Pharmacophore Modeling and Binding Affinity of Secondary Metabolites from Angelica keiskei to HMG Co-A Reductase.

Statins are cholesterol-lowering drugs with a mechanism of inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase, but long-term use can cause side effects. An example of a plant capable of reducing cholesterol levels is Angelica keiskei (ashitaba). Therefore, this study aimed to obtain suitable compounds with inhibitory activity against the HMG-CoA reductase enzyme from ashitaba through in silico tests. The experiment began with screening and pharmacophore modeling, followed by molecular docking on ashitaba's compounds, statins groups, and the native ligand was (3R,5R)-7-[4-(benzyl carbamoyl)-2-(4-fluorophenyl)-5-(1-methylethyl)-1H-imidazole-1-yl]-3,5-dihydroxyheptanoic acid (4HI). Based on the results of the molecular docking simulations, 15 hit compounds had a small binding energy (ΔG). Pitavastatin, as the comparator drug (ΔG = -8.24 kcal/mol; Ki = 2.11 µM), had a lower ΔG and inhibition constant (Ki) than the native ligand 4HI (ΔG = -7.84 kcal/mol; Ki = 7.96µM). From ashitaba's compounds, it was found that 4'-O-geranylnaringenin, luteolin, isobavachalcone, dorsmannin A, and 3'-carboxymethyl-4,2'-dihydroxy-4'-methoxychalcone have low ΔG of below -6 kcal/mol. The lowest ΔG value was found in 3'-carboxymethyl-4,2'-dihydroxy-4'-methoxy chalcone with a ΔG of -6.67 kcal/mol and Ki value of 16.66 µM, which was lower than the ΔG value of the other comparator drugs, atorvastatin (ΔG = -5.49 kcal/mol; Ki = 1148.17 µM) and simvastatin (ΔG = -6.50 kcal/mol; Ki = 22.34 µM). This compound also binds to the important amino acid residues, including ASN755D, ASP690C, GLU559D, LYS735D, LYS691C, and SER684C, through hydrogen bonds. Based on the results, the compound effectively binds to six important amino acids with good binding affinity and only requires a small concentration to reduce half of the enzyme activity.

An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types.

BACKGROUND: The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system. RESULTS: The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut. CONCLUSIONS: These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Chalcones from Angelica keiskei (ashitaba) inhibit key Zika virus replication proteins.

Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.

Silver nanoparticles decorated reduced graphene oxide: Eco-friendly synthesis, characterization, biological activities and embryo toxicity studies.

This study was aimed on the eco-friendly synthesis of silver nanoparticles (AgNPs), reduced graphene oxide (rGO) and AgNPs decorated rGO (rGO/AgNPs) nanocomposite and appraisal of their bioactivities and toxicity. As-prepared nanomaterials were established through high resolution X-ray diffraction (HR-XRD), high resolution transmission electron microscopy (HR-TEM), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, UV-Vis. spectroscopy and Fourier transform infrared spectroscopy (FT-IR). In this study, leaves extract, graphene oxide (GO) and rGO did not show antibacterial and anticancer activities; no significant embryo toxicity was recorded. On the other hand, AgNPs displayed good antibacterial and anticancer activities; however, higher toxic effects were observed even at the lowest test concentration (0.7 µg/ml). In case of rGO/AgNPs nanocomposite, significant antibacterial activity together with low cytotoxicity was noticed. Interestingly, the embryo toxicity of AgNPs was significantly reduced by rGO, implying the biocompatible nature of as-synthesized nanocomposite. Taken together, these results clearly suggest that rGO/AgNPs nano hybrid composite could be developed as the promising biomaterial for future biomedical applications.

Assessment of the Hypoglycemic and Hypolipidemic Activity of Flavonoid-Rich Extract from Angelica keiskei.

Angelica keiskei contains a variety of bioactive compounds including chalcone, coumarin, and phytochemicals, endowing it with pharmacological effects such as lipid-lowering activity, antitumor activity, liver protection, and nerve protection. This study aims to study the hypoglycemic and hypolipidemic effects of the flavonoid-rich extract from Angelica keiskei (FEAK) in an effort to exploit new applications of FEAK and increase its commercial value. In this paper, flavonoid compounds in Angelica keiskei were extracted using 50% ethanol, and the contents of the flavonoid compounds were analyzed by UPLC-MS/MS. Then, the hypoglycemic and hypolipidemic activities of the FEAK were investigated through in vitro enzyme activity and cell experiments as well as establishing in vivo zebrafish and Caenorhabditis elegans (C. elegans) models. The UPLC-MS/MS results show that the major flavonoid compounds in the FEAK were aureusidin, xanthoangelol, kaempferol, luteolin, and quercetin. The inhibitory rates of the FEAK on the activity of α-amylase and cholesterol esterase were 57.13% and 72.11%, respectively. In cell lipid-lowering experiments, the FEAK significantly reduced the total cholesterol (TC) and total triglyceride (TG) levels in a dose-dependent manner, with 150 µg/mL of FEAK decreasing the intracellular levels of TC and TG by 33.86% and 27.89%, respectively. The fluorescence intensity of the FEAK group was 68.12% higher than that of the control group, indicating that the FEAK exhibited hypoglycemic effects. When the concentration of the FEAK reached 500 µg/mL, the hypoglycemic effect on zebrafish reached up to 57.7%, and the average fluorescence intensity of C. elegans in the FEAK group was 17% lower than that of the control group. The results indicate that the FEAK had hypoglycemic and hypolipidemic activities. The findings of this study provide theoretical references for the high-value utilization of Angelica keiskei and the development of natural functional food with hypoglycemic and hypolipidemic activities.

Characterizing Tyrosinase Modulators from the Roots of Angelica keiskei Using Tyrosinase Inhibition Assay and UPLC-MS/MS as the Combinatorial Novel Approach.

In this study, an in vitro tyrosinase inhibition assay in combination with ultra performance liquid chromatography-orbitrap mass spectrometry (UPLC-orbitrap-MS) was developed for the rapid screening and identification of tyrosinase modulators from roots of Angelica keiskei. Of the 15 candidates considered, nine chalcones, xanthoangelols (1), B (2), D (3), E (4), G (5), H (6), 4-hydroxyderricin (7), xanthokeismin B (8) and (2E)-1-[4-hydroxy-2-(2-hydroxy-2-propanyl)-2,3-dihydro-1-benzofuran-7-yl]-3-(4-hydroxyphenyl)-2-propen-1-one (9), five coumarins, umbelliferone (10), selinidin (11), isopimpinellin (12), phellopterin (13) and xanthyletin (14), and one other compound, ashitabaol A (15), were distinguished between the test samples and the controls with statistical significance, and the structure of each compound was determined by comparing with in-house standards and the literature. Among these, six compounds, xanthoangelol (1), xanthoangelol D (3), xanthoangelol H (6), 4-hydroxyderricin (7), laserpitin (16) and isolaserpitin (17), were isolated from roots of A. keiskei. Of the compounds isolated, compounds 1, 7 and 16 were subjected to tyrosinase inhibitory assay, and the IC50 values were 15.87 ± 1.21, 60.14 ± 2.29 and >100 µM, respectively. The present study indicated that the combination of in vitro tyrosinase inhibition assay coupled with UPLC-MS/MS could be widely applied to the rapid screening of active substances from various natural resources.

Simultaneous Optimization of Ultrasound-Assisted Extraction for Flavonoids and Antioxidant Activity of Angelica keiskei Using Response Surface Methodology (RSM).

Angelica keiskei Koidzumi (A. keiskei), as a Japanese edible herbal plant, enjoys a variety of biological activities due to the presence of numerous active compounds, especially flavonoids. This study aims for the optimization of ultrasound-assisted extraction (UAE) for flavonoids in A. keiskei and their antioxidant activity by using the response surface methodology (RSM). Single-factor experiments and a four-factor three-level Box-Behnken design (BBD) were performed to explore the effects of the following parameters on flavonoid extraction and antioxidant activity evaluation: ultrasonic temperature (X1), ultrasonic time (X2), ethanol concentration (X3) and liquid-solid ratio (X4). The optimum conditions of the combination of total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (DPPH-RSC) and ferric-reducing antioxidant power (FRAP) were as follows: X1 = 80 °C, X2 = 4 min, X3 = 78%, X4 = 35 mL/g, respectively. The experimental results provide a theoretical basis for the extensive utilization of A. keiskei and flavonoids extraction from A. keiskei as a potential source of antioxidants.

Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation.

We isolated isobavachalcone (IBC) from Angelica keiskei (AK) as an anti-obesity component. IBC dose-dependently inhibited 3T3-L1 adipocyte differentiation by down-regulating adipogenic factors. At the mitotic clonal expansion stage (MCE), IBC caused cell cycle arrest in G0/G1 with decreased expression of cell cycle-regulating proteins. IBC also inhibited autophagic flux by inducing intracellular accumulation of LC3B and SQSTM1/p62 proteins while decreasing expression levels of regulating factors for autophagy initiation. In parallel with the inhibition of adipocyte differentiation, IBC decreased intrahepatic fat deposits and rescued the liver steatosis in high fat cholesterol diet-fed zebrafish. In this study, we found that IBC isolated from AK suppresses mitotic clonal expansion and autophagy flux of adipocytes and also shows anti-obesity activity in a high cholesterol-diet zebrafish model by decreasing intrahepatic fat deposits. These results suggest that IBC could be a leading pharmacological compound for the development of anti-obesity drugs.

Phytonutrient and anti-diabetic functional properties of flavonoid-rich ethanol extract from Angelica Keiskei leaves.

An extraction method was optimized to get a flavonoid-rich ethanol extract from Angelica keiskei leaves (FREE-AK). Trace elements and total flavonoid content of FREE-AK were identified, and the hypoglycemic and hypolipidemic effects of FREE-AK were studied in streptozotocin-induced diabetic mice. For FREE-AK extraction the optimal conditions were 65% ethanol, 45 °C and 15 min, resulting in a total flavonoid content up to 10.18% and K, Mg, Na, and Ca, content of about 36.59, 1.52, 14.51 and 7.486 mg/g, respectively. FREE-AK uptake could cause a marked decrease of fasting blood glucose and a significant improvement on glucose tolerance in diabetic mice. In addition, FREE-AK treatment with a dose of 800 mg/kg b.w. resulted in a reduction in the total triglyceride level (TC) in serum. Results demonstrated the effectiveness of FREE-AK for hypoglycemia and hypolipidemia in streptozotocin-induced mice and FREE-AK may be a potential dietary treatment for type 2 diabetes.

Minor phenolics from Angelica keiskei and their proliferative effects on Hep3B cells.

A new coumarin, (-)-cis-(3'R,4'R)-4'-O-angeloylkhellactone-3'-O-ß-d-glucopyranoside (1) and two new chalcones, 3'-[(2E)-5-carboxy-3-methyl-2-pentenyl]-4,2',4'-trihydroxychalcone (4) and (±)-4,2',4'-trihydroxy-3'-{2-hydroxy-2-[tetrahydro-2-methyl-5-(1-methylethenyl)-2-furanyl]ethyl}chalcone (5) were isolated from the aerial parts of Angelica keiskei (Umbelliferae), together with six known compounds: (R)-O-isobutyroyllomatin (2), 3'-O-methylvaginol (3), (-)-jejuchalcone F (6), isoliquiritigenin (7), davidigenin (8), and (±)-liquiritigenin (9). The structures of the new compounds were determined by interpretation of their spectroscopic data including 1D and 2D NMR data. All known compounds (2, 3, and 6-9) were isolated as constituents of A. keiskei for the first time. To identify novel hepatocyte proliferation inducer for liver regeneration, 1-9 were evaluated for their cell proliferative effects using a Hep3B human hepatoma cell line. All isolates exhibited cell proliferative effects compared to untreated control (DMSO). Cytoprotective effects against oxidative stress induced by glucose oxidase were also examined on Hep3B cells and mouse fibroblast NIH3T3 cells and all compounds showed significant dose-dependent protection against oxidative stress.

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro).

PTP1B inhibitors from stems of Angelica keiskei (Ashitaba).

Three new chalcones, xanthoangelols K-M (1-3), together with 19 known compounds were isolated from the stems of Angelica keiskei Koidzumi, a well-known rejuvenated and anti-diabetic plant originated from Japan. The structures of compounds 1-3 were elucidated on the basis of spectroscopic data and Mosher's method. All compounds were evaluated for their inhibitory activity against protein tyrosine phosphatase 1B (PTP1B). Among them, six chalcones, xanthoangelol K (1), xanthoangelol (4), xanthoangelol F (5), 4-hydroxyderricin (6), xanthoangelol D (7), xanthoangelol E (8), and a coumarin, methoxsalen (17), showed strong PTP1B inhibitory effect with IC50 values of 0.82, 1.97, 1.67, 2.47, 3.97, 1.43, and 2.53µg/mL, respectively. A kinetic study revealed that compound 1 inhibited PTP1B with characteristics typical of a competitive inhibitor. Molecular docking simulations elucidated that ring B of 1 may anchor in a pocket of PTP1B and the molecule is stabilized by hydrogen bonds with Arg47, Asp48, and π-π interaction with Phe182 of PTP1B.

Safety of ashitaba sap as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on ashitaba sap as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Ashitaba sap is collected from harvested stems of Angelica keiskei plants. The principal constituents of the sap with regard to the safety assessment are chalcones (1%-2.25%) and furanocoumarins (< 0.01%). The applicant proposed to use the NF in food supplements at a maximum dose of 780 mg per day. The target population is adults excluding pregnant and lactating women. Taking into consideration the composition of the NF and the proposed uses, the composition of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. Based on a 90-day oral toxicity study performed with the product as intended to be placed on the market (30% ashitaba sap powder and 70% cyclodextrins), the Panel establishes a safe dose of 0.5 mg/kg body weight (bw) per day for the product as it is intended to be placed on the market. For the target population, i.e. adults, this safe dose corresponds to 35 mg per day of the product as it is intended to be placed on the market and 137 mg per day of the NF, which is lower than the use level proposed by the applicant. The Panel concludes that the NF is safe for the target population at intake levels up to 137 mg per day.

Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

SCOPE: Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. METHODS AND RESULTS: In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. CONCLUSION: In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

Bioherbicides for sustainable barnyard grass management in paddy field: an in-silico perspective.

Paddy (Oryza sativa) yield is greatly influenced by the insidious presence of rice-mimicking weed, widely known as barnyard grass. This study explores the promising natural ACCase inhibitors that could enhance paddy yield by controlling weeds. A total of 2828 natural compounds were examined using diverse computational techniques. The results of this study depict that CNP0390839 (xanthoangelol) exhibited a better XP Gscore (-7.328 kcal/mol) and MM/GBSA score (-84.24 kcal/mol) than other investigated compounds. Importantly, ACCase-xanthoangelol complexes was thermodynamically stable with an RMSD value of ∼1.2 nm. Of note, 72% xanthoangelol resides in the Angelica keiskei plant root which exhibits 55% weed-inhibitory action. The A. keiskei plant mainly inhibits the hypocotyl (71.8 ± 5.4%) and root region (55.3 ± 4.7%) of weeds. Moreover, the existence of dihydroxyphenyl scaffold in xanthoangelol was also witnessed in literatures for weed inhibitory action. Overall, xanthoangelol might prove to be an effective ACCase herbicide in paddy weed management.

Angelica keiskei Impacts the Lifespan and Healthspan of Drosophila melanogaster in a Sex and Strain-Dependent Manner.

Angelica keiskei is a perennial plant, belonging to the Apiaceae family and originating from Japan. This plant has been reported to act as a diuretic, analeptic, antidiabetic, hypertensive, tumor, galactagogue, and laxative. The mechanism of action of A. keiskei is not known, but previous studies have suggested that it may act as an antioxidant. In this work, we used Drosophila melanogaster to evaluate the impact of A. keiskei on lifespan and healthspan and its potential anti-aging mechanism by conducting multiple assays on three fly strains: w1118, chico, and JIV. We observed that the extract extended lifespan and improved healthspan in a sex- and strain-dependent manner. A. keiskei extended lifespan and improved reproductive fitness in female flies and either had no effect or decreased survival and physical performance in males. The extract protected against the superoxide generator paraquat in both sexes. These sex-specific effects suggest that A. keiskei may act through age-specific pathways such as the insulin and insulin-like growth factor signaling (IIS) pathways. Upon examination, we found that the increased survival of A. keiskei-fed females was dependent on the presence of the insulin receptor substrate chico, supporting the role of IIS in the action of A. keiskei.

A Mixture of Morus alba and Angelica keiskei Leaf Extracts Improves Muscle Atrophy by Activating the PI3K/Akt/mTOR Signaling Pathway and Inhibiting FoxO3a In Vitro and In Vivo.

Muscle atrophy, which is defined as a decrease in muscle mass and strength, is caused by an imbalance between the anabolism and catabolism of muscle proteins. Thus, modulating the homeostasis between muscle protein synthesis and degradation represents an efficient treatment approach for this condition. In the present study, the protective effects against muscle atrophy of ethanol extracts of Morus alba L. (MA) and Angelica keiskei Koidz. (AK) leaves and their mixtures (MIX) were evaluated in vitro and in vivo. Our results showed that MIX increased 5-aminoimidazole-4-carboxamide ribonucleotide-induced C2C12 myotube thinning, and enhanced soleus and gastrocnemius muscle thickness compared to each extract alone in dexamethasone-induced muscle atrophy Sprague Dawley rats. In addition, although MA and AK substantially improved grip strength and histological changes for dexamethasone-induced muscle atrophy in vivo, the efficacy was superior in the MIX-treated group. Moreover, MIX further increased the expression levels of myogenic factors (MyoD and myogenin) and decreased the expression levels of E3 ubiquitin ligases (atrogin-1 and muscle-specific RING finger protein-1) in vitro and in vivo compared to the MA- and AK-alone treatment groups. Furthermore, MIX increased the levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR) that were reduced by dexamethasone, and downregulated the expression of forkhead box O3 (FoxO3a) induced by dexamethasone. These results suggest that MIX has a protective effect against muscle atrophy by enhancing muscle protein anabolism through the activation of the PI3K/Akt/mTOR signaling pathway and attenuating catabolism through the inhibition of FoxO3a.

Inhibitory activity of xanthoangelol isolated from Ashitaba (Angelica keiskei Koidzumi) towards α-glucosidase and dipeptidyl peptidase-IV: in silico and in vitro studies.

In Indonesia, the sap of Angelica keiskei Koidzumi has been utilized traditionally as a blood-sugar reducer, nonetheless, its molecular mechanism still needs to be studied. This study aimed to isolate xanthoangelol (XA) from the yellow sap of A. keiskei planted in Mount Rinjani, Indonesia, and to investigate its mechanism by in silico and in vitro methods towards α-glucosidase and dipeptidyl peptidase-IV (DPP-IV). The dried yellow sap was macerated using ethanol, subjected to liquid-liquid extraction using a different polarity of solvents, further gradient-eluted with column chromatography. The isolated compound, formed as yellow crystals, melting point 114-114.4 °C, λmax 368 nm, m/z 393.20 [M + H]+, was confirmed as XA. Acarbose, an α-glucosidase inhibitor, and sitagliptin, a DPP-IV inhibitor, respectively, were employed as the reference drugs for both the in silico and in vitro studies. XA interacts with essential amino acid residues 232-237 in the N-terminal N-loop of α-glucosidase by forming a hydrogen bond with Ala234, a salt-bridge with Asp232, and 9 hydrophobic interactions (binding energy -7.81 kcal/mol; Ki = 1.99 µM). These binding modes resemble those of acarbose. Moreover, XA forms hydrogen bonds with Glu205 and Glu206 in the subsite S2 and π-π interaction with Phe357 in the extensive subsite S2 of DPP-IV (binding energy -8.34 kcal/mol; Ki = 0.873 µM), which are similar to those of sitagliptin. XA inhibits both α-glucosidase (IC50 XA = 14.45 µM; IC50 acarbose = 207 µM) and DPP-IV (IC50 XA = 10.49 µM; IC50 sitagliptin = 0.87 µM). Taken together, XA isolated from the yellow sap of A. keiskei Koidzumi might possess the potential to be further developed as an inhibitor of α-glucosidase and DPP-IV.

Anti-obesity effect of Angelica keiskei Jiaosu prepared by yeast fermentation on high-fat diet-fed mice.

In this study, an Angelica keiskei (A. keiskei) Jiaosu (FAK) was prepared by yeast fermentation to investigate its anti-obesity effect on high-fat diet (HFD)-fed mice. 70 SPF grade male C57BL/6J mice were randomly divided into 7 groups (n = 10): blank control group (N), high-fat model group (M), positive control group (Orl), unfermented control group (NF), high-dose intervention group (FH), medium-dose intervention group (FM), and low-dose intervention group (FL). The results showed that FAK intervention significantly reduced the body weight, Lee's index and liver index of HFD-fed mice (P < 0.05). Compared with M group, the serum levels of triglyceride (TG), total cholesterol (TC), leptin and glucose (GLU) in FH group were remarkably decreased and that of interleukin-27 (IL-27) were increased (P < 0.05). The levels of TG, and TC in the liver of mice were also markedly decreased in the FH group (P < 0.05). HE staining results showed that the liver cells in the three intervention groups had less degeneration and fatty vacuoles in the cytoplasm, and the liver cords were orderly arranged compared with that of M group. Furthermore, FAK significantly inhibited epididymal adipose tissue cell expansion induced by HFD. FAK up-regulated the protein expression levels of p-AMPK and PPARα to promote lipolysis and down-regulated the expression of PPARγ to reduce lipid synthesis (P < 0.05). Additionally, the results of gut microbiota showed that after the intervention, a decrease trend of F/B value and Deferribacterota was noticed in the FH group compared with M group. At the genus level, FAK intervention significantly increased that of Ileiobacterium compared to the M group (p < 0.05). A rising trend of norank_f_Muribaculaceae, Lactobacillus, and Bifidobacterium were also observed in the HF group. Conclusively, these findings demonstrated that FAK intervention can effectively improve obesity in mice caused by HFD and the potential mechanisms was related to the regulation of serum levels of leptin and IL-27, lipogenesis and lipolysis in adipose tissue and gut microbiota composition.

In vitro embryotoxicity and mode of antibacterial mechanistic study of gold and copper nanoparticles synthesized from Angelica keiskei (Miq.) Koidz. leaves extract.

The present study demonstrated the in vitro embryotoxicity assessment of gold nanoparticles (AuNPs) and copper nanoparticles (CuNPs) prepared from the leaves extract of Angelica keiskei (Miq.) Koidz. and addressed their mode of antibacterial mechanisms. Both AuNPs and CuNPs were rapidly synthesized and the formations were observed within 1 h and 24 h, respectively. Further the morphological images of the nanoparticles were confirmed through transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). The high-resolution X-ray diffraction (HR-XRD) analysis of the biosynthesized AuNPs and CuNPs were matched with joint committee on powder diffraction standards (JCPDS) file no of 04-0784 and 89-5899, respectively. A strong prominent Au and Cu signals were observed through energy dispersive spectroscopy (EDS) analysis. Fourier transform infrared spectroscopy (FT-IR) analysis confirmed the responsible phytochemicals for the synthesis of AuNPs and CuNPs. In order to assess the toxic effects of AuNPs and CuNPs, bactericidal activity was performed against few of the test pathogens in which the effective inhibition was observed against Gram-negative bacteria than the Gram-positive bacteria. The mode of action and interaction of nanoparticles were performed on the bacterial pathogens and the results concluded that the interaction of nanoparticles initially initiated on the surface of the cell wall adherence followed by ruptured the cells and caused the cell death. In addition to the antibacterial activity, in vitro embryotoxicity studies were performed against zebrafish embryos and the results confirmed that 200 µg/ml concentration of AuNPs showed the embryotoxicity, whereas 2 µg/ml of CuNPs resulted the embryotoxicity. Furthermore, the morphological anomalies of zebrafish embryos revealed the toxic nature of the synthesized nanoparticles.