Evaluating the toxicity of the roots of Asarum heterotropoides var. mandshuricum extracted using the decoction method: Genotoxicity, single-dose toxicity, and 13-week repeated-dose toxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Asarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa (AR) is a traditional herbal medicine used across Asia, including Korea, China, and Japan. AR exhibits a range of biological activities, such as anti-inflammatory, anti-cancer, cold treatment, and anti-nociceptive effects. Various extraction methods, including decoction, which utilizes traditional knowledge and techniques. The AR decoction extract expected to contain fewer toxicants and have reduced toxicity due to the use of hot water in the extraction process. However, scientific evidence on the toxicity of AR decoction extracts is lacking, necessitating further studies for safe usage. AIM OF THE STUDY: This study aimed to evaluate the genotoxicity and toxicity of single and repeated administration of AR decoction extracts. MATERIALS AND METHODS: The genotoxicity was assessed using a bacterial reverse mutation (Ames test), an in vitro mammalian chromosome aberration test (CA test), and an in vivo micronucleus test (MN test) in Sprague-Dawley (SD) rats. The general toxicity was evaluated through single-dose and 13-week repeated-dose toxicity studies. In the single-dose toxicity study, 40 SD rats were orally administered AR decoction extract at doses of 1000, 2000, and 5000 mg/kg. In the 13-week repeated-dose toxicity study, 140 SD rats received daily oral doses of 0, 250, 500, 1000, 2000, and 5000 mg/kg of AR decoction extract. RESULTS: The genotoxicity tests revealed that AR decoction extract was not genotoxic. The single-dose toxicity study showed no changes in body weight, clinical pathology, or macroscopic findings, with the approximate lethal dose (ALD) exceeding 5000 mg/kg. The 13-week repeated-dose toxicity study demonstrated no treatment-related changes in body weight, general symptoms, hematology, clinical chemistry, or urinalysis. Histopathological findings revealed hyperplasia of squamous cells in the forestomach after AR decoction extract administration, a treatment-related effect that resolved during the recovery period. The no observed adverse effect level (NOAEL) for both male and female rats was estimated to be 2000 mg/kg. CONCLUSIONS: This study establishes the non-toxic dose of AR decoction extract, providing a foundation for further non-clinical and clinical evaluations AR safety.

Phosphorylation Modification, Structural Characterization, Antioxidant and DNA Protection Capacities of Polysaccharides from Asarum Sieboldii Miq.

Polysaccharide from Asarum sieboldii Miq (ASP) was extracted and five phosphorylation polysaccharides with different degree of substitution were obtained, namely ASPP1, ASPP2, ASPP3, ASPP4, and ASPP5 (ASPPs). The physical and chemical structure and biological activities were studied. The results suggested that the carbohydrate and protein content were reduced while uronic acid was increased after phosphorylation modification. The molecular weight of ASPPs was significantly lower than that of ASP. ASPPs were acidic heteropolysaccharides mainly composed of galacturonic acid, galactose, glucose, fructose, and arabinose. The UV-vis spectrum indicated that the polysaccharides did not contain nucleic acid or protein after modification. The Fourier transform infrared spectrum demonstrated that ASPPs contained characteristic absorption peaks of P=O and P-O-C near 1270 and 980 cm-1 . ASPPs presented a triple helix conformation, but it was not presented in ASP. The scanning electron microscopy analysis showed that the surface topography and particle structure of ASP were different after modification. Compared with ASP, ASPPs enhanced the activity to scavenge DPPH and ABTS free radicals and possessed more protective ability to DNA oxidation caused by OH⋅, GS⋅, and AAPH free radicals. These results suggest that chemical modification is beneficial for the exploitation and utilization of natural polysaccharides.

[Exploration of transcriptome SSR markers and its application in genetic diversity assessment of Asarum sieboldii].

To explore the genetic diversity of Asarum sieboldii this study developed SSR markers based on transcriptome sequencing results and five populations of A.sieboldii from different regions were used as samples for genetic diversity assessment using software such as GenALEx 6.5, NTSYS 2.1, and Structure 2.3.4. The results showed that 16 SSR markers with high polymorphism and good repeatability were selected from the A.sieboldii transcriptome. Primers designed based on the flanking sequences of these markers successfully amplified 56 polymorphic fragments from 150 individual samples of the five A.sieboldii populations. On average, each primer amplified 3.5 polymorphic fragments, ranging from 2 to 8. The mean values of expected heterozygosity(H_e), Shannon's diversity index(I), Nei's gene diversity index(H), and the polymorphic information content(PIC) were 0.172, 0.281, 0.429, and 0.382, respectively. The mean population differentiation coefficient(F_(ST)) was 0.588, consistent with the analysis of molecular variance(AMOVA) results, which indicated greater genetic variation among A.sieboldii populations(69%) than that within populations(31%). The percentage of polymorphic loci(PPL) ranged from highest to lowest as SNJ>LN>SY>SZ>TB. Principal coordinate analysis(PCoA) and UPGMA clustering analysis further revealed genetic clustering of A.sieboldii individuals based on their geographical distribution, consistent with the results of the structure clustering analysis. In summary, the SSR markers developed from the transcriptome effectively assessed the genetic differentiation and population structure of natural A.sieboldii populations, revealing a relatively low genetic diversity in A.sieboldii, with genetic variation primarily observed at the population level and a correlation between population differentiation and geographic distance.

Molecular Mechanism of the Asarum-Angelica Drug Pair in the Treatment of Periodontitis Based on Network Pharmacology and Experimental Verification.

(1) To examine the potential mechanism of the Asarum-Angelica drug pair against periodontitis and provide an experimental basis for the treatment of periodontitis with herbal medicine. (2) The core components and core targets of the Asarum-Angelica drug pair in the treatment of periodontitis were detected according to network pharmacology methods. Finally, the effect of the Asarum-Angelica drug pair on osteogenic differentiation was observed in mouse embryonic osteoblast precursor cells. (3) According to the results of network pharmacology, there are 10 potential active ingredients in the Asarum-Angelica drug pair, and 44 potential targets were obtained by mapping the targets with periodontitis treatment. Ten potential active ingredients, such as kaempferol and ß-sitosterol, may play a role in treating periodontitis. Cell experiments showed that the Asarum-Angelica drug pair can effectively promote the expression of osteoblast markers alkaline phosphatase (ALP), Runt-related Transcription Factor 2 (RUNX2), and BCL2 mRNA and protein in an inflammatory environment (p < 0.05). (4) Network pharmacology effectively analyzed the molecular mechanism of Asarum-Angelica in the treatment of periodontitis, and the Asarum-Angelica drug pair can promote the differentiation of osteoblasts.

Phytochemical investigation of Asarum sieboldii var. seoulense and the phenotypic profiles of its constituents against a Parkinson's Disease olfactory cell line.

Asarum sieboldii var. seoulense is a plant species under the family Aristolochiaceae and has been used for centuries as an ingredient in a well-known Traditional Chinese medicine (TCM), "Xixin", to treat symptoms of the neurodegenerative condition Parkinson's Disease (PD). Although there have been studies on the neuroprotective effect of this TCM, the phenotypic profiles of its chemical constituents against PD-implicated cellular organelles have not been reported. This research investigated the chemistry of A. sieboldii var. seoulense extract to identify the active small molecules that exhibited perturbation to the cellular compartments related to PD, potentially supporting its traditional application in treating this condition. 1H NMR-guided chemical investigation of this plant yielded twenty secondary metabolites which belong to isobutylamides, lignans and phenolics. The compounds were evaluated against an olfactory cell line derived from a PD patient using phenotypic assay. Several isolates, 2, 3, 7, 11, 13-16 and 18-20, were found to induce moderate perturbation to the staining of mitochondria, autophagosome and α-tubulin of the cells. Considering that PD pathogenesis is closely related to these cellular compartments, the results provided a rationale for the traditional application of Xixin in the treatment of PD.

Anti-inflammatory effect and metabolic mechanism of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on atopic dermatitis in vivo and in vitro.

BACKGROUND: Atopic dermatitis (AD) is a chronic, relapsing skin disease accompanied by itchy and dry skin. AD is caused by complex interactions between innate and adaptive immune response. AD treatment include glucocorticoids and immunosuppressants. However, long-term treatment can have serious side effects. Thus, an effective AD treatment with fewer side effects is required. Natural materials, including herbal medicines, have potential applications. PURPOSE: This study evaluated the in vivo and in vitro therapeutic effects of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on AD and investigated the underlying metabolic mechanisms. METHODS: The anti-inflammatory effects of BS012 were assessed using a mouse model of AD induced by 1­chloro-2,4-dinitrobenzene (DNCB) and in tumor necrosis factor-alpha/interferon-gamma (TNF-α/IFN-γ) stimulated normal human epidermal keratinocytes (NHEKs). In DNCB-induced mice, total dermatitis score, histopathological analysis, and immune cell factors were assessed to evaluate the anti-atopic activity. In TNF-α/IFN-γ-stimulated NHEKs, pro-inflammatory cytokines, chemokines, and related signaling pathways were investigated. Serum and intracellular metabolomics were performed to identify the metabolic mechanism underlying the therapeutic effects of BS012 treatment. RESULTS: In DNCB-induced mice, BS012 showed potent anti-atopic activity, including reducing AD-like skin lesions and inhibiting the expression of Th2 cytokines and thymic stromal lymphopoietin. In TNF-α/IFN-γ-stimulated keratinocytes, BS012 dose-dependently inhibited the expression of pro-inflammatory cytokines and chemokines by blocking nuclear factor-kappa B and signal transducer and activator of transcription signaling pathways. Serum metabolic profiles of mice revealed significant changes in lipid metabolism related to inflammation in AD. Intracellular metabolome analysis revealed that BS012 treatment affected the metabolism associated with inflammation, skin barrier function, and lipid organization of the stratum corneum. CONCLUSION: BS012 exerts anti-atopic activity by reducing the Th2-specific inflammatory response and improving skin barrier function in AD in vivo and in vitro. These effects are mainly related to the inhibition of inflammation and recovery of metabolic imbalance in lipid organization. BS012, a novel combination with strong activity in suppressing the Th2-immune response, could be a potential alternative for AD treatment. Furthermore, the metabolic mechanism in vivo and in vitro using a metabolomics approach will provide crucial information for the development of natural products for AD treatment.

Rapid simultaneous determination of thirteen aristolochic acids analogs in Aristolochiaceae plants by Ultra-High-Performance liquid Chromatography- tandem mass spectrometry in dynamic multiple reaction monitoring mode.

Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data.

Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam.

The essential oils (EOs) of the aerial parts of four Asarum species (A. geophilum, A. yentunensis, A. splendens and A. cordifolium) were isolated by steam distillation and analyzed by the GC/MS method. The A. cordifolium EO contains 33 constituents with the main component being elemicine (77.20%). The A. geophilum EO was contains 49 constituents with the main components being determined as 9-epi-(E)-caryophyllene (18.43%), eudesm-7(11)-en-4-ol (13.41%), ß-caryophyllene (8.05%) and phytol (7.23%). The A. yentunensis EO contains 26 constituents with the main components being safrole (64.74%) and sesquicineole (15.34%). The EO of A. splendens contains 41 constituents with the main components being 9-epi-(E)-caryophyllene (15.76%), eudesm-7(11)-en-4-ol (14.21%), ß-caryophyllene (9.52%) and trans-bicyclogermacrene (7.50%). For antimicrobial activity, the A. yentunensis EO exhibited the highest inhibition activity against Staphylococcus aureus and the A. cordifolium EO against Bacillus subtillis (MIC values of 100 µg/mL). For antioxidant activity, the A. geophilum EO showed the highest potential with an SC (%) value of 63.34 ± 1.0%, corresponding to an SC50 value of 28.57 µg/mL. For anti-inflammatory activity, the A. splendens EO exhibited the highest potential with an IC50 value of 21.68 µg/mL, corresponding to an inhibition rate of NO production of 69.58 ± 1.3% and the percentage of cell life was 81.85 ± 0.9%.

Long-term oral administration of Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. decoction and its aristolochic acid analogs do not cause renal toxicity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. (AH) is widely used to treat influenza, COVID-19, allergic rhinitis, headache, toothache, rheumatoid arthritis, and peptic ulcer. However, its clinical use is controversial due to the concern of aristolochic acid nephropathy (AAN) caused by its component aristolochic acid analogs (AAs). AIM OF THE STUDY: The chronic toxicity of AH decoction and its main components AA IVa (AA-IVa) and aristolactam I (AL-I) was evaluated in mice. MATERIALS AND METHODS: AAs contents in AH were quantitated by liquid chromatography-mass spectrometry. A parallel design was employed to examine the potential chronic toxicity of AH decoction at doses equivalent to 0.5, 1.6, and 5.0 g/kg AH (approximately 10-100 times the clinical doses for humans) and its major AA components at doses equivalent to that in 5.0 g/kg AH to mice after consecutive daily oral administration for 12 and 24 weeks, and at 32 weeks after withdrawal for 8 weeks. RESULTS: AH crude herb contained 2.18 µg/g of AA-I, 48.49 µg/g of AA-IVa, and 14.0 µg/g of AL-I. AH decoction contained 5.45 µg/g of AA-IVa and 2.71 µg/g of AL-I. None of AA-II and AA-IIIa were detected in AH. After long-term administration of AH decoction and its major components AA-IVa and AL-I, mice showed no signs of illness or body weight changes. In addition, biochemical and pathohistological examinations showed that long-term administration of AH decoction and its major components AA-IVa and AL-I did not alter 1) serum levels of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, alkaline phosphatase, creatinine, and urea nitrogen, 2) renal tissue mRNA expression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, and 3) pathological morphology in the mouse liver, kidney, stomach, and bladder. CONCLUSIONS: AH has no obvious toxicity to mice and is relatively safe when it is used in the form of decoction. AA-IVa and AL-I, the two major AAs in AH, are not toxic to mice at the dose equivalent to that in the high dose of AH decoction. Considering the limited toxicological data on AH, we recommend that AH decoction medication should not overdose and the duration should not be too long.

Molecular cloning and characterization of Cinnamoyl-CoA reductase promoter gene from Asarum sieboldii Miq.

Asarum sieboldii Miq., a perennial herb of the family Aristolochiaceae, is widely used in China to treat cold, fever, aphthous stomatitis, toothache, gingivitis, and rheumatoid arthritis. Methyleugenol is the most representative pharmacological constituent of this medicinal herb. Cinnamoyl-CoA reductase (CCR), which has been well known for occupying a critical position in the lignin biosynthesis pathway, is also shared with the biosynthesis of methyleugenol. To better understand the regulatory mechanisms of methyleugenol biosynthesis, a 1530-bp long promoter region of the AsCCR1 gene was isolated. PLACE and PlantCARE analysis affirmed the existence of the core promoter elements such as TATA and CAAT boxes, abiotic stress-responsive cis-regulation elements like abscisic acid-responsive element, G-box, and MBS in the isolated sequence. The histochemical assay suggested that it was a constitutive promoter, highly expressed in the root tissue. Moreover, the region of -200 bp to ATG (start codon) was enough to drive the expression of It GUS gene. Treatments with low temperature and high concentration of gibberellin or abscisic acid demonstrated the abiotic stress-induced expression of the AsCCR1 promoter. Overall, this study revealed the isolation and characterization of the AsCCR1 promoter. Moreover, it also provided a candidate gene for molecular breeding in A. sieboldii to enhance its pharmacological potential.

Evaluation of genotoxicity and 13-week subchronic toxicity of root of Asarum heterotropoides var. seoulense (Nakai) Kitag.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides var. seoulense (Nakai) Kitag is a traditional herbal medicine used in Korea and China. It is effective in aphthous stomatitis, local anesthesia, headache, toothache, gingivitis, and inflammatory diseases. However, information on the toxicity of the root of Asarum heterotropoides var. seoulense (Nakai) Kitag (AR) is limited. Therefore, preclinical toxicity studies on AR are needed to reduce the risk of excessive intake. AIM OF THE STUDY: We aimed to evaluate genotoxicity and the potential toxicity due to repeated administration of AR powder. MATERIALS AND METHODS: In vitro bacterial reverse mutation assay (Ames), in vitro chromosomal aberration assay (CA), and in vivo micronucleus (MN) assay in ICR mice were conducted. As positive results were obtained in Ames and CA assays, alkaline comet assay and pig-a gene mutation test were conducted for confirmation. For evaluating the general toxicity of AR powder, a 13-week subchronic toxicity test was conducted, after determining the dose by performing a single and a 4-week dose range finding (DRF) test. A total of 152 Sprague-Dawley (SD) rats were orally administered AR powder at doses of 0, 150, 350, 500, 1000, and 2000 mg/kg/day in the 13-week subchronic toxicity test. Hematology, clinical chemistry, urinalysis, organ weight, macro-, and microscopic examination were conducted after rat necropsy. RESULTS: AR powder induced genotoxicity evidenced in the Ames test at 187.5, 750, 375, and 1500 µg/plate of TA100, TA98, TA1537, and E. coli WP2uvrA in the presence and absence of S9, respectively; CA test at 790 µg/mL for 6 h in the presence of S-9; 75 µg/mL for 6 h in the absence of S-9, and 70 µg/mL for 22 h in the absence of S-9 in the stomach in the comet assay but not in MN and pig-a assays. In the 13-week subchronic toxicity study, clinical signs including irregular respiration, noisy respiration, salivation, and decreased body weight or food consumption were observed in males and females in the 2000 mg/kg/day group. In hematology tests, clinical chemistry, urinalysis, organ weight, and macroscopic examination, changes were observed in the dose groups of 500 mg/kg/day and above. Microscopic examination revealed hyperplasia of the stomach as a test-related change. Hepatocellular adenoma and changes in liver-related clinical chemistry parameters were observed. The rat No Observed Adverse Effect Level (NOAEL) was 150 mg/kg/day in males and <150 mg/kg/day in females. CONCLUSIONS: AR powder is potentially toxic to the liver and stomach and should be used with caution in humans. A long-term study on carcinogenicity is necessitated because DNA damage or changes in tissue lesions were observed in SD rats.

Dynamic seasonal changes in photosynthesis systems in leaves of Asarum tamaense, an evergreen understorey herbaceous species.

BACKGROUND AND AIMS: Evergreen herbaceous species in the deciduous forest understorey maintain their photosystems in long-lived leaves under dynamic seasonal changes in light and temperature. However, in evergreen understorey herbs, it is unknown how photosynthetic electron transport acclimates to seasonal changes in forest understorey environments, and what photoprotection systems function in excess energy dissipation under high-light and low-temperature environments in winter. METHODS: Here, we used Asarum tamaense, an evergreen herbaceous species in the deciduous forest understorey with a single-flush and long-lived leaves, and measured photosynthetic CO2 assimilation and electron transport in leaves throughout the year. The contents of photosynthetic proteins, pigments and primary metabolites were determined from regularly collected leaves. KEY RESULTS: Both the rates of CO2 assimilation and electron transport under saturated light were kept low in summer, but increased in autumn and winter in A. tamaense leaves. Although the contents of photosynthetic proteins including Rubisco did not increase in autumn and winter, the proton motive force and ΔpH across the thylakoid membrane were high in summer and decreased from summer to winter to a great extent. These decreases alleviated the suppression by lumen acidification and increased the electron transport rate in winter. The content and composition of carotenoids changed seasonally, which may affect changes in non-photochemical quenching from summer to winter. Winter leaves accumulated proline and malate, which may support cold acclimation. CONCLUSIONS: In A. tamaense leaves, the increase in photosynthetic electron transport rates in winter was not due to an increase in photosynthetic enzyme contents, but due to the activation of photosynthetic enzymes and/or release of limitation of photosynthetic electron flow. These seasonal changes in the regulation of electron transport and also the changes in several photoprotection systems should support the acclimation of photosynthetic C gain under dynamic environmental changes throughout the year.

[Effects of shading on photosynthetic physiology and energy metabolism of Asarum forbesii].

Light is the main source for plants to obtain energy.Asarum forbesii is a typical shade medicinal plant, which generally grows in the shady and wet place under the bushes or beside the ditches.It can grow and develop without too much light intensity.This experiment explores the effects of shading on the growth, physiological characteristics and energy metabolism of A.forbesii, which can provide reference and guidance for its artificial planting.In this experiment, A.forbesii was planted under 80%, 60%, 40%, 20% and no shade.During the vigorous growth period, the photosynthetic physiological characteristics such as fluorescence parameters, photosynthetic parameters, photosynthetic pigment content and ultrastructure, as well as the content of mitochondrial electron transport chain(ETC) synthase and nutrients were measured.The results showed that the photosynthetic pigment content, chlorophyll fluorescence parameters and net photosynthesis rate(P_n) decreased with the decrease of shading.Under 20%-40% shading treatment, the plants had damaged ultrastructure, expanded and disintegrated chloroplast, disordered stroma lamella and grana lamella, and increased osmiophi-lic granules and starch granules.The activities of nicotinamide adenine dinucleotide dehydrogenase(NADH), succinate dehydrogenase(SDH), cytochrome C oxidoreductase(CCO) and adenosine triphosphate(ATP) synthasewere positively related to light intensity.With the reduction of shading, the content of total sugar and protein in nutrients increased first and then decreased, and the content was the highest under 60% shade.In conclusion, under 60%-80% shading treatment, the chloroplast and mitochondria had more complete structure, faster energy metabolism, higher light energy-conversion efficiency, better absorption and utilization of light energy and more nutrient synthesis, which was more suitable for the growth and development of A.forbesii.

Comparative reproductive ecology of two sister Asarum species (Aristolochiaceae) in relation to the evolution of elongated floral appendage.

Genus Asarum (Aristolochiaceae) shows diverse floral morphology and is hypothesized to have diversified as a result of pollinator-mediated selection. Yet most aspects of their reproductive ecology, including pollinators, remain unclear. This study focuses on A. costatum and A. minamitanianum in Japan, a sister species pair having remarkable differences in calyx lobe length (10-20 mm and 70-180 mm, respectively). The objectives of this study are to elucidate multiple aspects of reproductive ecology of these two species and obtain evolutionary insights into floral organ elongation. We adopted combined approaches, including field observations, molecular analyses and cultivation experiments, such as pollinator observation for 3 years, fine-scale spatial genetic analysis of 769 individuals, paternity analysis based on 566 seeds over 4 years, and control pollination experiments. Both Asarum species had strong spatial genetic structures, indicating limited seed dispersal. Pollinator observation revealed that flies and ground-dwelling insects visited flowers of both species, but that the pollinator fauna differed between the species. The visitation rate of flies was extremely low but was more than twice as high in the species with an elongated floral appendage. Paternity analysis revealed A. minamitanianum was predominantly outcrossing, while A. costatum showed a wide range of selfing rates among fruits. These two Asarum species are likely adapted to fly pollination in the shady forest understorey, where available pollinator fauna is limited. In addition, although its function remains unclear, the elongated calyx lobe of A. minamitanianum could have evolved for effective pollen dispersal by attracting fly visitors.

The antinociceptive and anti-inflammatory potential and pharmacokinetic study of significant alkamides ingredients from Asarum Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: Asari Radix et Rhizoma (ARR), including 3 major plants of genus Asarum Linn, A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., A. sieboldii Miq. f. sieboldii and A. sieboldii Miq f. seoulense (Nakai) C. Y. Cheng et C. S. Yang, is one of the most important traditional herbal medicine in Asia with tremendous pharmacological activities. For a long time, researchers focus attention on studing asarinin and essential oils, the indicating ingredients of ARR, but paid less attention to another characteristic component, alkamides. The role of alkamides in the major efficacy of ARR medication remains to be elucidated. AIM OF THE STUDY: This study aims to investigate the contribution of alkamides in the efficacy of ARR according to the evaluation of antinociceptive and anti-inflammatory effects and in vivo pharmacokinetics processes. MATERIALS AND METHODS: For pharmacodynamic study, the analgesic and anti-inflammatory effects of alkamides-enriched fraction (ARRA) were comparatively evaluated by writhing test, hot plate test, and ear swelling test in mice after oral administration. For pharmacokinetic study, an UHPLC-MS/MS method was developed for the simultaneous determination of N-isobutyl-2E,4E,8Z,10Z/E-dodecatetraenamide (DDA) and other 6 major characteristic ingredients of ARR in rat plasma. The analytical method was validated and successfully applied to the pharmacokinetic study of ARR extract and DDA. RESULTS: Pharmacodynamic study show that the ARR and ARRA can significantly inhibit the writhing times of mice caused by acetic acid administration, increase the pain threshold of thermal stimulation, and inhibit xylene treated ear swelling degree by reduce PGE2 and TNF-α levels in the inflamed tissue. For pharmacokinetic study, the pharmacokinetic parameters of Vd/F and CL/F after intravenous administration in rats of DDA are 63.94 ± 32.12 L/kg and 0.33 ± 0.06 L/min/kg, respectively. The plasma drug concentration declined with the T1/2 value of 2.25 ± 0.96 h, and the MRT0-∞ was 2.23 ± 1.02 h. The absolute bioavailability of DDA after oral administration was calculated as 10.73%. DDA, methyleugenol, and asarinin have relatively high AUC0-∞ values when the ethanol and water extract of ARR is orally administered. CONCLUSIONS: ARRA is a kind of active ingredients with potential analgesic and anti-inflammatory effects that played a significant role in the major efficacy of ARR. DDA, the major compound of ARRA, has a high level of exposure in vivo, which could be is suitable for the pharmacokinetic marker or new quality marker of ARR.

Anti-Inflammatory Lignans from the Roots of Asarum heterotropoides var. mandshuricum and Their Mechanism of Action.

Bioassay-guided fractionation of Asarum heterotropoides var. mandshuricum F. Maekawa (Aristolochiaceae) root extract led to the isolation and characterization of one new ferulic acid glucose ester (1) and nine known lignans (2-10). Their structures were elucidated using extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra. The anti-inflammatory effects of the isolated compounds were investigated via their inhibition against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. Among them, compound 7 ((1R,2S,5R,6R)-5'-O-methylpluviatilol) showed the most effective inhibitory activity against NO production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein in an exceedingly dose-dependent manner. In addition, further study revealed that the mechanism of anti-inflammatory activity of the most active lignan (7) might be associated with the inhibition of extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) phosphorylation.

Systematic palynology in Korean Piperales with special focus on its exine surface ornamentation and orbicule morphology.

The pollen and orbicule morphology of the Korean Piperales (Aristolochia, Asarum, Houttuynia, Piper, and Saururus) were investigated via scanning electron microscopy. Piperales pollen is a monad, its size ranging from very small to large (P = 7.78-51.4 µm, E = 6.68-43.1 µm), and having a mainly circular to sub-circular shape. The aperture type is constant in the genus [inaperturate (Aristolochia), tri to pentaporate (Asarum), and monosulcate (Houttuynia, Piper, and Saururus)]. There are four distinct types of exine ornamentation: Fossulate with perforate, microreticulate with gemmae, microperforate with granula, and microechinate. The orbicules (minute sporopollenin granules) were observed in all studied taxa and thus, may be a possible symplesiomorphic characteristic of Piperales. Further, the observed orbicule surface ornamentation was similar to pollen exine patterns, for example muri, gemmae, or granula. This resemblance between orbicule and pollen exine ornamentation may imply a similar biosynthesis pattern of sporopollenin of pollen exine and orbicules. The phenogram resulting from a cluster analysis using palynological characters was generally consistent with the known molecular phylogeny of Piperales. This initial study will help understand the palynological diversity and provide detailed information of pollen and orbicule characteristics in Piperales.

Synthesis of Silver Nanoparticles from Extracts of Wild Ginger (Zingiber zerumbet) with Antibacterial Activity against Selective Multidrug Resistant Oral Bacteria.

Antibiotic resistance rate is rising worldwide. Silver nanoparticles (AgNPs) are potent for fighting antimicrobial resistance (AMR), independently or synergistically. The purpose of this study was to prepare AgNPs using wild ginger extracts and to evaluate the antibacterial efficacy of these AgNPs against multidrug-resistant (MDR) Staphylococcus aureus, Streptococcus mutans, and Enterococcus faecalis. AgNPs were synthesized using wild ginger extracts at room temperature through different parameters for optimization, i.e., pH and variable molar concentration. Synthesis of AgNPs was confirmed by UV/visible spectroscopy and further characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy analysis (EDXA), and Fourier-transform infrared spectroscopy (FTIR). Disc and agar well diffusion techniques were utilized to determine the in vitro antibacterial activity of plant extracts and AgNPs. The surface plasmon resonance peaks in absorption spectra for silver suspension showed the absorption maxima in the range of 400-420 nm. Functional biomolecules such as N-H, C-H, O-H, C-O, and C-O-C were present in Zingiber zerumbet (Z. zerumbet) (aqueous and organic extracts) responsible for the AgNP formation characterized by FTIR. The crystalline structure of ZZAE-AgCl-NPs and ZZEE-AgCl-NPs was displayed in the XRD analysis. SEM analysis revealed the surface morphology. The EDXA analysis also confirmed the element of silver. It was revealed that AgNPs were seemingly spherical in morphology. The biosynthesized AgNPs exhibited complete antibacterial activity against the tested MDR bacterial strains. This study indicates that AgNPs of wild ginger extracts exhibit potent antibacterial activity against MDR bacterial strains.

The genus Asarum: A review on phytochemistry, ethnopharmacology, toxicology and pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: In essentially every quadrant of the globe, many species of genus Asarum are used as a common herbal medicine and appear in many formulas or Kampo. Crude drug from several medicinal plants of genus Asarum (MA) known as Asari Radix et Rhizoma (ARR) has been proven to have the functions of dispelling cold, relieving pain, and reducing phlegm according to Traditional Chinese Medicine (TCM) theory for thousands of years. AIM OF THE STUDY: This article reviews the ethnopharmacology, phytochemistry, pharmacology, toxicology and metabolic kinetics related research of genus Asarum to evaluate its ethnopharmacology use and future opportunities for research. MATERIALS AND METHODS: Information on relevant studies of the genus Asarum was gathered via the Internet using Baidu Scholar, Web of Science, Elsevier, ResearchGate, ACS, Pudmed and Chinese National Knowledge Infrastructure (CNKI). Additionally, information was also obtained from some local books, PhD, MS's dissertations and Pharmacopeias. RESULTS: The genus Asarum has played an important role in herbal treatment. At present, more than 277 compounds have been isolated or identified from genus Asarum. Among them, volatile oil and lignans are the major active constituents and important chemotaxonomic markers. Modern pharmacological studies indicated that genus Asarum and its active compounds possess a wide range of pharmacological effects, especially analgesic, anti-inflammatory, neuroprotective, cardiovascular protection, antitussive, immunosuppressive, anti-tumor, and microbicidal activities. CONCLUSIONS: Based on this review, therapeutic potential of genus Asarum has been demonstrated with the pharmacological effects on inflammation, CNS, respiratory regulation, cardiovascular diseases, cancer and microbial infection. The available literature showed that the major activities of the genus Asarum can be attributed to the active lignans and essential oils. Further in-depth studies on the aspects of the genus for mechanism of actions, metabolism, pharmacokinetics, toxicology, drug interactions, and clinical trials are still limited, thereby intensive research and assessments should be performed.

A new sesquiterpene from South African wild ginger (Siphonochilus aethiopicus (Schweinf) B.L. Burtt).

A new eusdesmane sesquiterpenoid, characterised as 5-acetoxy-9aß-hydroxy-4aαH-3,5α, 8aß-trimethyl-4, 4a, 6, 7, 8a, 9-hexahydronaphtho-([2, 3 b]-dihydrofuran-2-one)-8-one or phaeusmane F acetate (1) has been isolated from the rhizomes of the South African variety of wild ginger (iphonochilus aethiopicus (Schweinf) B.L. Burtt). The compound was obtained after a series of column and gel filtration chromatography. Its structure was elucidated by NMR and Mass-Spectrometric analyses, including 1 D-, 2 D-NMR and HR-LCMS. This is an initial report of the compound from a Siphonochilus sp. Previously isolated similar compounds from the plant material were 4aαH-3,5α,8aß-trimethyl-4,4a,8a,9-tetrahydronaphtho[2,3b]-furan-8-one (siphonochilone) (2), 9aß-hydroxy-4aαH-3,5α,8aß-trimethyl-4,4a,8a,9-tetrahydronaphtho-([2,3b]-dihydrofuran-2-one)-8-one (3), 4aαH-3,5α,8aß-trimethyl-4,4a,8a,9-tetrahydronaphtho-([2,3b]-dihydrofuran-2-one)-8-one (4), 2-hydroxy-4aαH-3,5α,8aß-trimethyl-4,4a,8a,9-tetrahydro-naphtho[2,3b]- furan-8(5H)-one (5), 4aαH-3,5α,8aß-trimethyl-4,4a,8a-trihydronaphtho-([2,3b]-dihydrofuran-2-one)-8-one (6).[Formula: see text].