(-)-Asarinin alleviates gastric precancerous lesions by promoting mitochondrial ROS accumulation and inhibiting the STAT3 signaling pathway.

BACKGROUND: (-)-Asarinin (Asarinin) is the primary component in the extract of the herb Asarum sieboldii Miq. It possesses various functions, including pain relief, anti-viral and anti-tuberculous bacilli effects, and inhibition of tumor growth. Gastric precancerous lesion (GPL) is a common but potentially carcinogenic chronic gastrointestinal disease, and its progression can lead to gastric dysfunction and cancer development. However, the protective effects of asarinin against GPL and the underlying mechanisms remain unexplored. METHODS: A premalignant cell model (methylnitronitrosoguanidine-induced malignant transformation of human gastric epithelial cell strain, MC cells) and a GPL animal model were established and then were treated with asarinin. The cytotoxic effect of asarinin was assessed using a CCK8 assay. Detection of intracellular reactive oxygen species (ROS) using DCFH-DA. Apoptosis in MC cells was evaluated using an annexin V-FITC/PI assay. We performed western blot analysis and immunohistochemistry (IHC) to analyze relevant markers, investigating the in vitro and in vivo therapeutic effects of asarinin on GPL and its intrinsic mechanisms. RESULTS: Our findings showed that asarinin inhibited MC cell proliferation, enhanced intracellular ROS levels, and induced cell apoptosis. Further investigations revealed that the pharmacological effects of asarinin on MC cells were blocked by the ROS scavenger N-acetylcysteine. IHC revealed a significant upregulation of phospho-signal transducer and activator of transcription 3 (p-STAT3) protein expression in human GPL tissues. In vitro, asarinin exerted its pro-apoptotic effects in MC cells by modulating the STAT3 signaling pathway. Agonists of STAT3 were able to abolish the effects of asarinin on MC cells. In vivo, asarinin induced ROS accumulation and inhibited the STAT3 pathway in gastric mucosa of mice, thereby halting and even reversing the development of GPL. CONCLUSION: Asarinin induces apoptosis and delays the progression of GPL by promoting mitochondrial ROS production, decreasing mitochondrial membrane potential (MMP), and inhibiting the STAT3 pathway.

Altitudinal Variation on Metabolites, Elements, and Antioxidant Activities of Medicinal Plant Asarum.

Asarum (Asarum sieboldii Miq. f. seoulense (Nakai) C. Y. Cheng et C. S. Yang) is a medicinal plant that contains asarinin and sesamin, which possess extensive medicinal value. The adaptation and distribution of Asarum's plant growth are significantly affected by altitude. Although most studies on Asarum have concentrated on its pharmacological activities, little is known about its growth and metabolites with respect to altitude. In this study, the physiology, ionomics, and metabolomics were investigated and conducted on the leaves and roots of Asarum along an altitude gradient, and the content of its medicinal components was determined. The results showed that soil pH and temperature both decreased along the altitude, which restricts the growth of Asarum. The accumulation of TOC, Cu, Mg, and other mineral elements enhanced the photosynthetic capacity and leaf plasticity of Asarum in high-altitude areas. A metabolomics analysis revealed that, at high altitude, nitrogen metabolism in leaves was enhanced, while carbon metabolism in roots was enhanced. Furthermore, the metabolic pathways of some phenolic substances, including syringic acid, vanillic acid, and ferulic acid, were altered to enhance the metabolism of organic acids. The study uncovered the growth and metabolic responses of Asarum to varying altitudes, providing a theoretical foundation for the utilization and cultivation of Asarum.

Asarinin inhibits immunological rejection via the Toll-like receptor-myeloid differentiation factor 88 signaling pathway in vitro.

Asarinin has been found to prolong allograft survival and inhibit post-transplant immune rejection via the Toll-like receptor (TLR) signaling pathway. However, the underlying mechanism is not completely understood. Therefore, elucidating the possible pathophysiological role of asarinin in the TLR signaling pathway is essential. Here, dendritic cells were isolated from Sprague-Dawley® rats and cultured with splenocytes from Wistar rats treated with asarinin, lipopolysaccharide (LPS), and/or dimethyl sulfoxide. mRNA expression of TLR-2, TLR-4, myeloid differentiation factor 88 (MyD88), and nuclear factor kB (NF-kB) was determined using real-time polymerase chain reaction. Interleukin (IL)-6 and IL-12 levels were examined using an enzyme-linked immunosorbent assay. LPS resulted in an increase in the expression of TLR-2 rather than TLR-4 and MyD88. Furthermore, it inhibited the secretion of IL-6 and IL-12. MyD88 can be silenced after lentiviral transduction, and LPS can activate MyD88, whereas asarinin can inhibit this kind of activation. The effect of LPS and asarinin on TLR-4 could only be achieved when MyD88 was not silenced by lentivirus transduction. Therefore, asarinin might suppress TLR-4-mediated activation via the MyD88-dependent pathway. Overall, asarinin has a pre-application effect in inhibiting graft rejection.

Kinetic modeling of the sesamin conversion into asarinin in the presence of citric acid loading on Hß.

In the present work, effects of reaction temperature, reactant concentration, catalyst loading, and rotation speed on the kinetics of sesamin conversion in a sesame oil system were studied by using citric acid loading on Hß zeolite (CA/Hß) as a catalyst. A kinetic model was built for sesamin conversion. The kinetic model fits correctly the experimental concentration of sesamin and asarinin ( R S ⁢ e ⁢ s ⁢ a ⁢ m ⁢ i ⁢ n 2 = 0.93 and R A ⁢ s ⁢ a ⁢ r ⁢ i ⁢ n ⁢ i ⁢ n 2 = 0.97). The sesamin conversion is an endothermic reaction (△H rIso = 3 4.578kJ/mol). The CA/Hß catalyst could be easily regenerated by calcination, and there was no obvious loss of catalytic activity when reused. Knowledge of the sesamin conversion is of great significance for guiding production and improving the value and nutrition of sesame oil. In a word, this study lays the foundation for the scale-up of the production of asarinin from sesame oil using CA/Hß as the catalyst.

Preparation and Characterization of Solid Acid Catalysts for the Conversion of Sesamin into Asarinin in Sesame Oil.

Asarinin, an isomer of sesamin, has attracted attention because it has stronger biological properties than sesamin. The research on the conversion of sesamin into asarinin is limited. In this study, solid acid catalysts were screened and applied to promote the conversion of sesamin into asarinin in sesame oil. The results showed that citric acid loaded on zeolite beta (CTAH) was the optimal catalyst for asarinin production among the prepared catalysts. Characterization showed that CTAH had the greatest pore volume, largest surface area and strongest acid content. Response surface methodology (RSM) was applied to optimize the reaction conditions for asarinin yield using CTAH. The optimal reaction conditions were as follows: temperature, 85 °C; time, 2.7 h; catalyst amount, 1.6%. The predicted and experimental values of asarinin yield were 50.79 and 51.80 mg/100 g, respectively. The peroxide value and color in sesame oil samples treated with CTAH were clearly improved. In short, CTAH is a solid acid catalyst with potential application in the industrial conversion of sesamin into asarinin and in the improvement of sesame oil.

Effect of Asarinin on survival time after heart transplantation and anti-immune rejection of spleen and peripheral blood in rats / 中华器官移植杂志

Objective:To investigate the effect of Asarinin on the survival time of transplanted heart after allogeneic heterotopic heart transplantation and to further verify the anti-immune rejection effect of Asarinin in spleen and peripheral blood.Methods:Using 64 Wistar rats as donors, 64 SD rats as recipients to establish the allogeneic heterotopic heart transplantation model in rats.After successful transplantation, 64 rats were use simple randomization divided into control group, cyclosporine A(CsA) group, Asarinin group and half CsA + half Asarinin group with 16 rats in each group.CsA group was given 5 mg/kg by gavage; Asarinin group was given 25 mg/kg; half dose group was given CsA 2.5 mg/kg+ Asarinin 12.5 mg/kg and the control group was given the same volume of normal saline by gavage.After administration for 1 week, half of them were used to observe the survival time.The other half of the rats were fully anesthetized with chloral hydrate, spleen and peripheral blood were taken.Half of the spleen was taken to observe the slices under the microscope.The other half of spleen was used RT-PCR to detect the relative expression of IFN-γ and IL-4.The expression of co-stimulatory molecules CD80, CD86 and CD40 in peripheral blood were detected by flow cytometry.Results:Survival time of transplanted heart was control group (8.4±0.9), CsA group (30.5±8.3), Asarinin group (16.5±4.3) and half-dose group (26.1±5.2) days.Compared with control group, survival time of heart transplantation became prolonged in all groups and the difference was statistically significant ( P<0.05). HE staining of splenic tissue showed that, as compared with control group, the injury of each group was alleviated.The relative expression of IFN-γ in spleen was control group (1.055±0.083), CsA group (0.396±0.038), Asarinin group (0.833±0.094) and half-dose group (0.862±0.104). The last three groups were lower than control group and the difference was statistically significant ( P<0.05). The relative expression of IL-4 in spleen was control group (1.429±0.234), CsA group (3.808±0.729), Asarinin group (2.209±0.306) and half-dose group (2.323±0.321). The last three groups all spiked as compared with control group and the difference was statistically significant ( P<0.05). The expressions of CD80, CD86 and CD40 in peripheral blood were control group (98.21±0.54), (85.78±0.89) and (96.36±0.66), CsA group (89.26±0.36), (56.86±2.32) and (88.11±1.61), Asarinin group (94.19±0.47), (79.01±1.12) and (87.86±1.67) and half-dose group (94.87±0.74), (80.81±0.98) and (89.71±0.97) respectively.The last three groups were lower than control group and the difference was statistically significant ( P<0.05). Conclusions:Asarinin can prolong the survival time of transplanted heart after allogeneic heterotopic heart transplantation in rats, inhibit the immune injury of spleen after allogeneic heterotopic heart transplantation in rats, decrease IFN-γ in spleen, increase IL-4 in spleen and inhibit the expression of peripheral blood costimulatory molecules CD80, CD86 and CD40.

In Silico Investigation of Phytoconstituents of Medicinal Herb 'Piper Longum' Against SARS-CoV-2 by Molecular Docking and Molecular Dynamics Analysis.

Unavailability of treatment for the SARS-CoV-2 virus has raised concern among the population worldwide. This has led to many attempts to find alternative options to prevent the infection of the disease, including focusing on vaccines and drugs. The use of natural products and herbal extracts can be a better option in beating the virus and boosting up immunity. In the present paper, we have done a systematic in silico study of papain-like protease of COVID-19 virus with the chemical constituents of herbal plant Piper Longum. Screening of the pharmacokinetic properties is done with thirty-two phytoconstituents of Piper Longum which help us in selecting the most active components of the plant. After selection molecular docking is performed with Aristololactam (C17H11NO4), Fargesin (C21H22O6), l-asarinin (C20H18O6), Lignans Machilin F (C20H22O5), Piperundecalidine (C23H29NO3), and Pluviatilol (C20H20O6). Molecular dynamic (MD) is also performed with the inhibitor-receptor complex which suggest significant inhibition and a stable complex of I-Asarinin with PLpro. Docking scores and simulation results suggest that I-Asarinin can act as a potential drug like candidate against COVID-19.

A novel study on the immunomodulatory effect of umbilical cord derived mesenchymal stem cells pretreated with traditional Chinese medicine Asarinin.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the key for the treatment of malignant hematological diseases, and acute graft-versus-host disease (aGVHD) that might occur after allogenic transplantation can be life threatening and promote disease recurrence. GVHD damages the various parts of the body by upregulating T helper 1 cytokines (Th1) cytokines and stimulating CD4、CD8 + T cells. GVHD can exhibit significant immunoregulatory effects, but could be easily affected by the mesenchymal stem cells (MSC) environment, and hence the MSC immunosuppressive effects on GVHD remain unpredictable. Hence, to better understand the role of MSC in the prevention and treatment of GVHD, umbilical cord derived mesenchymal stem cells (UC-MSC) were pre-treated with Chinese medicine Asarinin and IFN-γ. In the mix lymphocyte reaction, we found that Asarinin pre-treated UC-MSC can exert significantly greater inhibition towards the proliferation of CD4 and CD8 + T cells, down-regulate Th1 type cytokines, up-regulate Th2 type cytokines, and reduce the inflammatory damage to liver, lung and intestine of aGVHD mice model. Moreover, Asarinin can cooperate with IFN-γto promote UC-MSC to secrete indoleamine 2,3-dioxygenase (IDO). Our findings establish that Asarinin pre-treated UC-MSC can significantly promote the immunosuppressive effects of MSC on aGVHD after hematopoietic stem cell transplantation.

Full-length transcriptome analysis and identification of genes involved in asarinin and aristolochic acid biosynthesis in medicinal plant Asarum sieboldii.

Asarum sieboldii, a well-known traditional Chinese medicinal herb, is used for curing inflammation and ache. It contains both the bioactive ingredient asarinin and the toxic compound aristolochic acid. To address further breeding demand, genes involved in the biosynthetic pathways of asarinin and aristolochic acid should be explored. Therefore, the full-length transcriptome of A. sieboldii was sequenced using PacBio Iso-Seq to determine the candidate transcripts that encode the biosynthetic enzymes of asarinin and aristolochic acid. In this study, 63 023 full-length transcripts were generated with an average length of 1371 bp from roots, stems, and leaves, of which 49 593 transcripts (78.69%) were annotated against public databases. Furthermore, 555 alternative splicing (AS) events, 10 869 long noncoding RNAs (lncRNAs) as well as their 11 291 target genes, and 17 909 simple sequence repeats (SSRs) were identified. The data also revealed 97 candidate transcripts related to asarinin metabolism, of which six novel genes that encoded enzymes involved in asarinin biosynthesis were initially reported. In addition, 56 transcripts related to aristolochic acid biosynthesis were also identified, including CYP81B. In summary, these transcriptome data provide a useful resource to study gene function and genetic engineering in A. sieboldii.

Simultaneous determination of asarinin, ß-eudesmol, and wogonin in rats using ultraperformance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies following administration of standards and Gumiganghwal-tang.

Asarinin, ß-eudesmol, and wogonin have common antiangiogenic activities and have the potential for use in chemotherapy. Besides, they are multivalent substances that are combined in various herbal medicines. The purpose of this study was to develop a method for simultaneous analysis of asarinin, ß-eudesmol, and wogonin, which are representative pharmacological components of Asarum heterotropoides, Atractylodes lancea, and Scutellaria baicalensis, respectively, in rat biosamples using ultraperformance liquid chromatography-tandem mass spectrometry. The three components were separated using 5 mm aqueous ammonium acetate containing 0.1% formic acid and acetonitrile as a mobile phase, equipped with a KINETEX core-shell C18 column. The analysis was quantitated on a triple-quadrupole mass-spectrometer employing electrospray ionization, and operated in the multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma, urine, and feces constituents. The developed analytical method satisfied international guidance criteria and could be successfully applied to the pharmacokinetic (PK) studies evaluating oral bioavailability of asarinin, ß-eudesmol, and wogonin after oral and intravenous administration and their urinary and fecal excretion ratios after oral administration to rats. Furthermore, the analysis was extended to PK studies following oral administration of Gumiganghwal-tang. This study was the first simultaneous analysis of the aforesaid three constituents in rat plasma, urine, and feces that also determined their PK parameters.

TlR2 and TlR4 are involved in the treatment of rheumatoid arthritis synovial fibroblasts with a medicated serum of asarinin through inhibition of Th1/Th17 cytokines.

Asarinin is one of the main active chemical components isolated from Xixin, a Chinese medicine. To investigate the role of asarinin in rheumatoid arthritis (RA), the present study investigated the effect of an asarinin-medicated serum on human fibroblast-like synoviocytes in vitro. An asarinin-medicated serum was generated and analyzed by high-performance liquid chromatography. Fibroblast-like synoviocytes were isolated from patients with osteoarthritis and RA. The third generation of the rheumatoid synoviocytes was used in the experimental research and the third generation of osteoarthritic synoviocytes was used as control cells. Trypan blue staining was performed to detect the viability of RA synovial fibroblasts (RASFs). ELISA, reverse transcription-quantitative (RT-q) PCR and western blotting were also performed to detect the expression of various cytokines. Additionally, RT-qPCR was employed to detect Toll-like receptor (TLR) 2 and TLR4. The results revealed that medicated asarinin serum inhibited the viability of RASFs in a dose- and time-dependent manner. The serum also suppressed the expression of interleukin (IL)-17A, tumor necrosis factor-α, interferon-γ, IL-6, TLR2 and TLR4. The inhibitory effect of asarinin drug serum on RASFs may be achieved by inhibition of T helper cell (Th)1/Th17 cytokines through suppression of TLR2 and TLR4.

(-)-Asarinin inhibits mast cells activation as a Src family kinase inhibitor.

As one of the major global health issues, allergic disease represents a considerable burden both on individual patients and public health. (-)-Asarinin (Asa), a lignan isolated from the roots of Asiasari radix, was reported to be associated with anti-allergic effect, but its efficacy and mechanism of action remain unclear. This study investigated the inhibitory effect of Asa on allergic reaction and its mechanism of action. Asa significantly suppressed Ag-sensitized human mast cell line LAD2 calcium mobilization, degranulation, and secretion. It also could reduce OVA-induced local and system anaphylaxis of mice in vivo. Further experiments revealed that Asa inhibit the mast cell activation by preventing the phosphorylation of Src family kinases. Moreover, after the IgE-dependent murine model of allergic rhinitis was treated with Asa, not only the concentration of histamine, total IgE, and IL-4 decreased, but also the inflammatory infiltrates and nasal mucosa incrassation were attenuated significantly. Meanwhile, Asa also inhibited the activation of mast cells induced by Compound48/80 in vivo and in vitro. In conclusion, Asa may serve as a potential novel Src family kinase inhibitor to inhibit IgE-dependent andIgE-independent allergic reaction and treat anaphylactic disease.

Optimization of Ultrasonic-assisted Extraction of Antioxidant Compounds from Asari Radix et Rhizoma Using Response Surface Methodology / 中国药学杂志

OBJECTIVEP: To investigate ultrasonic-assisted estraction(UAE) and response surface methodology(RSM) for the extraction of asarinin from Asari Radix et Rhizoma(ARR). METHODS: The RSM was based on a three-level, four-variable Box-Behnken design (BBD). The independent variables were ultrasonic time, liquid to solid ratio, ultrasonic temperature, and ultrasonic power, the dependent variable was extraction rate of asarinin, which was used to estimate the relationship between independent and dependent variables. Box-Behnken design and RSM were used to optimize the process of extraction. The prediction was carried out through comparing the observed and predicted values. Antioxidant activity of the extract of ARR was determined by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH) and 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate(ABTS) radical scavenging assays in vitro, and good correlation between extraction rate of asarinin and antioxidant activity was observed. RESULTS: The results indicated that ultrasonic time, liquid to solid ratio, ultrasonic temperature, and ultrasonic power had a significant effect on extraction rate of asarinin. Overall process intensification was achieved with ultrasonic time of 56 min, liquid to solid ratio of 17:1 mL•g-1, ultrasonic temperature of 52℃, and ultrasonic power of 180 W by UAE method. Under optimal conditions, the yield of asarinin was (1.55±0.32) mg•g-1 (n=3), which was in accordance with the predicted yield of 1.58 mg•g-1. The IC50 values of the extract of ARR sample were 29.701 and 64.643 mg•mL-1, respectively. The antioxidant results indicate that the extract of ARR has excellent ability to scavenge free radicals and antioxidant capacity and is expected to be used as a natural antioxidant in industrial applications. CONCLUSION: The extraction technology is simple, reliable and highly predictive.The UAE method is effective for extraction of asarinin from ARR.

Analysis and evaluation on main active ingredients of Asarum heterotropoides var. mandshuricum from different habitats / 中草药

Objective: Active ingredients of 47 Asarum samples from different habitats with different phenotypes were analyzed in this paper in order to reveal the relationship between the formation of active ingredients and genetics and geography, and provide some scientific basis for the breeding and untilizing of Asarum. Methods: Ethanol extract and volatile oil were extracted according to the methods of Chinese Pharmacopoeia. Asarinin was detected by HPLC. Volatile oil compositon was determined by GC-MS. The between-groups linkage clustering method was carried out. Results: Volatile oil content of 47 samples was 0.81%-3.32%, and five samples’ volatile oil content exceeded 3.0 %. Ethanol extract content of samples was 9.87%-29.40%, and ethanol extract content of 30 resources accounting for 63.8 percent of all samples exceeded 20%. Asarinin content of 26 resources was more than 0.25%. Totally 48-77 compounds from the volatile oil could be determined and relative content of each compound was different. Forty-seven samples from different habitats with different phenotypes were divided to ten groups by between-groups linkage clustering method according to the active ingredients. All the accessions could be classified clearly. The cluster of accessions was associated with resource region. Asarum heterotropoides var. mandshuricum and Asarum sieboldii var. seoulense from the same origin were grouped together. Conclusion: There were much more differences in active ingredients content among Asarum resources which were in different regions cultivated with same way and environment with different phenotypes. We could select the perfect fundamental material according to the phenotypes and introduce germplasm from different regions for breeding and utilizing of Asarum. Most of samples clustered together associated with their origins instead of phenotype or specie.

Amelioration of CIA by Asarinin Is Associated to a Downregulation of TLR9/NF-κB and Regulation of Th1/Th2/Treg Expression.

To study the role of asarinin on collagen-induced arthritis (CIA) and its treatment mechanism on dendritic cells (DCs) and T cells. Before the onset of arthritis, asarinin were given orally to CIA mouse. Macroscopic scoring and micrometer caliper measurement were used to assess arthritis. The occurrence of cartilage destruction and bone erosion were assessed by histology of knee. Sandwich enzyme-linked immunosorbent assay (ELISA) and PCR were used to assess the level of cytokines in hindpaw and arthritic joint. The CD11c MicroBeads were employed to isolate CD11c+ cells from the spleen. Quantitative PCR was used to determine DCs surface molecules of spleen. Macroscopic score and the frequency of arthritis were inhibited by asarinin. Swelling of hindpaws, inflammatory cell infiltration in the synovium, cartilage destruction, and bone erosion were delayed with asarinin. Asarinin treatment suppressed the expression of T helper type 1 (Th1) cytokines and increased the levels of Th2 cytokines (interleukin (IL)-10), transforming growth factor (TGF)-ß and Foxp3 in the synovium and hindpaw, however T-bet mRNA levels in synovium decreased. Lower expression of toll-like receptor 9 (TLR9) and nuclear factor-kappaB (NF-κB) were found in DCs after asarinin treatment. There was no difference in the expression of intercellular cell adhesion molecule-1(ICAM-1), OX40-L, and 4-1BBL in spleen DCs between the asarinin group and model control group. Asarinin can treat CIA. TLR9/NF-κB pathway may be involved in the asarinin treatment of CIA by skewing the balance of Th1/Th2/regulatory T (Treg) to a Th2 type.

Zanthoxylum armatum DC.: Current knowledge, gaps and opportunities in Nepal.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC. possesses several medicinal properties and has been commonly used in different indigenous medicinal practices to cure several diseases because of its stomachic, carminative and anthelmintic properties. AIM: This review paper aims to provide an update on and analysis of information about the ecology, uses, phytochemistry, pharmacology, trade opportunities, policy gaps for the commercialization of this species forming a basis for further scientific innovations MATERIALS AND METHODS: Information was gathered through a search of different books, journals, articles, annual reports, proceedings and web-based materials. RESULT: Alkaloids, sterols, phenolics, lignins coumarins, terpenoids and flavonoids have been identified from leaves, fruits, stem, bark and seeds. Its trade value is also very high with its manifold applications in Ayurveda, allopathy, general pharmacy, and other industries. Antimicrobial, antiviral, antioxidant, anti-inflammatory, cytotoxic, hepato-protective, insecticidal/larvicidal effects are of particular relevance. CONCLUSION: It is one of the prioritized medicinal plants for economic development in Nepal. Owing to its diverse applications, the species can be developed as an important commodity for alleviation of poverty in rural areas. The various ethno-pharmacological applications of Zanthoxylum armatum have been verified by several related researches. More extensive study on the individual specific phyto-component can lead to novel innovations for the well-being of mankind.

Analysis of Main Components in Asarum heterotropoides var. mandshuricum Seedling at Different Growth Periods and Maturation Period / 中国实验方剂学杂志

Objective: To analyze the change rule of main chemical components in Asarum heterotropoidesvar.mandshuricum seedling during the growing process.Method:Whole seedling samples (one week and two weeks) and the mature plant (three months) of A.heterotropoidesvar.mandshuricum were collected and every sample was divided to aerial part (stems and leaves) and underground part (roots).The secondary metabolites were qualitatively identified by HPLC-TOF-MS and the quantitative identification was carried out at the same time with asarinin as index component.Result: A total of 6 known compounds were identified from the underground part of A.heterotropoidesvar.mandshuricum as α-asarone (1),N-isobutyl-2,4,8,10-dodecatetraenamide (2),9-methoxyaristolactam Ⅳ(3),asarinin (4),caulesnarinside (6) and chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7),respectively,the peak area values showed that the contents of these compounds increased gradually with the growth time.A total of 4 known compounds were identified from the aerial part of this herb as N-isobutyl-2,4,8,10-dodecatetraenamide (2),caulesauroneside (5),caulesnarinside (6) or chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7) and peonidin 3-caffeoylgentiobioside (8).Asarinin was identified only in the underground part of mature plant,its content was 155.4 μg·g-1.Conclusion: The species and contents of secondary metabolites are quite different in the aerial and underground parts of A.heterotropoidesvar.mandshuricum.At different growth stages of A.heterotropoides var.mandshuricum seedling,the types and contents of secondary metabolites in the same site are also different,while the contents of main components show an increasing trend with the growth time.

(-)-Asarinin from the Roots of Asarum sieboldii Induces Apoptotic Cell Death via Caspase Activation in Human Ovarian Cancer Cells.

Two tetrahydrofurofurano lignans (1 and 2), four phenylpropanoids (3⁻6), and two alkamides (7 and 8) were isolated from the EtOAc-soluble fraction of the roots of Asarum sieboldii. The chemical structures of the isolates were identified by analysis of spectroscopic data measurements, and by a comparison of their data with published values. The isolates (1, 2, 4⁻8) were evaluated for their cytotoxicity against human ovarian cancer cells (A2780 and SKOV3) and immortalized ovarian surface epithelial cells (IOSE80PC) using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Of the isolates, (-)-asarinin (1) exhibited the most potent cytotoxicity to both A2780 and SKOV3 cells. A propidium iodide/annexin V-fluorescein isothiocyanate (V-FITC) double staining assay showed that (-)-asarinin (1) induces apoptotic cell death in ovarian cancer cells. In addition, (-)-asarinin (1) increased the activation of caspase-3, caspase-8, and caspase-9 in ovarian cancer cells. Pretreatment with caspase inhibitors attenuated the cell death induced by (-)-asarinin (1). In conclusion, our findings show that (-)-asarinin (1) from the roots of A. sieboldii may induce caspase-dependent apoptotic cell death in human cancer cells.

Effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine-induced cytotoxicity in PC12 cells.

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25-50 µM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 µM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 µM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 µM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 µM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.

In vitro Transdermal Absorption of Xiaochuan Ointment / 中国药师

Objective:To study the transdermal absorption in vitro of sinapine thiocyanate,tetrahydropalmatine and asarinin in Xiaochuan ointment so as to offer reference for its clinical application.Methods:A Franz diffusion cells method with isolated rat skins was carried out to study the percutaneous rate of sinapine thiocyanate,tetrahydropalmatine and asarinin determined by LC-MS/MS.Results:With the increase of administration dosage,the cumulative penetration of sinapine thiocyanate,tetrahydropalmatine and asarinin showed few changes.The results showed that the transdermal behavior of sinapine thiocyanate fit to a Higuchi equation,and that of tetrahydropalmatine and asarinin fit zero-order process.The penetration rate of tetrahydropalmatine and asarinin respectively was 0.362×10-1 and 0.330×10-2 μg·cm-2·h-1.Conclusion:Xiaochuan ointment exhibits transdermal penetration and absorption properties,which provides evidence for its transdermal administration.