynia injection, pulse-activating injection in 1. 5 times clinical concentration and Qingkailing injection in 3.3 times clinical concentration caused mild pseudo-allergic reactions mainly for vascular permeability, no pseudo-allergic reactions occurred when the concentration of the two injections was below the concentration metioned above; 5 minutes after intravenous injection of the three TCM injections into ICR mice with the highest dose, the levels of histamine in plasma of pulse-activating injection and Qingkailing injection groups were increased significantly, 30 minutes later, the levels of VEGF, TNF-α and IL-10 in the serum of all groups were no significant change compared to normal group. The mouse of pulse-activating and Qingkailing injection groups showed inflammatory changes in ear and lung tissues. It can be conluded that when the dose or concentration increased to a certain extent, pulse-activating and Qingkailing injection could induce pseudo-allergic reactions on ICR mice, the increased histamine realease maybe the main reason for pseudo-allergic reactions of the two traditional Chinese medicine injections. In addition the author preliminary thought that inflammatory mechanisms leading to hyperpermeabilities were the main reason of the traditional Chinese medicine injection's pseudo-allergic reaction.

[Influence of solvent and drug preparation time on Shuanghuanglian injections induce pseudo-allergic reaction].

Choosing the right solvent and timely use is the basis of rational drug use and the most direct and efficient way to improve the safety of traditional Chinese medicine injections. This study aimed to evaluate the influence of solvent and drug preparation time on Shuanghuanglian injection inducing pseudo-allergic reactions with mouse mode. The two tests were carried out: (1) Comparative experiment between different solvent: Shuanghuanglian injection preparation to the appropriate concentration with 0.9% sodium chloride injection and 5% dextrose injection, mixed with Evans blue, at one time intravenous injected into mice, 30 minutes later, the mouse ears vascular permeability were observed and compared. (2) Comparative experiment among different preparation time: placed 10 min, 2.5 h, 6 h and 24 h after Shuanghuanglian injection were prepared and then to detect the pseudo-allergic reactions in mice using the same methods as in (1). The results showed that there was no significant difference in the pseudo-allergic reactions in mice which induced by the same dose of Shuanghuanglian injection, respectively with 0.9% sodium chloride injection and 5% dextrose injection preparation, and with the extension of preparation time, the degree of pseudo-allergic reactions of Shuanghuanglian injection was gradually severe.

[Preclinical evaluation of pseudoallergic reactions on Chinese herbal injections: study on animal strain and gender difference].

Pseudoallergic reactions occured after the first administration of patients, and the pathogenic mechanisms of them were different from the allergic reactions which needed excitation after antigen sensitization. To provide a basis for evaluation, clinical use and drug development of pseudoallergic reactions, the models were established by two kinds of Chinese herbal injections (CHI) both on different strain or gender mice. With the use of ICR, Kunming, BALB/C, C57 mice, pseudoallergic tests of two CHI were conducted to compare the sensitivity of four strains mice, and compared the differences in male and female animals. Test substances contain 0.8% Evans blue (EB) were intravenously injected into different strain and gender mice. Scores of ear blue staining and quantitation of ear EB exudation were the parameters for pseudoallergic reaction. Results of strain difference indicated that both CHI A and B could cause severe pseudoallergic reactions indicated by obvious vascular hyperpermeability on ICR mice. The pseudoallergic reactions in ICR mice are more obvious under the the same dose of injection, which stated the sensibility of ICR mice. And the reactions of KM mice and BALB/C mice were slightly reduced which compared to ICR mice, even alomost nothing on C57 mice. Comparison results of gender difference showed that one CHI was not have significant difference in male and female animals, but male animals were more susceptible than females on another CHI. Therefore, ICR mice were preferable experimental strain on the model of pseudoallergic reactions induced by CHI A and B. Because of female animals were easily influenced by estrous cycle, the pseudoallergic reactions induced by CHI A and B select and use male mice befittingly.

[Study of screening pseudoallergenic substances of Shuanghuanglian injection].

In this study, chemistry, biology and pharmacology were combinated to screen pseudoallergenic substances of Shuang-huanglian injection (SHLI) so that to establish a scientific and systematic approach to screen pseudoallergenic substances of traditional Chinese medicine injections. The mouse pseudoallergic reaction models were used to screen the pseudoallergic reaction of SHLI's intermediate extract and the intermediate extract's component or ingredient. Among the three intermediates of Shuanghuanglian injection (extract of Scutellaria baicalensis, extract of Lonicera japonica, extract of Forsythia suspensa) , pseudoallergic action of Forsythia suspensa was the strongest, Forsythia suspesnsa's pseudoallergic reaction mainly associated with the composition with largerchemical polarity. Further it was found that forsythiaside A and arctiin which existed in the the composition with largerchemical polarity caused obvious pseudoallergic reactions. SHLI with removal forsythoside A with the technology of HPLC-MS displayed reduced pseudoallergic reaction and a significant improved safety. This study provided a scientific basis for SHLI process improvements and also offered idea and research foundation for screening pseudoallergenic substances injections in other TCM injections.

[Experimental study on pseudoallergic reaction of Qingkailing injection].

Pseudoallergic reactions of Qingkailing injection (QKLI) was assessed by vascular hyperpermeability which were indicated by ear blue staining in ICR mice after single intravenous injection of QKLI mixed with Evans blue (EB) and skin blue spot formation in SD rats after intradermal injection of QKLI and intravenous injection of EB. In addition, QKLI-induced histamine, VEGF, TNF-alpha release was measured after ICR mice received the single dosing of QKLI iv. The mild vascular hyperpermeability characterized by ear blue staining could be observed in mice after intravenous injection of QKLI and EB. Intracutaneous injection of 50 micro L of test solution containing QKLI (25,50 microL) in rat back skin caused obvious local vascular hyperpermeability at the injection sites so as to result the larger diameters of blue spots than that in negative control group (P <0. 01). QKLI induced a significant increase of VEGF and a slight elevation of histamine in mice after intravenous administration, while TNF-alpha showed no change after QKLI iv. The results in this study indicated that both intravenous injection and intracutanous injection of QKLI could induce vascular hyperpemeability so as to cause pseudoallergic reaction in mice and rats. QKLI-induced pseudoallergic reaction may be associated with the release of histamine and VEGF.

[Thought and method of reproductive toxicity research in traditional Chinese medicine].

Reproductive toxicity research takes an important place in traditional Chinese medicine pre-clinical safety evaluation. Modern reproductive toxicity experiment includes drug-related miscarriage, fetal death, teratism, and adverse effects on fertility, genital system, embryonic development and fetus, which is different from contraindicated in pregnancy in traditional Chinese medicine theory. Now the three-phases reproductive toxicity study is the method mainly applied in traditional Chinese medicine reproductive toxicity evaluation. Besides that, alternative methods of whole embryos culture and embryonic stem cell test are also used in traditional Chinese medicine embryo toxicity evaluation. This article reviews research progress and pre-clinical evaluation on reproductive toxicity of traditional Chinese medicine.