Identification of Angelica acutiloba, A. sinensis, and other Chinese medicinal Apiaceae plants by DNA barcoding.

Crude drug Angelicae acutilobae radix is one of the most important crude drugs in Japanese traditional medicine and is used mainly for the treatment of gynecological disorders. In the listing in the Japanese Pharmacopoeia XVIII, Angelicae acutilobae radix is defined as the root of Angelica acutiloba (Apiaceae), which has long been produced on an industrial scale in Japan. With the aging of farmers and depopulation of production areas, the domestic supply has recently declined and the majority of the supply is now imported from China. Due to having only slightly different morphological and chemical characteristics for the Apiaceae roots used to produce dried roots for Chinese medicines, the plant species originating the crude drug Apiaceae roots may be incorrectly identified. In particular, Angelicae sinensis radix, which is widely used in China, and Angelicae acutilobae radix are difficult to accurately identify by morphology and chemical profiles. Thus, in order to differentiate among Angelicae acutilobae radix and other radixes originated from Chinese medicinal Apiaceae plants, we established DNA markers. Using DNA sequences for the chloroplast psbA-trnH intergenic spacer and nuclear internal transcribed spacer regions, Angelicae acutilobae radix and other Chinese Apiaceae roots, including Angelicae sinensis radix, can be definitively identified.

Angelica acutiloba Kitagawa flower induces A549 cell pyroptosis via the NF-κB/NLRP3 pathway for anti-lung cancer effects.

Angelica acutiloba Kitagawa, a traditional medicinal herb of the Umbelliferae family, has been demonstrated to have anticancer activity. In this study, we investigated the anti-lung cancer effects of two compounds extracted from A. acutiloba flowers: kaempferol-3-O-α-L-(4″-E-p-coumaroyl)-rhamnoside (KAE) and platanoside (PLA). MTT, cell colony formation, and cell migration (scratch) assays revealed that both KAE (100 µM) and PLA (50 µM and 100 µM) inhibited the viability, proliferation, and migration of A549 cells. Dichlorodihydrofluorescein diacetate assays showed that KAE and PLA also induced the generation of reactive oxygen species in A549 cells. Morphologically, A549 cells swelled and grew larger under treatment with KAE and PLA, with the most significant changes at 100 µM PLA. Fluorescence staining and measurement of lactate dehydrogenase release showed that the cells underwent pyroptosis with concomitant upregulation of interleukin (IL)-1ß and IL-18. Furthermore, both KAE and PLA induced upregulation of NF-κB, PARP, NLRP3, ASC, cleaved-caspase-1, and GSDMD expression in A549 cells. Subsequent investigations unveiled that these compounds interact with NLRP3, augment NLRP3's binding affinity with ASC, and stimulate the assembly of the inflammasome, thereby inducing pyroptosis. In conclusion, KAE and PLA, two active components of A. acutiloba flower extract, had significant anti-lung cancer activities exerted through regulation of proteins related to the NLRP3 inflammasome pathway.

Didymella acutilobae sp. nov. Causing Leaf Spot and Stem Rot in Angelica acutiloba.

During disease surveys of Angelica acutiloba plants in Korea, leaf spot symptoms were observed in a field in Andong in July 2019, and stem rot symptoms in vinyl greenhouses in Yangpyeong in April 2020. Incidence of leaf spot and stem rot of the plants ranged from 10 to 20% and 5 to 30%, respectively. Morphological and cultural characteristics of fungal isolates from the leaf spot and stem rot symptoms fitted into those of the genus Phoma. Molecular phylogenetic analyses of two single-spore isolates from the symptoms using concatenated sequences of LSU, ITS, TUB2, and RPB2 genes authenticated an independent cluster from other Didymella (anamorph: Phoma) species. Moreover, the isolates showed different morphological and cultural characteristics in comparison to closely related Didymella species. These discoveries confirmed the novelty of the isolates. Pathogenicity of the novel Didymella species isolates was substantiated on leaves and stems of A. acutiloba through artificial inoculation. Thus, this study reveals that Didymella acutilobae sp. nov. causes leaf spot and stem rot in Angelica acutiloba.

Population genetic structure of wild Angelica acutiloba, A. acutiloba var. iwatensis, and their hybrids by atpF-atpA intergenic spacer in chloroplast DNA and genome-wide SNP analysis using MIG-seq.

Sampling surveys of Angelica acutiloba and A. acutiloba var. iwatensis, which are medicinal plants endemic to Japan, were conducted in the Chubu region in the central area of the main island of Japan. A. acutiloba grows in riverbeds in mountainous areas, while A. acutiloba. var. iwatensis grows on slopes near mountain ridges at 1000 m above sea level or on constantly collapsing rocky slopes and bare fields on developed land along asphalt roads in valleys of mountainous areas. Specimens of two wild Angelica species collected in this region were examined for maternal lineage by DNA polymorphism analysis of the atpF-atpA region for chloroplast DNA using direct sequencing and genomic component analysis by genome-wide SNP using MIG-seq. In this study area, while all A. acutiloba populations were monophyletic in both maternal and ancestral lineages, A. acutiloba var. iwatensis were genetically heterogeneous due to being composed of three maternal and three ancestral lineages to various degrees. In addition, a natural hybrid population with maternal lineage presumed to be A. acutiloba and paternal lineage A. acutiloba var. iwatensis was also found. In the present study, we report that the combined method of atpF-atpA and MIG-seq analyses is a useful tool for determining the population genetic structure of two wild Angelica species and for identifying hybrids.

Network medicine framework identified drug-repurposing opportunities of pharmaco-active compounds of Angelica acutiloba (Siebold & Zucc.) Kitag. for skin aging.

Increasing incidence of skin aging has highlighted the importance of identifying effective drugs with repurposed opportunities for skin aging. We aimed to identify pharmaco-active compounds with drug-repurposing opportunities for skin aging from Angelica acutiloba (Siebold & Zucc.) Kitag. (AAK). The proximity of network medicine framework (NMF) firstly identified 8 key AAK compounds with repurposed opportunities for skin aging, which may exert by regulating 29 differentially expressed genes (DGEs) of skin aging, including 13 up-regulated targets and 16 down-regulated targets. Connectivity MAP (cMAP) analysis revealed 8 key compounds were involved in regulating the process of cell proliferation and apoptosis, mitochondrial energy metabolism and oxidative stress of skin aging. Molecular docking analysis showed that 8 key compounds had a high docked ability with AR, BCHE, HPGD and PI3, which were identified as specific biomarker for the diagnosis of skin aging. Finally, the mechanisms of these key compounds were predicted to be involved in inhibiting autophagy pathway and activating Phospholipase D signaling pathway. In conclusion, this study firstly elucidated the drug-repurposing opportunities of AAK compounds for skin aging, providing a theoretical reference for identifying repurposing drugs from Chinese medicine and new insights for our future research.

Angelica acutiloba Exerts Antihypertensive Effect and Improves Insulin Resistance in Spontaneously Hypertensive Rats Fed with a High-Fat Diet.

INTRODUCTION: Angelica acutiloba is one of the crude drugs used in Chinese herbal medicine, and its intake is expected to improve metabolic syndrome-associated disorders. Here, we examined the effects of A. acutiloba extract (AAE) on hypertension and insulin resistance induced by the treatment of high-fat diet (HFD) to spontaneously hypertensive rats (SHRs). Then, we investigated the mechanisms associated with the effects of AAE. METHODS: AAE was administered to HFD-fed SHRs. Systolic blood pressure (SBP), sympathetic nerve activity, hypothalamic angiotensin-converting enzyme (ACE) activity, blood glucose level, plasma insulin concentration, visceral fat mass, and gene expression of tumor necrosis factor-alpha (TNF-α) in the visceral fat were evaluated. RESULTS: AAE reduced the increases in SBP and hypothalamic ACE activity observed in the HFD-fed SHRs, whereas the suppressive effect on sympathetic nerve activity was slight. Environmental stress-induced pressure and sympathetic overactivity were suppressed by the treatment of AAE. It also decreased the increase in the blood glucose level, plasma insulin concentration, homeostasis model assessment for the insulin resistance, and TNF-α gene expression in the visceral fat, but not the increase in the visceral fat mass. CONCLUSION: AAE has an antihypertensive effect, suppresses stress-induced hypertension, and improves insulin resistance in HFD-fed SHRs. The suppression of brain ACE activity, sympathetic nerve activity, and inflammation are partly involved in the effects of AAE.

Efficient fertilization in the cultivation of Angelica acutiloba (Siebold & Zucc.) Kitag. in Hokkaido: Effect of amount of supplied nitrogen on growth, yield, and quality of A. acutiloba.

Angelica acutiloba (Siebold & Zucc.) Kitag., the source plant of the crude drug Angelicae acutilobae radix, is traditionally cultivated in western regions of Japan. Since A. acutiloba is now also grown in Hokkaido, the northernmost prefecture of the country, the cultivation method, especially the quantity of fertilizer, needs to be adapted because of the cooler climate and shorter growing period. In this study, we compared plant growth and harvest yield of A. acutiloba cultivated with different amounts of nitrogen (N) fertilizer. When plants were fertilized with 24 kg N/10 a, the aerial part was lush, and the diameter at the top of the root was about 1.3 times thicker than that in plants treated with 12 kg N/10 a. On the other hand, the weight of the harvested root grown with 24 kg N/10 a was slightly, although not significantly, less than that in plants grown with 12 kg N/10 a. In addition, we found that the content of (Z)-ligustilide, a major essential oil contained in A. acutiloba root, is affected by nitrogen application. Interestingly, it increased with increasing amounts of supplied nitrogen. However, the importance of Angelicae acutilobae radix as the crude drug is not limited to its (Z)-ligustilide content, and there are also other crucial quality features, such as having thick lateral roots, which were generated in this study with a moderate nitrogen application. We conclude that about 12 kg N/10 a is the optimal amount of nitrogen for healthy growth of A. acutiloba in Hokkaido.

Studies on cultivation of Angelica acutiloba using paper pot (Part 2): effect of difference in taproot base diameter of seedlings on growth after planting and quality of toki produced from paper pot seedlings.

By using Angelica acutiloba seedlings produced by short-period raising in paper pot (PP), we investigated the effect of taproot base diameter on the growth after planting and evaluated the quality of toki (, Angelicae Acutilobae Radix) produced from PP-grown seedlings (PT). In this study, PP-grown seedlings of seven classes which are sorted difference taproot base diameter (P1-P7) were transplanted. As a result, the survival rates were not less than 90% for P6 (taproot base diameter: 10.0-12.7 cm) and P7 (taproot base diameter: 12.8-17.3 cm), and the bolting rates were 20.5% and 24.1% for P6 and P7, respectively. The traditionally grown seedling (TS) showed the highest available harvest rate of 82.3%, followed by P6 (71.8%) and P7 (69.0%). P7 showed the highest fresh weight and dry weight per 1a (100 m2), followed by P6 and TS. However, PP-grown seedlings are prone to bolting, and it is expected the yield can be increased by preventing bolting. In addition, PT satisfied the regulations of JP17 in terms of, for example, dilute ethanol-soluble extract content, total ash content, and acid-insoluble ash content. It was also found that PT has a higher proportion of lateral roots with respect to the total weight than toki produced from traditionally grown seedlings (TT). The total (Z)-ligustilide contents were 0.07% in PT and 0.12% in TT. It was revealed that it is possible to produce toki that satisfies JP17 regulations; moreover, the period required for toki production was shortened by 6 months with PP-grown seedlings.

Quality evaluation of Angelicae acutilobae radix: individual differences and localization of (Z)-ligustilide in Angelica acutiloba root.

It has been difficult to evaluate the quality of Angelicae acutilobae Radix (Toki) because of large differences in the contents of its chemical constituents. In this research, we revealed individual differences and localization of (Z)-ligustilide in Toki cultivated and processed under the same conditions. Thirteen Toki samples (dry weight: 68.2 g-132.3 g) were divided and categorized into 13 parts and the (Z)-ligustilide content of each part was quantified. Total (Z)-ligustilide content ranged from 0.08% to 0.22% and the maximum difference between samples was approximately 2.8-fold. In addition, the localization of (Z)-ligustilide was examined. (Z)-Ligustilide content was the highest in lateral root at 0.19%, followed by main root at 0.13%, and the lowest in root head at 0.09%. Furthermore, the content tended to increase as the root became thinner. In particular, the difference in content between the inner side of upper root head (removed 5 mm from the epidermis 0.06%) and the 1.1-3.0 mm in diameter lateral root (0.24%) was largest at approximately 4.1-fold. We revealed that not only differences among individuals but also localization is a factor affecting the quality of Toki. In contrast, individuals with higher root part (main root + lateral root) weight ratio in whole root dry weight had higher (Z)-ligustilide content. The difference in (Z)-ligustilide content among individuals is due to the balance between root head part and root other than head part. It is possible to predict (Z)-ligustilide content from weight ratio of root part to whole root.

Comprehensive quality evaluation and comparison of Angelica sinensis radix and Angelica acutiloba radix by integrated metabolomics and glycomics.

Angelica radix (Danggui in Chinese) used in China and Japan is derived from two species of Angelica, namely Angelica sinensis and Angelica acutiloba, respectively. The differences in quality between A. sinensis radix (ASR) and A. acutiloba radix (AAR) should be therefore investigated to guide the medicinal and dietary applications of these two species. Secondary metabolites and carbohydrates have been demonstrated to be the two major kinds of bioactive components of Danggui. However, previously, quality comparison between ASR and AAR intensively concerned secondary metabolites but largely overlooked carbohydrates, thus failing to include or take into consideration an important aspect of the holistic quality of Danggui. In this study, untargeted/targeted metabolomics and glycomics were integrated by multiple chromatography-based analytical techniques for qualitative and quantitative characterization of secondary metabolites and carbohydrates in Danggui so as to comprehensively evaluate and compare the quality of ASR and AAR. The results revealed that not only secondary metabolites but also carbohydrates in ASR and AAR were different in type and amount, which should collectively contribute to their quality difference. By providing more comprehensive chemical information, the research results highlighted the need to assess characteristics of both carbohydrates and secondary metabolites for overall quality evaluation and comparison of ASR and AAR.

Determination of (E)-ferulic acid content in the root of Angelica acutiloba: a simple chemical evaluation method for crude drug quality control.

The root of Angelica acutiloba Kitagawa is an important crude drug in Kampo medicines (traditional Japanese medicine). Chemical evaluation of crude drugs is crucial to ensuring the safety and efficacy of herbal medicine; however, there is currently no chemical standard for the A. acutiloba crude drug in Japanese pharmacopoeia. (E)-ferulic acid (FA) is an important active ingredient of Angelica spp., including A. sinensis (Oliv.) Diels, and has been suggested as a marker for quality evaluation of those crude drugs. However, it has been controversial whether FA is a reliable marker constituent of A. acutiloba. To achieve effective extraction of FA from A. acutiloba, we compared three different extraction methods: alkaline hydrolysis, ethanol extraction, and hexane extraction. FA levels in these extracts were assessed using high performance liquid chromatography (HPLC), and alkaline hydrolysis was found to be the most effective. Furthermore, in the hydrolysate, FA was distinctly identified by thin layer chromatography (TLC) analysis. These results provide useful information for the quality control of the A. acutiloba crude drug.

Oviposition inhibitor in umbelliferous medicinal plants for the common yellow swallowtail (Papilio machaon).

Umbelliferous medicinal plants, such as Angelica acutiloba Kitagawa and Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier, account for a large percentage of crude drug consumption in Japan. The most serious problem in the cultivation of umbelliferous medicinal plants is the feeding damage caused by the common yellow swallowtail (Papilio machaon hippocrates C. & R. Felder, 1864). When we compared the numbers of eggs laid by P. machaon on six umbelliferous medicinal plants, the eggs on A. acutiloba, A. dahurica, and Glehnia littoralis Fr. Schmidt ex Miquel were the most numerous, those on Saposhnikovia divaricata Schischkin and Cnidium officinale Makino were rare, and Bupleurum falcatum Linné was not oviposited at all. To identify oviposition inhibitors for P. machaon in B. falcatum, S. divaricata, and C. officinale, the volatile chemical constituents of these umbelliferous medicinal plants were compared with GC-MS. We carried out multivariate analysis of gas chromatographic data and concluded that germacrene D, α-humulene, and trans-caryophyllene play important roles in protecting plants from oviposition by P. machaon. Their oviposition repellent activity was confirmed by the fact that the number of eggs laid on the leaves around a repellent device containing a mixture of germacrene D, α-humulene, and trans-caryophyllene was reduced by 40% compared to a control.

Preclinical safety assessment of Angelica acutiloba using a 13-week repeated dose oral toxicity study in rats.

Angelica acutiloba (AA), a Japanese species of Danggui, has been used worldwide as a traditional herbal medicine with several bioactivities including anti-diabetic, anti-allergic, anti-inflammatory, anti-tumor, and anti-obesity. However, there is lack of toxicological data available to evaluate potential long-term toxicity and the no-observed-adverse-effect level (NOAEL) of AA extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In the 14-day repeat-dose toxicity study, no adverse effects on mortality, body weight change, clinical signs, and organ weights was found following repeat oral administration to rats for 14 days (125, 250, 500, 1000, and 2000 mg/kg body weight), leading that 2000 mg/kg is the highest recommended dose of AA extract for the 13-week repeat-dose oral toxicity study. In the 13-week repeat-dose oral toxicity study, the AA extract was orally administered to groups of rats for 13 weeks (125, 250, 500, 1000, and 2000 mg/kg body weight) to compare between control and AA extract groups. The administration of AA extract did not produce mortality or remarkable clinical signs during this 13-week study. And, the data revealed that there were no significant differences in food/water consumption, body weight, hematological parameters, clinical chemistry parameters, gross macroscopic findings, organ weight and histopathology in comparison to the control group. On the basis of these results, the subchronic NOAEL of the AA extract was more than 2000 mg/kg/day when tested in rats. And, the AA extract is considered safe to use orally as a traditional herbal medicine.

Evaluation of in vitro and in vivo genotoxicity of Angelica acutiloba in a standard battery of assays.

Among three representative species of Angelica found in Asian countries, including Korea, China, and Japan, Angelica acutiloba (AA) has been used as traditional herbal medicine with antitumor, anti-inflammatory, anti-obesity, and anti-diabetes activities. In this study, the potential genotoxicity and mutagenicity of the AA extract were examined in a battery of in vitro and in vivo tests (bacterial reverse mutation assay, in vitro chromosomal aberrations assay, and in vivo micronucleus assay) in accordance with the test guidelines for toxicity testing developed by the Organization for Economic Cooperation and Development. Upon testing in the bacterial mutation assay (Ames test) using five Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537, no significant increase the number of revertant colonies in the metabolic activation system and non-activation system was noted in the AA extract groups. Also, in the chromosome aberration test, the AA extract did not cause chromosomal aberration with or without metabolic activation by S9 mix. A bone marrow micronucleus test of mice demonstrated that the incidence of micronucleated polychromatic erythrocytes in the AA extract groups (500, 1000 and 2000 mg/kg BW) was equivalent to that of the negative control group. Based on these results from a standard battery of assays, the AA extract was concluded to have no genotoxic at the proper dose.

Anti-Photoaging Effects of Angelica acutiloba Root Ethanol Extract in Human Dermal Fibroblasts.

The effects that ultraviolet rays elicit on collagen synthesis and degradation are the most common causes of wrinkle formation and photo-aging in skin. The objectives of this study were to evaluate the effects of Angelica acutiloba root ethanol extract (AAEE) to promote collagen synthesis and inhibit collagen degradation in human dermal fibroblasts. By examining total polyphenol and flavonoid contents, electron donating ability, radical scavenging activity, and superoxide dismutase-like activity, we found that AAEE exhibited fairly good antioxidant activity. Treatment with AAEE significantly increased type I procollagen production by cultured fibroblasts, as well as reduced ultraviolet-induced matrix metalloproteinase-1 (MMP-1) expression and MMP-2 activity in a dose-dependent manner (p < 0.05). In addition, AAEE significantly increased TIMP-1 mRNA expression (p < 0.05), although without an associated dose-dependent increase in TIMP-1 protein expression. In summary, we suggest that AAEE may be a potentially effective agent for the prevention or alleviation of skin-wrinkle formation induced by ultraviolet rays.

Anti-inflammatory Activity of Constituents Isolated from Aerial Part of Angelica acutiloba Kitagawa.

Recently, the resources of medicinal plants have been exhausting. The root of Angelica acutiloba is one of the most important ingredients in Japanese Kampo medicine for the treatment of gynecological diseases. In our search for alternative medicinal plant resources of the root of A. acutiloba, we found that its aerial part has the anti-inflammatory potency as well as the root. Phytochemical investigation of the aerial part resulted in the isolation of four compounds including a new dimeric phthalide, namely tokiaerialide (2), along with Z-ligustilide (1), falcarindiol (3), and bergaptol (4). Next, we investigated the in vitro anti-inflammatory activity of 1-4 in lipopolysaccharide-stimulated RAW264 macrophages. Among the isolated compounds, 1 exhibited the most potent inhibition against lipopolysaccharide-induced production of prostaglandin E2 , nitric oxide, and pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α). Compounds 3 and 4 also inhibited all inflammatory mediators, but their inhibitory abilities were weaker than those of 1. Furthermore, 1, 3, and 4 strongly also induced heme oxygenase-1. These results suggest that 1, 3, and 4 potentially exert anti-inflammatory activity, and the aerial part of A. acutiloba may be considered to be a useful medicinal resource for inflammatory diseases.

Angelica acutiloba root attenuates insulin resistance induced by high-fructose diet in rats.

Angelica acutiloba root (Japanese Dong Quai), used for treatment of gynecological disorders, is currently cultivated in Taiwan. The present study evaluated the preventative effect of Angelica acutiloba root (Japanese Dong Quai) on the induction of insulin resistance. Insulin resistance was induced in rats by feeding a high fructose diet for 6 weeks. Thereafter, the rats were maintained on the same diet and treated with oral A. acutiloba root extract or pioglitazone once daily for 8 weeks. At the end of treatment, the degree of basal insulin resistance was measured by homeostasis model assessment (HOMA-IR). Insulin sensitivity was calculated using the composite whole body insulin sensitivity index (ISIcomp). Protein expression was evaluated by immunoblotting. A. acutiloba (300 mg/kg/day) displayed similar characteristics to pioglitazone (20 mg/kg/day) in reducing HOMA-IR and elevating ISIcomp. Elevated glycosylated hemoglobin levels and hyperinsulinemia were ameliorated by A. acutiloba treatment without hepatotoxic or nephrotoxic effects. A. acutiloba treatment improved dyslipidemia, induced lipoprotein lipase activity and enhanced hepatic glycogen accumulation. Further, A. acutiloba treatment enhanced the action of insulin on muscle glucose transporter subtype 4 translocation and attenuated hepatic phosphoenolpyruvate carboxykinase expression. The findings suggest that A. acutiloba may be an effective ethnomedicine for improving insulin sensitivity.