Metabolomics based comprehensive investigation of Gardeniae Fructus induced hepatotoxicity.

Gardeniae Fructus (Zhizi in Chinese, ZZ in brief), a commonly used herbal medicine, has aroused wide concern for hepatotoxicity, but the mechanism remains to be investigated. This study was aimed at investigating the mechanism of ZZ-induced liver injury in vivo and in vitro based on metabolomics and evaluating the hepatotoxicity prediction ability of the in vitro model. SD rats were administered with extracted ZZ and HepG2 cells were treated with genipin, the major hepatotoxic metabolite of ZZ. Liver, plasma, intracellular and extracellular samples were obtained for metabolomics analysis. As a result, ZZ caused plasma biochemical and liver histopathological alterations in rats, and induced purine and amino acid metabolism disorder in the liver and pyrimidine, primary bile acids, amino acid metabolism and pantothenate and CoA biosynthesis disorder in the plasma. Pyrimidine, purine, amino acid metabolism and pantothenate and CoA biosynthesis were also found to be disturbed in the genipin-treated HepG2 cells, which exhibited similarity with the result in vivo. This study comprehensively illustrates the underlying mechanism involved in ZZ-related hepatotoxicity from the aspect of metabolome, and provides evidence that identifying hepatotoxicity can be achieved in cells, representing a non-animal alternative for systemic toxicology.

Genipin induces developmental toxicity through oxidative stress and apoptosis in zebrafish.

Genipin, an iridoid substance, is mainly derived from Gardenia jasminoides Ellis of the traditional Chinese medicine and is widely used in raw materials for the food additive gardenia blue and biological materials. The developmental toxicity of genipin has not been investigated, and its underlying mechanism is unclear. Therefore, in this study we attempt to investigate the potential developmental toxicity of genipin in zebrafish embryos/larvae. The results showed zebrafish embryos treated with 50 µg/ml dose of genipin display inhibited hatching rates and body length. The pericardial edema was observed. It was also found that genipin could induce cardio-toxicity, hepatotoxicity and nephrotoxicity in zebrafish larvae. After genipin treatment, the suppression of antioxidant capacity and increase of oxidative stress were showed for the triggered generation of ROS and MDA, and decreased activity of SOD. Compared with the 0.5% DMSO group, a number of apoptotic cells in zebrafish were increased after genipin exposure. By measuring marker gene expression with the using of qRT-PCR, we proposed that developmental toxicity after genipin treatment might be associated with oxidative stress and apoptosis increase. Our research offers a better understanding for developmental toxicity of genipin.

Therapeutic potential of genipin in various acute liver injury, fulminant hepatitis, NAFLD and other non-cancer liver diseases: More friend than foe.

Genipin is an aglycone derived from the geniposide, the most abundant iridoid glucoside constituent of Gardenia jasminoides Ellis. For decades, genipin is the focus of studies as a versatile compound in the treatment of various pathogenic conditions. In particularly, Gardenia jasminoides Ellis has long been used in traditional Chinese medicine for the prevention and treatment of liver disease. Mounting experimental data has proved genipin possesses therapeutic potential for cholestatic, septic, ischemia/reperfusion-triggered acute liver injury, fulminant hepatitis and NAFLD. This critical review is a reflection on the valuable lessons from decades of research regarding pharmacological activities of genipin. Of note, genipin represents choleretic effect by potentiating bilirubin disposal and enhancement of genes in charge of the efflux of a number of organic anions. The anti-inflammatory capability of genipin is mediated by suppression of the production and function of pro-inflammatory cytokines and inflammasome. Moreover, genipin modulates various transcription factor and signal transduction pathway. Genipin appears to trigger the upregulation of several key genes encoding antioxidant and xenobiotic-metabolizing enzymes. Furthermore, the medicinal impact of genipin extends to modulation of regulated cell death, including autophagic cell death, apoptosis, necroptosis and pyroptosis, and modulation of quality of cellular organelle. Another crucial effect of genipin appears to be linked to dual role in targeting uncoupling protein 2 (UCP2). As a typical UCP2-inhibiting compound, genipin could inhibit AMP-activated protein kinase or NF-κB in circumstance. On the contrary, reactive oxygen species production and cellular lipid deposits mediated by genipin through the upregulation of UCP2 is observed in liver steatosis, suggesting the precise role of genipin is disease-specific. Collectively, we comprehensively summarize the mechanisms and pathways associated with the hepatoprotective activity of genipin and discuss potential toxic impact. Notably, our focus is the direct medicinal effect of genipin itself, whereas its utility as a crosslinking agent in tissue engineering is out of scope for the current review. Further studies are therefore required to disentangle these complicated pharmacological properties to confer this natural agent a far greater potency.

Safety Evaluations of Single Dose of the Olive Secoiridoid S-(-)-Oleocanthal in Swiss Albino Mice.

Epidemiological and clinical studies compellingly showed the ability of Mediterranean diet rich in extra-virgin olive oil (EVOO) to reduce multiple diseases such as cancer, cardiovascular diseases, and aging cognitive functions decline. The S-(-)-Oleocanthal (OC) is a minor phenolic secoiridoid exclusively found in extra-virgin olive oil (EVOO). OC recently gained notable research attention due to its excellent in vitro and in vivo biological effects against multiple cancers, inflammations, and Alzheimer's disease. However, OC safety has not been comprehensively studied yet. This study reports for the first time the detailed safety of oral single OC dose in Swiss albino mice, applying the OECD 420 procedure. Male and female Swiss albino mice (n = 10) were orally treated with a single OC dose of either 10, 250, or 500 mg/kg bodyweight or equivalent volumes of distilled water. Mice fed a regular diet, and carefully observed for 14 days. Further, mice were then sacrificed, blood samples, and organs were collected and subjected to hematological, biochemical, and histological examinations. OC 10 mg/kg oral dose appears to be without adverse effects. Further, 250 mg/kg OC, p.o., is suggested as a possible upper dose for preclinical studies in the future.

[Marker genes of geniposide-induced hepatotoxicity based on genomic strategy].

The aim of this paper was to screen out relevant genes of geniposide-induced hepatotoxicity based on genomics,in order to provide a scientific basis for the non-clinical evaluation of drugs containing Gardeniae Fructus and geniposide. Fifty-five SD rats were randomly divided into normal control group,24 h group and 72 h group. The changes of appearance,behavior and weight of rats were observed after administration by gavage for 3 days. The activities of ALT and AST were detected. Molecular mechanism of geniposideinduced hepatotoxicity was investigated by Affymetrix miRNA 4. 0 and Affymetrix Rat Gene 2. 0 to examine the gene expression levels in Sprague-Dawley rat livers at 24 h and 72 h after administration of overdose-geniposide( 300 mg·kg-1 daily),and then verified by Realtime quantitative PCR. Compared with the normal control group,the activities of ALT and AST were markedly increased. In addition,experimental results indicated that 324 genes were differentially expressed,among which 259 were up-regulated and 65 down-regulated.Nine candidate genes were verified by qRT-PCR,including Bcl2,Il1 b,Tpm3,MMP2,Col1α1,Ifit1,Aldob,Nr0 b2,Cyp2 c23. And Bcl2,Col1α1,Aldob,Nr0 b2 and Cyp2 c23 were found to be correlated with geniposide-induced hepatotoxicity. This study provides an important clue for mechanism of geniposide-induced hepatotoxicity.

The Effects of Different Varieties of Aurantii Fructus Immaturus on the Potential Toxicity of Zhi-Zi-Hou-Po Decoction Based on Spectrum-Toxicity Correlation Analysis.

In accordance with the provision in China Pharmacopoeia, Citrus aurantium L. (Sour orange-SZS) and Citrus sinensis Osbeck (Sweet orange-TZS) are all in line with the requirements of Aurantii Fructus Immaturus (ZS). Both kinds of ZS are also marketed in the market. With the frequent occurrence of depression, Zhi-Zi-Hou-Po decoction (ZZHPD) has attracted wide attention. Currently, studies have shown that ZZHPD has a potential toxicity risk, but the effect of two commercial varieties of ZS on ZZHPD has not been reported. In this study, the toxicity differences of ZZHPD prepared by SZS and TZS were revealed through repeated administration experiments in rats. This indicated that different varieties of ZS could affect the toxicity of the prescription. In order to further study the chemical material basis of the toxicity difference, the fingerprints of ZZHPD prepared by different varieties of ZS were established by high-performance liquid chromatography (HPLC). Five different characteristic peaks were screened by non-target chemometrics. They were identified as geniposide, neoeriocitrin, naringin, hesperidin, and neohesperidin using an HPLC-time-of-flight mass spectrometry analyzer (TOF/MS) and an HPLC-triple stage quadrupole mass spectrometry analyzer (QqQ-MS/MS). Combined with a quantitative analysis and previous studies on promoting the intestinal absorption of geniposide, it is speculated that the synergistic effects of the components may be the main reason for the difference of toxicity among the different medicinal materials. This study provides a reference for the clinical, safe use of ZZHPD, and also provides a new perspective for the study of the potential toxic substances of traditional Chinese medicine compound preparations.

Sustained scleral stiffening in rats after a single genipin treatment.

Scleral stiffening has been proposed as a therapy for glaucoma and myopia. Previous in vivo studies have evaluated the efficacy of scleral stiffening after multiple treatments with a natural collagen crosslinker, genipin. However, multiple injections limit clinical translatability. Here, we examined whether scleral stiffening was maintained after four weeks following a single genipin treatment. Eyes from brown Norway rats were treated in vivo with a single 15 mM genipin retrobulbar injection, sham retrobulbar injection, or were left naive. Eyes were enucleated either 1 day or four weeks post-injection and underwent whole globe inflation testing. We assessed first principal Lagrange strain of the posterior sclera using digital image correlation as a proxy for scleral stiffness. Four weeks post-injection, genipin treatment resulted in a 58% reduction in scleral strain as compared to controls (p = 0.005). We conclude that a single in vivo injection of genipin effectively stiffened rat sclera for at least four weeks which motivates further functional studies and possible clinical translation of genipin-induced scleral stiffening.

Role of intestinal microbiota-mediated genipin dialdehyde intermediate formation in geniposide-induced hepatotoxicity in rats.

Geniposide is a natural hepatotoxic iridoid glycoside. Its hydrolysate of intestinal microbiota, genipin, is thought to be responsible for the hepatotoxicity. However, the underlying mechanism that genipin contributes to the hepatotoxicity of geniposide is not well understood. In this study, we found that genipin spontaneously converted into a reactive dialdehyde intermediate and covalently bond to the primary amine group of free amino acids in both of the phosphate buffers and geniposide-treated rats. Furthermore, genipin dialdehyde can form the covalent linkage to the primary amino group (ε) of lysine side chains of the hepatic proteins in geniposide-treated rats. Pretreatment with ß-glucosidase or antibiotics significantly modulated the genipin dialdehyde formation and protein modification, revealing the essential role of microbial glycosidases. The levels of protein adduct were that mapped onto the hepatotoxicity of geniposide. In summary, this study demonstrates that the intestinal microbiota mediated covalent modification of the hepatic protein by genipin dialdehyde may play a crucial role in the liver injury of geniposide. The study is also helpful for understanding the contribution of intestinal microbiota to the metabolic activation of xenobiotics.

The Short-Term Safety Evaluation of Corneal Crosslinking Agent-Genipin.

BACKGROUND: Genipin (GP) is a safe method for corneal crosslinking, even for very thin corneas. However, there have been no reports on the optimal GP concentration range to use in vivo for corneal crosslinking. OBJECTIVES: To investigate the safety of corneal crosslinking after a 24-h incubation with different concentrations of GP. METHODS: Twenty New Zealand white rabbits were divided into a phosphate-buffered saline (PBS) group, 0.2% GP crosslinking (GP-CXL) group, 0.25% GP-CXL group, and 0.3% GP-CXL group. Before and after surgery, the operated eyes of each group were characterized by confocal microscopy, and corneal buttons were excised for endothelium staining and electron microscopy. RESULTS: The keratocyte structures in each GP group appeared to be similar to those in the PBS group. Through the confocal microscopy, the changes in corneal endothelial cell density also did not significantly differ among groups. There was a significant difference in apoptosis between the 0.3% GP-CXL and PBS groups (p < 0.05) and between the 0.3% GP-CXL and 0.25% GP-CXL groups (p < 0.05), but there were no significant differences between the 0.2 and 0.25% GP-CXL groups compared to the PBS group. Transmission electron microscopy showed endothelial cell damage in the 0.3% GP-CXL group, with minimal endothelial cell damage in the other groups. CONCLUSIONS: Treatment of rabbit corneas with ≤0.25% GP resulted in minimal toxicity to keratocytes and endothelial cells, suggesting that it is a safe crosslinking agent at those concentrations.

Oral Acute and Repeated-Doses Toxicity Study of Valepotriates from Valeriana glechomifolia (Meyer) in Mice.

BACKGROUND: Species of Valeriana show sedative, hypnotic, anxiolytic, antidepressant and anti-inflammatory properties, which are associated with valepotriates. However, data about toxicity and safety of these compounds are still limited. The aim of this study was to investigate the toxicity of a valepotriate-enriched fraction (VAL) from Valeriana glechomifolia Meyer based on the Organization for Economic Cooperation and Development (OECD) guidelines 423 and 407. METHODS: In the acute study, CF1 mice were treated with a single dose of VAL (2000 mg/kg, p.o.) and observed for 14 days. In the repeated dose study, CF1 mice received single daily doses of VAL (30, 150 or 300 mg/kg, p.o.) or vehicle for 28 days. These doses were chosen based on previous results by our group and according to Guideline 407- OECD. RESULTS: The acute study allowed to classify VAL in the hazard category 5. The repeat-dose study has shown that VAL 300 mg/kg delayed weight gain and reduced food consumption in the first week, probably due to transient sedative effects. The other doses had no effect on animals' ponderal evolution. At the end of the treatment, all groups had equal body weight and food consumption. None of the doses altered any behavioral, urinary, biochemical, hematological, anatomic or histological parameters. CONCLUSION: A valepotriate-enriched fraction from Valeriana glechomifolia presents relatively low oral acute toxicity and does not induce evident toxicity after oral repeated treatment (at least up to 300 mg/kg) in mice.

Marker genes of geniposide-induced hepatotoxicity based on genomic strategy / 中国中药杂志

The aim of this paper was to screen out relevant genes of geniposide-induced hepatotoxicity based on genomics,in order to provide a scientific basis for the non-clinical evaluation of drugs containing Gardeniae Fructus and geniposide. Fifty-five SD rats were randomly divided into normal control group,24 h group and 72 h group. The changes of appearance,behavior and weight of rats were observed after administration by gavage for 3 days. The activities of ALT and AST were detected. Molecular mechanism of geniposideinduced hepatotoxicity was investigated by Affymetrix miRNA 4. 0 and Affymetrix Rat Gene 2. 0 to examine the gene expression levels in Sprague-Dawley rat livers at 24 h and 72 h after administration of overdose-geniposide( 300 mg·kg-1 daily),and then verified by Realtime quantitative PCR. Compared with the normal control group,the activities of ALT and AST were markedly increased. In addition,experimental results indicated that 324 genes were differentially expressed,among which 259 were up-regulated and 65 down-regulated.Nine candidate genes were verified by qRT-PCR,including Bcl2,Il1 b,Tpm3,MMP2,Col1α1,Ifit1,Aldob,Nr0 b2,Cyp2 c23. And Bcl2,Col1α1,Aldob,Nr0 b2 and Cyp2 c23 were found to be correlated with geniposide-induced hepatotoxicity. This study provides an important clue for mechanism of geniposide-induced hepatotoxicity.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.

Oral chronic toxicity study of geniposide in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Geniposide, the major active constituent of Fructus Gardeniae (FG), has been widely used to treat various diseases in China. AIM OF THE STUDY: This chronic toxicity study was conducted to investigate the safety of geniposide. MATERIALS AND METHODS: Geniposide was administered to Sprague-Dawley (SD) rats of both sexes by oral gavage at dosages of 25, 50, or 100mg/kg in a volume of 10mL/kg once daily for 26 weeks. Endpoints included clinical observations, food consumption, body weights, blood biochemistry, haematology, and histomorphological observations. RESULTS: The administration of geniposide did not influence animal mortality, the general conditions of the animals, body weights or food consumption. After 4 weeks of administration, significant toxicity was not observed. However, in the 13th week of the toxicity study, a few haematological parameters and some relative organ weights of male rats in the 50 and 100mg/kg geniposide groups were significantly increased. The percentage of reticulocytes (Retic %) was significantly increased in male and female rats administered 100mg/kg geniposide. In addition, two female rats in the 100mg/kg geniposide group showed slight pathological changes in hepatic and renal tissues. Furthermore, in a chronic (26 weeks) toxicity study, differences were detected in alanine aminotransferase (ALT), aspartate aminotransferase (AST), sodium (Na+), potassium (K+), white blood cell (WBC), red blood cell (RBC), and haemoglobin (HGB) levels and the relative weights of the liver and spleen in male rats administered 100mg/kg geniposide. In addition, differences were detected in Retic % and the relative weights of the liver, thymus, and kidneys in female rats administered 100mg/kg geniposide. Urinalysis results from male and female rats in the 100mg/kg geniposide group revealed noticeable changes. The histopathological structures of hepatic and renal tissues in the high-dose geniposide group exhibited serious abnormalities and pigment deposition. CONCLUSION: Geniposide affected serum biochemistry, urinalysis, and haematological parameters as well as relative organ weights. The treatment also caused noticeable pathological abnormalities in liver and kidney tissues. Therefore, administration of a high dose of geniposide (100mg/kg) for 26 weeks could induced obvious liver and kidney damage.

Physicochemical and Toxic Properties of Novel Genipin Drug Delivery Systems Prepared by Mechanochemistry.

BACKGROUND: Complexes of Genipin and different water-soluble adjuvant polysaccharides, such as arabinogalactane, hydroxyethyl starch, fibergum, and oligosaccharides ß-CD and HP-ß-CD, were synthesized as drug delivery system using mechanochemical technology. METHOD: We have investigated physicochemical properties, stability, and hepatotoxicity of the synthesized complexes in solid state and aqueous solution. The formation of the complexes was evidenced by different physical and spectroscopy assays, and the stability constants of our synthesized Genipin-based complexes were also calculated. RESULTS: The HP-ß-CD inclusion complex showed the highest characteristics. We have found that the molecule of Genipin was completely included in the cyclodextrin cavity of the HP-ß-CD. This complex of Genipin has shown a 6.14-fold increase of solubility compared with the original Genipin, and more stable in solvent and solid states. CONCLUSION: The hepatotoxicity assays showed that our investigated complexes of Genipin are much safer than the original Genipin. These results suggest that new Genipin-based preparations can be synthesized with advantageous of higher stability and safety.

Examination of toxicity and collagen linearity after the administration of the protein cross-linker genipin in equine tendon and dermis: a pilot study.

OBJECTIVE: Collagen cross-linking is an attractive therapeutic route aimed at supplementing natural collagen stabilisation. In this study the toxicity of the cross-linker genipin (GP) was examined in avascular (tendon) and vascular (dermis) tissue. METHODS: High doses of GP were injected intratendinously into three yearling horses and evaluated at various time points up to 30 days. A second group of three yearlings were injected into the dermis and evaluated at various time points up to 1 year. Metrics used included lameness, circumferential swelling, ultrasound evaluation, microscopic morphology, collagen production and systemic effect on blood parameters. RESULTS: The tendon injection sites exhibited mild lameness and swelling with no apparent systemic toxicity or stabilisation defects. Treated tendons exhibited increased linear collagen microscopically. Dermal injections showed similar results, with mild swelling at the injection site. Microscopic morphology resulted in a decrease in dermal collagen at 30 days post-injection. Dermis injected at the high dose of 355 mmol/L examined 1 year post-treatment appeared similar to the untreated biopsies; however, there was an increase in mature collagen. CONCLUSION: GP injection appeared to be well tolerated, with transient lameness and mild circumferential swelling when injected into the tendon and local tissue swelling when injected into the dermis. No systemic hypersensitivities or toxicities were observed. Microscopically, GP resulted in increased linear collagen in tendons at 30 days post-injection and overall increased collagen in dermal tissue when evaluated 1 year post-injection.

Biofabrication of genipin-crosslinked peptide hydrogels and their use in the controlled delivery of naproxen.

The synthesis and optimization of peptide-based hydrogel materials have gained growing interest in the last years, thanks to their properties, that make them appealing for diverse biotechnological applications, with a particular focus in the field of biomedicine. The self-assembling abilities of low molecular weight peptides make them ideal for designing advanced materials using mild reaction conditions. In this work, a biocatalytic approach has been used for the synthesis of an Fmoc-tripeptide that is able to self-assemble in water affording a self-supporting hydrogel. The mechanical properties of this material have been enhanced through chemical crosslinking by using a natural compound, genipin, that allows to minimize cytotoxic effects. Moreover, we have tested the potential of the prepared materials to be employed as drug delivery systems using naproxen as an anti-inflammatory model drug, and studying its release kinetics in aqueous medium. The cytotoxicity of the hydrogels has been evaluated, and their mechanical and morphological properties have been studied by rheology and SEM microscopy.

A New Iridoid Dimer and Other Constituents from the Traditional Kurdish Plant Pterocephalus nestorianus Nábelek.

Accompanied by other rare compounds, a new iridoid dimer, named kurdnestorianoside (1), showing an unprecedented secologanol configuration, has been isolated for the first time from the Kurdish medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for treating oral diseases and inflammation. The structure of 1 was established from 1D- and 2D-NMR spectroscopic data. Kaempferol 3-O-[3,6-di-O-(E)-p-coumaroyl]-ß-d-glucopyranoside (7) showed a remarkable antiproliferative activity against several human tumor cell lines.

Evaluation of safety of iridoids rich fraction from Valeriana jatamansi Jones: Acute and sub-chronic toxicity study in mice and rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana jatamansi Jones named Zhizhuxiang in China is one of the most popular traditional medicine for varied ailments related to malaise, abdominal distention, insomnia, and rheumatism. AIM OF THE STUDY: Evaluate the safety of iridoids rich fraction from V. jatamansi Jones (IRFV) and to provide data for clinical application. MATERIALS AND METHODS: The acute and sub-chronic toxicity of IRFV were investigated by employing established methods. The acute toxicity study was conducted through oral administration of a single dose (3,200 mg/kg body weight) of IRFV to adult mice. The vehicle used for dilution of the IRFV was a mixture of 0.5% CMC-Na and 99.5% water. The weight, diet, toxic reaction, and death after 14 days were observed. In the sub-chronic toxicity study, low doses (240 mg/kg bw), middle doses (960 mg/kg bw), and high doses (1,200 mg/kg bw) of IRFV were administered daily to adult rats for 6 days a week (except Sunday) for 3 months. The general behavior of the rats was observed and recorded daily. The weight and food consumption of rats were tested weekly. The effect on organs, the hematological and blood biochemical parameters, and the histopathology were assessed after 1.5 months (five males and five females) and after 3 months (10 males and 10 females).The remaining 10 rats (five males and five females) in each group were fed for 2 weeks to observe reversible and delayed toxicity after the medicine was administered. RESULTS: In the acute toxicity study, no significant difference was found in the body weight of the mice in the control group and those in the drug group (p>0.05). The maximum tolerated dose of IRFV on mice was 3,200 mg/kg, which is 2666 times of the clinical adult daily dose. In the sub-chronic toxicity study on rats, the daily single oral doses of the IRFV did not result in death nor affected the general behavior, including appearance, activities, discharge, and waste at all tested doses. Moreover, no significant difference was found (p>0.05) between the body weights of the rats from the drug groups and those from the control group. Food consumption was significantly affected (p<0.05) only in the first 3 weeks. No statistically significant differences (p>0.05) were observed in the hematological and blood biochemical parameters, and no abnormality of other organs were noted in both gross and histopathological examinations, except several animal transients (p<0.05) or spontaneous lesions (abnormality). CONCLUSION: IRFV is extremely safe in the usual clinical dose, and may not have any single dose toxicity. The lethal dose with 50% mortality rate (LD50) on mice is over 2,000 mg/kg bw. The no-observed adverse effects level is 1,200 mg/kg/day for rats. No direct correlation was found between the hematology, blood biochemical indexes, and organ coefficient of tested rats and the toxicity of IRFV.

Dose- and time-dependent effects of genipin crosslinking on cell viability and tissue mechanics - toward clinical application for tendon repair.

The crosslinking agent genipin is increasingly invoked for the mechanical augmentation of collagen tissues and implants, and has previously been demonstrated to arrest mechanical damage accumulation in various tissues. This study established an in vitro dose-response baseline for the effects of genipin treatment on tendon cells and their matrix, with a view to in vivo application to the repair of partial tendon tears. Regression models based on a broad range of experimental data were used to delineate the range of concentrations that are likely to achieve functionally effective crosslinking, and predict the corresponding degree of cell loss and diminished metabolic activity that can be expected. On these data, it was concluded that rapid mechanical augmentation of tissue properties can only be achieved by accepting some degree of cytotoxicity, yet that post-treatment cell survival may be adequate to eventually repopulate and stabilize the tissue. On this basis, development of delivery strategies and subsequent in vivo study seems warranted.

[Application of genipin for modification of natural biomaterials as a crosslinking agent].

OBJECTIVE: To review the application of genipin for the modification of natural biomaterials as a crosslinking agent and progress in research. METHODS: Domestic and foreign literature on application of genipin for the modification of natural biomaterials as a crosslinking agent was thoroughly reviewed. Results Genipin is an effective natural crosslinking agent with a very low level of cytotoxicity compared with conventional synthetic crosslinking agents. Tissues fixed with genipin can maintain a high level of stability as well as resistance to enzymatic degradation. CONCLUSION: Genipin is a promising substitute for conventional synthetic crosslinking agents, which has offered an alternative for modification of natural biomaterials for tissue engineering.