Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Ameliorative Effect of Areca Nut Polyphenols on Adverse Effects Induced by Lipopolysaccharides in RAW264.7 Cells.

In Asian regions, areca nuts are tropical fruits that are extensively consumed. The areca nut contains a lot of polyphenols and its safety is unknown. In this research, we investigated the effects of lipopolysaccharides (LPS) and areca nut polyphenols (ANP) on normal RAW264.7 cells. The results showed that LPS stimulated adverse effects in normal cells by affecting cytokine production. The GO analysis results mainly affected DNA repair, cell division, and enzyme activities. In the KEGG analysis results, the NOD-like receptor signaling pathway, which is related to NF-κB, MAPK, and the pro-inflammatory cytokines, is the most significant. In the protein-protein interaction network (PPI) results, significant sub-networks in all three groups were shown to be related to cytokine-cytokine receptor interaction. Collectively, our findings showed a comprehensive understanding of LPS-induced toxicity and the protective effects of ANP by RNA sequencing.

Bioactive Components of Areca Nut: An Overview of Their Positive Impacts Targeting Different Organs.

Areca catechu L. is a widely cultivated tropical crop in Southeast Asia, and its fruit, areca nut, has been consumed as a traditional Chinese medicinal material for more than 10,000 years, although it has recently attracted widespread attention due to potential hazards. Areca nut holds a significant position in traditional medicine in many areas and ranks first among the four southern medicines in China. Numerous bioactive compounds have been identified in areca nuts, including alkaloids, polyphenols, polysaccharides, and fatty acids, which exhibit diverse bioactive functions, such as anti-bacterial, deworming, anti-viral, anti-oxidant, anti-inflammatory, and anti-tumor effects. Furthermore, they also display beneficial impacts targeting the nervous, digestive, and endocrine systems. This review summarizes the pharmacological functions and underlying mechanisms of the bioactive ingredients in areca nut. This helps to ascertain the beneficial components of areca nut, discover its medicinal potential, and guide the utilization of the areca nut.

Towards a replacement therapy for stimulant betel quid dependence: A proof of concept study.

Stimulant betel quid (SBQ) containing Piper betle leaf (L), green unripe Areca catechu nut (AN) and the alkalizing agent, slaked lime, is an addictive, carcinogenic stimulant, with no pharmacotherapy, chewed by millions of people in the Asia/Pacific region. We compared the in vivo physiological profile of chewing (1) non-stimulant P. betle leaf+AN (LAN), (2) SBQ utilizing slaked lime and (3) a novel SBQ utilizing Mg(OH)2 , as an alkalizing agent, by measuring physiological parameters of intoxication and these were correlated with in vitro levels of alkaloids measured by UHPLC-MS/MS. Chewing LAN, which contains high levels of arecoline, had no stimulatory physiological effect. Chewing SBQ containing slaked lime or novel SBQ containing Mg(OH)2 , induced equivalent stimulatory physiological responses. In vitro, slaked lime hydrolyzed muscarinic esters in LAN while Mg(OH)2 did not. The physiological stimulation induced by chewing both SBQ and the lack of physiology to chewing LAN can be explained by changes in lipid solubility of phytochemicals induced by mouth pH during chewing of basic SBQ or acidic LAN. Since antiquity people have added slaked lime to SBQ to enhance absorption of phyto-chemicals across oral membranes to stimulate physiology. The same physiological changes can be induced by substituting slaked lime for less physically and chemically destructive bases. If attitudes regarding SBQ dependence can advance towards the more progressive attitudes already used to help smokers quit tobacco, modern chemistry has the potential to make chewing SBQ safer and quitting programs may become more accessible and efficacious.

Comparative metabolomics study on areca nut from China and Southeast Asia (Thailand and Indonesia).

INTRODUCTION: Areca nut is an economic crop and an important component in traditional Chinese medicine (TCM) and ethnomedicine. The crop is rich in alkaloids and flavonoids. Most previous studies have focused on the chemical components, especially alkaloids, in crops from certain areca nut-producing areas. OBJECTIVE: The purpose of this study was to compare the differences in areca nut seeds in two main cultivation areas, identify differential metabolites, and evaluate seed quality in different production areas. METHODS: A widely targeted metabolomics method based on ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS), combined with the TCM systems pharmacology (TCMSP) database and multivariate statistical analysis, was used in this study to maximise the differentiation between quality characteristics of areca nut seeds from China and Southeast Asian regions. RESULTS: Altogether, 1031 metabolites were identified in areca nut seeds; by querying the TCMSP database, 375 metabolites were identified as the main active ingredients. Moreover, the research showed that the metabolic profiles of areca nut seeds from China (ASCN) and Southeast Asia (ASSA) exhibit significant differences, and the difference is mainly reflected in 318 compounds. The relative content of 146 metabolites in ASCN was significantly higher than that in ASSA. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) comparative analysis, areca nut seed metabolites in Chinese production areas were determined to have a wider metabolic pathway. CONCLUSION: The areca nut seeds from cultivation areas possess many metabolites that are beneficial for health, including alkaloids, amino acids, phenolic acids, and lipids. Thus, compared with ASSA, ASCN have a higher medicinal value. This study provides a direction for the subsequent development and utilisation of areca nut seeds.

The Controversial Roles of Areca Nut: Medicine or Toxin?

Areca nut (AN) is used for traditional herbal medicine and social activities in several countries. It was used as early as about A.D. 25-220 as a remedy. Traditionally, AN was applied for several medicinal functions. However, it was also reported to have toxicological effects. In this review article, we updated recent trends of research in addition to acquire new knowledge about AN. First, the history of AN usage from ancient years was described. Then, the chemical components of AN and their biological functions was compared; arecoline is an especially important compound in AN. AN extract has different effects caused by different components. Thus, the dual effects of AN with pharmacological and toxicological effects were summarized. Finally, we described perspectives, trends and challenges of AN. It will provide the insight of removing or modifying the toxic compounds of AN extractions for enhancing their pharmacological activity to treat several diseases in future applications.

Assessment of Areca Nut Bioactivities in Western Diet-Induced Mice NAFLD Model.

The areca nut is often consumed as a chewing food in the Asian region. Our previous study revealed that the areca nut is rich in polyphenols with high antioxidant activity. In this study, we further assessed the effects and molecular mechanisms of the areca nut and its major ingredients on a Western diet-induced mice dyslipidemia model. Male C57BL/6N mice were divided into five groups and fed with a normal diet (ND), Western diet (WD), WD with areca nut extracts (ANE), areca nut polyphenols (ANP), and arecoline (ARE) for 12 weeks. The results revealed that ANP significantly reduced WD-induced body weight, liver weight, epididymal fat, and liver total lipid. Serum biomarkers showed that ANP ameliorated WD-enhanced total cholesterol and non-high-density lipoprotein (non-HDL). Moreover, analysis of cellular signaling pathways revealed that sterol regulatory element-binding protein 2 (SREBP2) and enzyme 3-hydroxy-3-methylglutaryld coenzyme A reductase (HMGCR) were significantly downregulated by ANP. The results of gut microbiota analysis revealed that ANP increased the abundance of beneficial bacterium Akkermansias and decreased the abundance of the pathogenic bacterium Ruminococcus while ARE shown the opposite result to ANP. In summary, our data indicated that areca nut polyphenol ameliorated WD-induced dyslipidemia by increasing the abundance of beneficial bacteria in the gut microbiota and reducing the expressions of SREBP2 and HMGCR while areca nut ARE inhibited this improvement potential.

Systematic Analysis of NRAMP Family Genes in Areca catechu and Its Response to Zn/Fe Deficiency Stress.

Areca catechu is a commercially important medicinal plant widely cultivated in tropical regions. The natural resistance-associated macrophage protein (NRAMP) is widespread in plants and plays critical roles in transporting metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu is quite limited. In this study, we identified 12 NRAMPs genes in the areca genome, which were classified into five groups by phylogenetic analysis. Subcellular localization analysis reveals that, except for NRAMP2, NRAMP3, and NRAMP11, which are localized in chloroplasts, all other NRAMPs are localized on the plasma membrane. Genomic distribution analysis shows that 12 NRAMPs genes are unevenly spread on seven chromosomes. Sequence analysis shows that motif 1 and motif 6 are highly conserved motifs in 12 NRAMPs. Synteny analysis provided deep insight into the evolutionary characteristics of AcNRAMP genes. Among the A. catechu and the other three representative species, we identified a total of 19 syntenic gene pairs. Analysis of Ka/Ks values indicates that AcNRAMP genes are subjected to purifying selection in the evolutionary process. Analysis of cis-acting elements reveals that AcNRAMP genes promoter sequences contain light-responsive elements, defense- and stress-responsive elements, and plant growth/development-responsive elements. Expression profiling confirms distinct expression patterns of AcNRAMP genes in different organs and responses to Zn/Fe deficiency stress in leaves and roots. Taken together, our results lay a foundation for further exploration of the AcNRAMPs regulatory function in areca response to Fe and Zn deficiency.

Transcriptome and targeted metabolomic integrated analysis reveals mechanisms of B vitamin accumulation in Areca catechu nut development.

Areca catechu is well known as a medicinal plant that has high nutritional and medicinal benefits. However, the metabolism and regulatory mechanism of B vitamins during areca nut development remain largely unclear. In this study, we obtained the metabolite profiles of six B vitamins during different areca nut developmental stages by targeted metabolomics. Furthermore, we obtained a panoramic expression profile of genes related to the biosynthetic pathway of B vitamins in areca nuts at different developmental stages using RNA-seq. In total, 88 structural genes related to B vitamin biosynthesis were identified. Furthermore, the integrated analysis of B vitamin metabolism data and RNA-seq data showed the key transcription factors regulating thiamine and riboflavin accumulation in areca nuts, including AcbZIP21, AcMYB84, and AcARF32. These results lay the foundation for understanding metabolite accumulation and the molecular regulatory mechanisms of B vitamins in A. catechu nut.

Optimization of synergic antibacterial activity of Punica granatum L. and Areca nut (P.G.L.A.N) extracts through response surface methodology.

The primary objective of this study was to evaluate the use of natural compounds as opposed to chemical preservatives. This study employed response methodology to evaluate the synergistic antibacterial effect of Areca nut and Punica granatum L. extract. Independent variables included extract type (Punica granatum L., Areca nut, and their mixture), solvent (water, ethanol, methanol), bacterial type (S. aureus, Salmonella, E. coli), and extract concentration (1, 10, 100 mg/L). The sensitivity was determined using the disk diffusion method, and the diameter of the inhibitory zone was measured. On the specified bacteria, the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of each extract were ascertained using the serial dilution method. This study revealed the existence of beneficial synergistic effects between the two extracts. Results indicated that the ethanolic extracts of Punica granatum L. and Areca nut had a synergistic effect on E. coli.

Integrated metabolomic and transcriptomic analysis revealed the flavonoid biosynthesis and regulation in Areca catechu.

INTRODUCTION: Flavonoids are active substances in many herbal medicines, and Areca catechu fruit (AF), an important component in traditional Chinese medicine (TCM), is rich in flavonoids. Different parts of AF, Pericarpium Arecae (PA) and Semen Arecae (SA), have different medicinal effects in prescription of TCM. OBJECTIVE: To understand flavonoid biosynthesis and regulation in AF. METHODOLOGY: The metabolomic based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PA and SA. RESULTS: From the metabolite dataset, we found that 148 flavonoids showed significant differences between PA and SA. From the transcriptomic dataset, we identified 30 genes related to the flavonoid biosynthesis pathway which were differentially expressed genes in PA and SA. The genes encoding the key enzymes in the flavonoid biosynthesis pathway, chalcone synthase and chalcone isomerase (AcCHS4/6/7 and AcCHI1/2/3), were significantly higher expressed in SA than in PA, reflecting the high flavonoid concentration in SA. CONCLUSIONS: Taken together, our research acquired the key genes, including AcCHS4/6/7 and AcCHI1/2/3, which regulated the accumulation of flavonol in AF. This new evidence may reveal different medicinal effects of PA and SA. This study lays a foundation for investigating the biosynthesis and regulation of flavonoid biosynthesis in areca and provides the reference for the production and consumption of betel nut.

Rapidly analyzing of ingredients during chewing and processing of areca nut using feature-based molecular networking.

As a traditional herbal medicine and food in China and many other Asian countries, the areca nut (Areca catechu L.) is not only widely used for the treatment of various diseases, but also popular as a chewing hobby. However, as a first-class carcinogen designated by IARC, clinical studies have shown that long-term chewing of areca nut is associated with oral mucosal diseases and even oral cancer. Moreover, the incidence of these diseases varies regionally, suggesting that it may be related to edible methods in different regions. In this study, UPLC-Q-TOF-MSE was combined with feature-based molecular networking to systematically characterise the chemical ingredients of areca nut. Based on these results, the ingredients of different edible parts and edible methods was rapidly compared. The compositional changes during the production process were also analysed. The obtained results provide a foundation for the scientific utilisation of areca nut.

Microwave-assisted synthesis of biodiesel by a green carbon-based heterogeneous catalyst derived from areca nut husk by one-pot hydrothermal carbonization.

In this study, we have synthesized a solid acid catalyst by areca nut husk using low temperature hydrothermal carbonization method. The fabricated catalyst has enhanced sulfonic actives sites (3.12%) and high acid density (1.88 mmol g-1) due to -SO3H, which are used significantly for effective biodiesel synthesis at low temperatures. The chemical composition and morphology of the catalyst is determined by various techniques, such as Fourier transform infrared (FTIR), powder X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET), Scanning electron microscope (SEM), Energy disruptive spectroscopy (EDS), Mapping, Thermogravimetric analysis (TGA), CHNS analyzer, Transmission electron microscopy (TEM), particle size analyzer, and X-ray photoelectron spectroscopy (XPS). Acid-base back titration method was used to determine the acid density of the synthesized material. In the presence of the as-fabricated catalyst, the conversion of oleic acid (OA) to methyl oleate reached 96.4% in 60 min under optimized conditions (1:25 Oleic acid: methanol ratio, 80 °C, 60 min, 9 wt% catalyst dosage) and observed low activation energy of 45.377 kJ mol-1. The presence of the porous structure and sulfonic groups of the catalyst contributes to the high activity of the catalyst. The biodiesel synthesis was confirmed by gas-chromatography mass spectrometer (GC-MS) and Nuclear magnetic resonance (NMR). The reusability of the catalyst was examined up to four consecutive cycles, yielding a high 85% transformation of OA to methyl oleate on the fourth catalytic cycle.

Centrifugation-Assisted Solid-Phase Extraction Coupled with UPLC-MS/MS for the Determination of Mycotoxins in ARECAE Semen and Its Processed Products.

Mycotoxins can occur naturally in a variety of agriculture products, including cereals, feeds, and Chinese herbal medicines (TCMs), via pre- and post-harvest contamination and are regulated worldwide. However, risk mitigation by monitoring for multiple mycotoxins remains a challenge using existing methods due to their complex matrices. A multi-toxin method for 22 mycotoxins (aflatoxin B1, B2, G1, G2, M1, M2; ochratoxin A, B, C; Fumonisin B1, B2, B3; 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, diace-toxyscirpenol, HT-2, T-2, deepoxy-deoxynivalenol, deoxynivalenol, neosolaniol, zearalenone, and sterigmatocystin) using centrifugation-assisted solid-phase extraction (SPE) clean-up prior to ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis for Arecae Semen and its processed products was developed and validated. Several experimental parameters affecting the extraction and clean-up efficiency were systematically optimized. The results indicated good linearity in the range of 0.1-1000 µg/kg (r2 > 0.99), low limits of detection (ranging from 0.04 µg/kg to 1.5 µg/kg), acceptable precisions, and satisfactory recoveries for the selected mycotoxins. The validated method was then applied to investigate mycotoxin contamination levels in Areca catechu and its processed products. The mycotoxins frequently contaminating Areca catechu were aflatoxins (AFs), and the average contamination level and number of co-occurring mycotoxins in the Arecae Semen slices (Binlangpian) were higher than those in commercially whole Arecae Semen and Arecae Semen Tostum (Jiaobinlang). Sterigmatocystin was detected in 5 out of 30 Arecae Semen slices. None of the investigated mycotoxins were detected in Arecae pericarpium (Dafupi). The results demonstrated that centrifugation-assisted SPE coupled with UHPLC-MS/MS can be a useful tool for the analysis of multiple mycotoxins in Areca catechu and its processed products.

Quantitative determination of major alkaloids in areca nut products by high-performance liquid chromatography coupled with ion mobility spectrometry.

Areca nut is a popular and addictive food as well as a traditional herbal medicine in many countries. Areca nut contains alkaloids including arecoline, guvacine, and arecaidine, which are the major bioactive compounds in areca products. Areca alkaloids can be carcinogenic, and thus sensitive and specific analytical methods are urgently desired for the identification and quantification of these compounds. High-performance liquid chromatography-based methods are often preferred, but areca alkaloids do not have chromophores, and detection using a traditional UV detector can be difficult. The complexity of areca sample extracts can also lead to the co-elution of peaks leading to poor quantitative performance. We report here high-performance liquid chromatography coupled with an ion mobility spectrometer for sensitive determination of areca alkaloids in various products including areca nut, areca nut products, and herbal oral liquid. An X-Bridge reversed-phase C18 column was used in the experiment and was combined with high-performance liquid chromatography coupled to an ion mobility spectrometer system. A custom-made adjustable post-column splitter acted as an interface between the high-performance liquid chromatography and the ion mobility spectrometer; it also acted as the electrospray ionization source. The mobile phase was methanol and 0.5% ammonium hydroxide. The results demonstrate that the splitter can afford a wide range of split ratios that match the ion mobility spectrometer ionization source while keeping the separation efficiency of high-performance liquid chromatography. Three major alkaloid compounds were then accurately determined using the resulting method without dativization steps. Many coeluted high-performance liquid chromatography peaks are effectively separated in the ion mobility spectrometer dimension, which in turn improved the quantification accuracy.

Genome-wide analysis of DNaseI hypersensitivity unveils open chromatin associated with histone H3 modifications after areca nut with lime exposure.

Research over the years revealed that precocious anaphase, securin overexpression, and genome instability in both target and nontarget cells are significantly associated with the increased risk of areca nut (AN) and lime-induced oral, esophageal, and gastric cancers. Further, hyperphosphorylation of Rb and histone H3 epigenetic modifications both globally and in the promoter region of the securin gene were demonstrated after AN + lime exposure. This study aims whether the extract of raw AN + lime relaxes chromatin structure which further facilitates the histone H3 epigenetic modifications during the initial phase of carcinogenesis. Three groups of mice (10 in each group) were used. The treated group consumed 1 mg/day/mice of AN extract with lime ad libitum in the drinking water for 60 days. The dose was increased by 1 mg every 60 days. Isolated nuclei were digested with DNaseI and 2 kb and below DNA was eluted from the agarose gel, purified and PCR amplified by using securin and GAPDH primers. Securin and E2F1 expression, pRb phosphorylation, and histone epigenetic modifications were analyzed by immunohistochemistry. The number of DNA fragments within 2 kb in size after DNaseI treatment was higher significantly in AN + lime exposed tissue samples than in the untreated one. The PCR result showed that the number of fragments bearing securin gene promoter and GAPDH gene was significantly higher in AN + lime exposed DNaseI-treated samples. Immunohistochemistry data revealed increased Rb hyperphosphorylation, upregulation of E2F1, and securin in the AN + lime-treated samples. Increased trimethylation of histone H3 lysine 4 and acetylation of H3 lysine 9 and 18 were observed globally in the treated samples. Therefore, the results of this study have led to the hypothesis that AN + lime exposure relaxes the chromatin, changes the epigenetic landscape, and deregulates the Rb-E2F1 circuit which might be involved in the upregulation of securin and some other proto-oncogenes that might play an important role in the initial phases of AN + lime mediated carcinogenesis.

Phenethoxy Derivatives with Anti-inflammatory Activities from the Betelnut Endophytic Trichoderma asperellum G10.

Eight new phenethoxy derivatives, trichoasperellins A-H (1-8), were isolated from the endophytic fungus Trichoderma asperellum G10 isolated from the medicinal plant Areca catechu L. The structures of these compounds were elucidated from spectroscopic data, J-based configurational analysis, and Mosher's methods. Compounds 1-4 and 6-8 bear one or two multioxidized C7 moieties with the same carbon skeleton. The carbon skeletons of compounds 6-8 are new, all containing three moieties connected via two acetal carbons similar to those of disaccharide glycosides. Compound 4 inhibited nitric oxide production with an IC50 value of 48.3 µM, comparable to that of the positive control indomethacin (IC50, 42.3 µM).

[Water migration and kinetics of Arecae Semen during moistening process].

In this study, low-field nuclear magnetic resonance(LF-NMR) and magnetic resonance imaging(MRI) were employed to analyze the water distribution, status, and migration in the moistening process of Arecae Semen. Peleg model was adopted to study the water absorption kinetics of Arecae Semen moistened at different water temperatures(10, 30, and 50 ℃). The Arecae Semen samples soaked at different water temperatures all contained four water states: binding water T_(21), non-flowing water T_(22), free water T_(23), and unbound water T_(24). Non-flowing water had the largest increase in peak area during the moistening process, followed by free water. The peak areas of non-flowing water, free water, and total water were correlated with the water content(P<0.01). Therefore, LF-NMR can quickly and non-destructively predict the water content of Arecae Semen during moistening. The peak area of non-flowing water and the content of free water were correlated with the content of arecoline in the soaking solution(P<0.01), which indicated that the faster flow of non-flowing water and more free water corresponded to more arecoline dissolved. The MRI images showed that the water migration pathway varied at different soaking temperatures, and the moistening degree obtained by this means was consistent with that obtained based on traditional experience. The rate constant K_1 fitted by Peleg model decreased with the increase in water temperature, while the capacity constant K_2 showed an opposite trend. The Arrhenius equation fitting of K_1 with temperature showed that the activation energy of Arecae Semen in the moistening process was 32.98 kJ·mol~(-1). LF-NMR/MRI can be used to analyze the water status and content and determine the end moisturing point of Arecae Semen. Peleg model can accurately describe the water absorption properties of Arecae Semen in the moistening process. The findings of this study can guide the moistening optimization and mechanism research of other seed Chinese medicinal materials.

Mongolian Medicine Areca Thirteen Pill (GY-13) Improved Depressive Syndrome via upregulating cAMP/PKA/CREB/BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Areca Thirteen Pill, also called Gao You-13 (GY-13), is a traditional Mongolian herbal formula and has been extensively used to treat depression in Mongolian areas, which belongs to Heyi disease in Mongolian medicine. Major depressive disorder is a serious psychiatric disease, only one-third of individuals with depression are responsive to current antidepressants in clinic. Growing attention has been attracted by traditional herbal medicines in fighting depression because they are considered safer alternatives to pharmacotherapy. AIM OF THE STUDY: To reveal the mechanism of GY-13 in the treatment of depression. MATERIALS AND METHODS: The rat depression model was established by chronic unpredictable mild stress (CUMS), and primary hippocampal neurons were used to construct a glutamate-induced excitotoxicity model. The antidepressant effect of GY-13 was then assessed by performing sucrose preference tests, open field tests, and body weight measurements on rats. The expression of cAMP and PKA, mRNA levels of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB), and hippocampal neuronal apoptosis were measured. RESULTS: The results indicate that GY-13 significantly improves depression-like behavior, rescues decreased cAMP， PKA, recovers the mRNA levels of CREB and BDNF, and increases the proliferative activity of hippocampus. In addition, blockade of PKA reverses the effects of GY-13 treatment on CREB mRNA, BDNF mRNA levels. In vitro, GY-13 treatment increased hippocampal proliferative activity and attenuated Glu-induced apoptosis of hippocampal neurons as well as reduced CREB mRNA and BDNF mRNA expression levels. CONCLUSIONS: Our research demonstrated that GY-13 treatment exerted a potent antidepressant action via activation of cAMP/CREB/BDNF signaling pathway, promoting proliferation, and suppressing apoptosis. This research provides molecular biological ground for developing GY-13 into a potent alternative for the intervention of depression.