Exploring the Mechanism of Hepatotoxicity Induced by Dictamnus dasycarpus Based on Network Pharmacology, Molecular Docking and Experimental Pharmacology.

The root bark of Dictamnus dasycarpus Turcz is a traditional Chinese medicine, Dictamni Cortex (DC), which is mainly used in the clinical treatment of skin inflammation, eczema, rubella, rheumatism, and gynecological inflammation. Unexpectedly, there are some cases of liver injury after the administration of DC. However, the mechanism of hepatotoxicity remains ambiguous. The aim of this study was to explore the mechanism and substance bases of DC hepatotoxicity based on network pharmacology and molecular docking, verified through pharmacological experiments. Partial prototype components and metabolites in vivo of quinoline alkaloids from DC were selected as candidate compounds, whose targets were collected from databases. Network pharmacology was applied to study the potential hepatotoxic mechanism after correlating the targets of candidate compounds with the targets of hepatotoxicity. Molecular docking was simulated to uncover the molecular mechanism. Furthermore, the hepatotoxicity of the extract and its constituents from DC was evaluated in vivo and in vitro. We constructed the "potential toxic components-toxic target-toxic pathway" network. Our results showed that the targets of DC included CYP1A2 and GSR, participating in heterologous steroid metabolism, REDOX metabolism, drug metabolism, heterocyclic metabolic processes, the synthesis of steroid hormone, cytochrome P450 metabolism, chemical carcinogens and bile secretion pathways. In vitro and in vivo experiments displayed that DC could result in a decrease in GSH-Px and oxidative stress, simultaneously inhibiting the expression of CYP1A2 and inducing hepatotoxicity. These results further indicated the mechanism of hepatotoxicity induced by Dictamnus dasycarpus, providing a basic theory to explore and prevent hepatotoxicity in the clinical usage of Dictamnus dasycarpus.

Antimicrobial alkaloids from the root bark of Dictamnus dasycarpus.

Two new furoquinoline alkaloids, named 1'-oxo-isoplatydesmine (1) and demethoxyacrophylline (2), as well as 11 known alkaloids (3-13) were isolated from the root bark of Dictamnus dasycarpus Turcz. The structures of 1 and 2 were established by detailed spectroscopic elucidation, such as 1 D & 2 D NMR and HRMS, etc. The unexpected autoracemization of 1 was discussed based on the stereochemistry of reported dihydrofuroquinolines. Compounds 3-5 exhibited moderate inhibitory activities against Bacillus subtilis, Candida albicans, and Pseudomonas aeruginosa with MICs 32-64 µg/ml, revealing the active principles of D. dasycarpus for treating skin diseases in its traditional usage.

[Chemical constituents and pharmacological effects of Dictamni Cortex: a review].

Dictamni Cortex, the dried root bark of Dictamnus dasycarpus, has many chemical constituents, such as alkaloids, limonoids, flavonoids, sesquiterpenoids, glycosides, and steroids.It has the effects of anti-inflammation, anti-fungi, anti-arteriosclerosis, stopping bleeding, anti-cancer, neuroprotection, and antioxidation.The chemical constituents of Dictamni Cortex are the important material basis for its medicinal effects.This paper reviewed the chemical constituents and pharmacological activities of Dictamni Cortex and analyzed the research trend and present research progress on this medicinal, with a view to its further development and utilization.

Cytochrome P450-Mediated Bioactivation: Implication for the Liver Injury Induced by Fraxinellone, A Bioactive Constituent from Dictamni Cortex.

Fraxinellone, a furanoid, is one of the bioactive and potentially hepatotoxic constituents from Dictamnus dasycarpus Turcz, which is extensively spread throughout Asian countries. This herb was reported to cause liver injury in clinical application. However, the mechanism behind is still not fully understood. This study mainly focused on the hepatotoxicity of fraxinellone and the underlying mechanism. The current study demonstrated that fraxinellone resulted in a significant elevation of serum alanine aminotransferase and aspartate aminotransferase in a dose-dependent manner in mice after oral administration. Pretreatment with ketoconazole for three successive days could significantly alleviate the hepatotoxicity of fraxinellone. Considering that fraxinellone has a structural alert of furan ring, it is believed that the hepatotoxicity caused by fraxinellone required cytochrome P450-mediated bioactivation. Bioactivation studies were subsequently carried out in vitro and in vivo. Fraxinellone was metabolized into cis-enedial intermediate, an electrophile that was prone to react with glutathione or N-acetyl-lysine through 1,2- or 1,4-addition to form stable conjugates. Ketoconazole significantly inhibited the formation of the glutathione conjugates (M1 and M2) in microsomal incubation and similar finding was obtained in vivo. Phenotyping study indicated that CYP3A4 was the principal enzyme responsible for the bioactivation of fraxinellone. This study suggested that CYP3A4-mediated bioactivation plays an indispensable role in fraxinellone-induced hepatotoxicity. The work performed herein enables us to better understand the hepatotoxicity of fraxinellone as well as the mechanism behind.

Three new compounds from Dictamnus dasycarpus and their anti-inflammatory activities.

Three new compounds (1-3), named dasycarine G (1), dasycarether (2), and dasycarester (3), along with seven known compounds (4-10) obtained from the genus Dictamnus for the first time, were isolated from the root bark of Dictamnus dasycarpus. Their structures were elucidated on the basis of spectroscopic data (UV, IR, HR-ESI-MS, 1D and 2D NMR, and CD). In the in vitro assay, compounds 1, 5, 6, 9, and 10 exhibited NO inhibitory effects of LPS-induced BV-2 cells with IC50 values in the range of 10.4 µM to 27.2 µM.

[Network pharmacology study on potential active components in volatile oil of Dictamni Cortex].

There are many chemical components in the volatile oil of Dictamni Cortex. The complex network relationship of "component-target-disease" can be revealed by using the network pharmacology method, and the mechanism of the efficacy of Dictamni Cortex can be revealed. In this study, we used Swiss Target Prediction database to predict the target of action, STRING database to build protein interaction network, and Cytoscape software to build "component-target-disease" network. The results showed that the antibacterial, anti-inflammatory and antiallergic effects of Dictamni Cortex were closely related to the components of thymol methyl ether, elemenol, anethole, and the related targets of each component were cross-linked to play a multi-target pharmacodynamic role. This study laid a foundation for the study of the effective substance basis and quality control evaluation of the Dictamni Cortex, and provided a scientific basis for further revealing its mechanism.

[Clinical cases and experimental evaluation of liver injury related to anti-psoriasis Keyin Pills].

Keyin Pills is a kind of traditional Chinese medicine for the treatment of psoriasis, but it has been reported that it can cause serious liver injury. In this paper, we used the integrated evidence chain method to retrieve and reevaluate the adverse drug reaction database, CNKI literature and cases of liver injury relating to Keyin Pills in specialist hepatology hospitals. We screened out 23 cases with the causal relationship of the possible grade and above. Among them, 11 cases showed the positive causal relationship only with Keyin Pills, accounting for 47.83%, suggesting that there was objective liver injury caused by Keyin Pills. The incubation period of liver injury caused by Keyin Pills is 1-90 days, and the cumulative dosage span is 20-1 800 g. There were obvious individual diffe-rences. There was no relationship between liver injury as well as dose and course of treatment, suggesting that Keyin Pills could induce immune idiosyncratic liver injury. Furthermore, based on the liver injury model induced by immunological stress, it was confirmed that Keyin Pills could induce acute liver injury in a dose-dependent manner in rats with immunological stress. The toxic dose(14 g·kg~(-1)) of a single dose was 6.7 times of the clinical equivalent dose, and had no significant effect on the biochemical index of liver function and histopathology in normal rats. Decomposition experiments showed that Dictamnus dasycarpus in Keyin Pills is the main medicinal flavor that causes special liver injury, and the other three medicines had neither liver injury nor compatibility attenuation effect. The results suggest that clinical medication shall pay attention to the risk of liver injury caused by Keyin Pills in patients with immunological stress.

Anti-inflammatory effects of a methanol extract of Dictamnus dasycarpus Turcz. root bark on imiquimod-induced psoriasis.

BACKGROUND: The root bark of Dictamnus dasycarpus Turcz. has been successfully used for the treatment of inflammatory skin conditions such as eczema and pruritus. However, the anti-psoriatic effect of this plant has not until now been investigated. METHODS: The aim of this project was to investigate whether a methanol extract of Dictamnus dasycarpus Turcz. root bark (MEDD) can be used as a therapeutic agent for psoriasis in C57BL/6 mice model of imiquimod (IMQ)-induced psoriasis. IMQ and MEDD was applied to mouse skin continuously for 7 days. The skin phenotype and the levels of inflammatory cytokines, such as interferon (IFN)-γ and interleukin (IL)-17, were analyzed. The immune cell population was determined by flow cytometry, and STAT1 and 3 protein levels were measured. RESULTS: An alleviation of scaly skin phenotype, immune cell infiltration in the dermis, and epidermal hyperplasia was observed after daily MEDD treatment in the lesion-affected area. It was also found that MEDD reduced IL-17 cytokine levels decreased by 44.37% (p < 0.05), the number of IL-17-producing Th17 cells and γδT cells, and the size of the Th1 population secreting IFN-γ decreased by 45.98, 62.21, and 44.42%, respectively (p < 0.05), compared with the vehicle control group. STAT3 signals, associated with IL-17 are also reduced by MEDD. CONCLUSIONS: An anti-psoriatic effect of MEDD was observed, as determined by decreased skin inflammation, reduced number of inflammatory cytokines, and a smaller population of inflammatory cells. These results contribute to the validation of the use of MEDD in the treatment of psoriasis.

[Analysis of epidemiological characteristics of drug induced liver injury associated with Baixianpi Preparations].

A retrospective study was performed in drug-induced liver injury(DILI) cases associated with Dictamni Cortex(Baixianpi,BXP) Preparations,which were treated at grade Ⅲ class A liver disease hospitals from 2008 to 2016 and spontaneously reported for adverse reactions between 2012 and 2016 at HILI Cloud(hilicloud.net). The results showed 25 DLII cases associated with BXP Preparations treated at grade Ⅲ class A liver disease hospitals during the 9 years,including only 14 cases in line with the clinical diagnostic criteria of Guidelines for the Diagnosis and Treatment of Herb-Induced Liver Injury. And 74 DILI cases associated with BXP Preparations spontaneously reports adverse reactions,and 18. 92% of them had unreasonable medication,including polypharmacy(21. 43%),overdose(28. 57%) and repeated dosage(50%). And 47 DILI cases used BXP Preparations to treat psoriasis and vitiligo(a total of59. 57%). The time range of taking BXP Preparations until liver injury occurred was 1-366 d,with the median of 18 d. The dose of BXP Preparations was estimated to be 0. 09-12 g·d-1. And the cumulative dosage of taking drugs until liver injury occurred was 1. 1-336 g. Obvious associations with time-toxicity as well as quantity-toxicity could not be found based on the wide range of time-toxicity relations and quantity-toxicity relations. On the basis of the study,we found that DILI cases associated with BXP Preparations commonly occurred in patients with immune diseases,such as psoriasis and vitiligo,indicating specific individual differences. The results suggested that DILI cases associated with BXP Preparations would be correlated with the property of idiosyncratic drug-induced liver injury. In conclusion,the risk of liver injury clinically caused by BXP Preparations should be paid more attention,and the studies on the mechanism of idiosyncratic drug-induced liver injury must be enhanced,and those on risk factors,like irrational drug use,should be strengthened. Moreover,the evaluation of the risk-to-benefit ratio is supposed to be performed for the sake of improving the risk prevention and control standards for BXP preparations,and ensuring safe and rational clinical application of BXP Preparations.

The inhibitory effect of kokusaginine on the growth of human breast cancer cells and MDR-resistant cells is mediated by the inhibition of tubulin assembly.

The emergence of multidrug resistance (MDR) is a significant challenge in breast carcinoma chemotherapy. Kokusaginine isolated from Dictamnus dasycarpus Turcz. has been reported to show cytotoxicity in several human cancer cell lines including breast cancer cells MCF-7. In this study, kokusaginine showed the potent inhibitory effect on MCF-7 multidrug resistant subline MCF-7/ADR and MDA-MB-231 multidrug resistant subline MDA-MB-231/ADR. Kokusaginine markedly induced apoptosis in a concentration-dependent manner in MCF-7/ADR cells. Furthermore, kokusaginine reduced P-gp mRNA and protein levels, and suppressed P-gp function especially in MCF-7/ADR cells. In addition, kokusaginine showed to inhibit tubulin assembly and the binding of colchicine to tubulin by binding directly to tubulin and affects tubulin formation in vitro. Taken together, these results support the potential therapeutic value of kokusaginine as an anti-MDR agent in chemotherapy for breast carcinoma.

Eudesmane-Type Sesquiterpene Glycosides from Dictamnus dasycarpus Turcz.

Eudesmane-type sesquiterpenes have been reported to exhibit varieties of biological activities. During the process of investigating this kind of natural product from the root bark of Dictamnus dasycarpus Turcz., 13 eudesmane-type sesquiterpene glycosides including six new isolates, named as dictameudesmnosides A1 (1), A2 (2), B (3), C (4), D (5), and E (6), together with seven known ones (7-13), were obtained. Herein, their structures were determined by the analysis of physical data, spectroscopic analysis, and chemical methods. The existence of α-configuration glucose units in their structures (1-5, 8) is not very common in natural glycosidic components. Meanwhile, compounds 3-5, 7, and 9-13 displayed TG accumulation inhibitory effects on HepG2 cells.

Subchronic Toxicity Studies of Cortex Dictamni Extracts in Mice and Its Potential Hepatotoxicity Mechanisms in Vitro.

Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages (2.3, 4.6, or 9.2 g/kg/day, p.o.) in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines.

[Chemical constituents from root barks of Dictamnus dasycarpus and their cytotoxic activities].

Nineteen compounds, including kihadanin D (1), obacunone (2), kihadanin A (3), kihadanin B (4), kihadanin C (5), limonin (6), evodol (7), fraxinellone (8), furo[2,3-b]quinolin-4-ol (9), preskimmianine (10), ifflaiamine (11), dictamnol (12), naringenin (13), diosmetin (14), wogonin (15), scopoletin (16), cleomiscosin A (17), apocynin (18), and methyl pyroglutamate (19), were isolated from the methanol extract of the root barks of Dictamnus dasycarpus by using various column chromatographies. Their chemical structures were extensively determined on basis of UV, IR, NMR, MS, and CD spectroscopic data analyses. Among them, 1 is a new limonoid, 9 was isolated from plant kingdom for the first time, 11, 13-14 and 17-19 were obtained from the genus Dictamnnus for the first time. Cytotoxicities of compounds 1-18 were tested, and the results indicated that 1 exhibited cytotoxicities against three human cancer cell lines MDA-MB-231, A549 and HT29 with IC58 values of 16.22, 21.72 and 31.06 µmol·L⁻¹, respectively.

[Mass spectrometry guided strategy based on feature fragment ions for guided-separation on quinoline alkaloids from root barks of Dictamnus dasycarpus].

The root bark of Dictamnus dasycarpus is one of common traditional Chinese medicines (TCMs). Quinoline alkaloids are one of the main active substances in this TCM and possess many biological activities including anti-titumor, anti-inflammation, anti-bacteria, anti-oxidation, and anti-platelet aggregation activities. In this study, eight quinoline alkaloids 1-8 were firstly separated from the root barks of D. dasycarpus. It was difficult to isolate more quinoline alkaloids from the remaining fraction 8 in D. dasycarpus by this conventional chemical separation, so the target analysis method combined LC-MS guided-separation of quinoline alkaloids from fraction 8 was established. MS/MS fragmentation patterns of eight quinoline alkaloids reference standard compounds 1-8 were studied by ultra-performance liquid chromatography-electrospary ionization-mass spectrometry (UPLC-ESI-MS/MS). Based on the feature fragment ion m/z 200, the parent ion scan mode was established for the target analysis of quinoline alkaloids in fraction 8. Finally, 8-methoxyflindersine (9) and N-metilatanina (10) were discovered and isolated quickly from fraction 8 guided by LC-MS, and their structures were identified by NMR and MS. Among them, compound 10 was isolated from the genus Dictamnus for the first time. These results indicated that this method is not only quick and sensitive for analyzing the quinoline alkaloids, but also to effectively guided-separate this kind of alkaloids in the root barks of D. dasycarpus.

Rapid identification and global characterization of multiple constituents from the essential oil of Cortex Dictamni based on GC-MS.

The root of Dictamnus dasycarpus Turcz., also known as Cortex Dictamni, is a Chinese herbal medicine that has been commonly used in the treatment of inflammation, microbial infection, cancer, and other diseases in China for thousands of years. Recently, the essential oil of Cortex Dictamni has been widely studied, and a large number of volatile constituents have been discovered. However, the research of the essential oil of Cortex Dictamni in vivo remains unknown, especially the constituents absorbed into blood after oral administration. Hence, a sensitive and rapid method using gas chromatography with mass spectrometry combined with MassHunter software and the National Institute of Standards and Technology 2014 database was used to investigate the absorbed components in rat serum after oral administration of the essential oil of Cortex Dictamni. With the established method, a total of 36 compounds were screened and identified in the essential oil of Cortex Dictamni based on the mass spectrometry data and compound database. Among them, eight compounds, elemol, thymol methyl ether, ß-eudesmol, ß-cyclocostunolid, guaiazulene, trans-4-hydroxystilbene, ethyl oleate, and monoelaidin, were tentatively characterized in rat serum. This work demonstrated that the established method proved to be a powerful technique for rapid, simple, reliable, and automated identification of bioactive components of herbal medicine.

Radicol, a Novel Trinorguaiane-Type Sesquiterpene, Induces Temozolomide-Resistant Glioma Cell Apoptosis via ER Stress and Akt/mTOR Pathway Blockade.

Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system (CNS) in adults. Multidrug resistance (MDR) results in undesirable prognosis during GBM chemotherapy. In this study, we determined that Radicol (RAD), a novel trinorguaiane-type sesquiterpene originally isolated from the root of Dictamnus radicis Cortex, exhibited potently cytotoxic effect on temozolomide (TMZ)-resistant GBM cell lines in a dose-dependent manner. Radicol-induced apoptosis was confirmed with Hoechst 33342/propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labelling (TUNEL) staining. Studies investigating the mechanism revealed that RAD triggered an attenuation of protein disulphide isomerase (PDI) and induced the unmitigated unfolded protein response (UPR) and lethal endoplasmic reticulum (ER) stress. Simultaneously, we further demonstrated that RAD suppressed the activation of Akt/mTOR/p70S6K phosphorylation by up-regulating the induction of glycogen synthase kinase-3ß (GSK-3ß). These results established a link between RAD-induced ER stress and inhibition of the Akt/mTOR/p70S6K pathway, and the attenuation of PDI and activation of GSK-3ß might be the synergistic target of antineoplastic effects during RAD-induced apoptosis. These findings suggested that RAD, possessing multiple cytotoxicity targets, low molecular weight and high lipid solubility, could be a promising agent for the treatment of malignant gliomas. Copyright © 2017 John Wiley & Sons, Ltd.

[Dictamni Cortex powder-induced liver injury based on integrated evidence chain].

A typical clinical case of taking Dictamni Cortex(Baixianpi) powder was analyzed to study liver damage caused by Dictamni Cortex. Liver damage was diagnosed according to the integrated evidence chain method recommended by the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury. By analyzing clinical history and biochemistry and imaging examinations, underlying diseases, such as viral hepatitis, autoimmune liver disease and alcoholic liver disease, were excluded. Through the investigation of medication history, we made it clear that the patient only took Dictamni Cortex powder during the period, and thus suspected that the liver injury was induced by Dictamni Cortex. Furthermore, the quality of the drug was tested, and the results showed it was consistent with the quality standard of Chinese Pharmacopoeia. DNA barcoding showed that the drug was 100% similar with Dictamnus dasycarpus. Moreover, exogenous harmful substances and chemical drug additions were tested, and the results showed that the content of heavy metal, pesticide residues and microbial toxin were consistent with the required standards, and no chemical drug additions were found in Agilent Fake TCM-Drugs database. In summary, we confirmed that the clinical case of drug-induced liver injury was induced by D. dasycarpus with the dose of 15 g•d⁻¹, which exceeded the prescribed amount of Chinese Pharmacopoeia. According to the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury, the case of drug-induced liver injury induced by D. dasycarpus was confirmed, which provided a direct and reliable evidence for the study of risk of liver injury induced by D. dasycarpus and its relevant preparations.

Antiproliferative glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus.

Four new glabretal-type triterpenoids, dictabretols A-D (1-4), were isolated by activity-guided fractionation from the root bark of Dictamnus dasycarpus T. (Rutaceae) using an in vitro antiproliferative assay on T cells using splenocytes. The structures of these compounds were determined by spectroscopic methods, including 2D NMR experiments. Compounds were evaluated for their immunosuppressive activity on T cells and demonstrated inhibition of proliferation of activated T cells, up to IC50 of 1.5µM.

Contact Toxicity and Repellency of the Essential Oil of Dictamnus dasycarpus Roots from China against Two Stored-Product Insects.

During our screening program for new agrochemicals from traditional medicinal herbs, Dictamnus dasycarpus was found to possess contact toxicity and repellent activity against the cigarette beetle Lasioderma serricorne and the booklouse Liposcelis bostrychophila. The essential oil obtained by hydrodistillation of D. dasycarpus roots was characterized by GC-FID and GC/MS analyses, and the main components identified were syn-7-hydroxy-7-anisylnorbornene (1, 49.9%), 1,3,4,5,6,7-hexahydro-2H-inden-2-one (2, 11.6%), 5,6-diethenyl-1-methylcyclohexene (3, 7.4%), and 3,9-dimethyltricyclo[4.2.1.1(2,5)]dec-3-en-9-ol (4, 6.35%). The D. dasycarpus root oil exhibited strong contact toxicity against L. serricorne adults and L. bostrychophila, with LD50 values of 12.4 µg/adult and 27.2 µg/cm(2), respectively. Moreover, the essential oil also showed strong repellency against both stored-product insects tested.

Limonin, a Component of Dictamni Radicis Cortex, Inhibits Eugenol-Induced Calcium and cAMP Levels and PKA/CREB Signaling Pathway in Non-Neuronal 3T3-L1 Cells.

Limonin, one of the major components in dictamni radicis cortex (DRC), has been shown to play various biological roles in cancer, inflammation, and obesity in many different cell types and tissues. Recently, the odorant-induced signal transduction pathway (OST) has gained attention not only because of its function in the perception of smell but also because of its numerous physiological functions in non-neuronal cells. However, little is known about the effects of limonin and DRC on the OST pathway in non-neuronal cells. We investigated odorant-stimulated increases in Ca(2+) and cAMP, major second messengers in the OST pathway, in non-neuronal 3T3-L1 cells pretreated with limonin and ethanol extracts of DRC. Limonin and the extracts significantly decreased eugenol-induced Ca(2+) and cAMP levels and upregulated phosphorylation of CREB and PKA. Our results demonstrated that limonin and DRC extract inhibit the OST pathway in non-neuronal cells by modulating Ca(2+) and cAMP levels and phosphorylation of CREB.