First report of post-harvest tuber rot of Apios americana Medikus caused by Penicillium expansum in South Korea.

Apios americana Medikus, a perennial vine legume native to North America, is known as 'Indian potato' or 'Apios' for their underground tubers that are used for human consumption in Korea (Choi et al., 2017). These tubers are known to be rich in isoflavones as well as other secondary products to have known several medicinal properties (Chu et al., 2019). The harvested tubers in 2020 were observed to rot during storage of tubers for 4 months at 4°C in the genetic resource storage room at the Bioenergy Crop Research Institute, National Institute of Crop Science, Muan, Jeollanam-do, South Korea. The incidence of rot symptoms with blue mold was less than 1% per 20 kg box. Ten infected tubers were collected from several boxes and the lesions were cut into small pieces, and then surface sterilized in 1.5% NaClO for 2 minutes, followed by rinsing 3 times with sterilized water. To investigate their morphological characteristics, ten isolates were cultured in Malt Extract Agar (MEA) medium at 25° C for 5 days (Pitt and Hocking, 1988). The surface morphology of the mycelium had white or light green fluffy, and completely blue spores were formed after about 5 days. The conidia were one-stage branched with an elliptical shape, about 3.5 to 4.3 × 2.9 to 3.6 (mean 3.8 ± 0.3 × 3.2 ± 0.2) ㎛ in diameter (n=30). Genomic DNAs of the isolates were extracted using Solgent DNA Extraction Kit (Solgent, Daejeon, Korea), and then PCR products of the internal transcribed spacer (ITS1/ITS4) region and the beta-tubulin gene (Bt2a/Bt2b) were sequenced and analyzed (Glass and Donaldson, 1995). The BLASTn showed that the representative isolate had 99% homology with reference Penicillium expansum strain ICMP 2708 (ITS region and TUB2 gene) in NCBI GenBank. The sequences of the isolate were deposited in GenBank as accession numbers MZ636667 and MZ702813 for ITS and TUB2 genes, respectively. Based on the morphological characteristics and molecular analysis, the isolate was identified as P. expansum. Pathogenicity assays of the isolate were also performed using three tubers in three replicates inoculated with spore suspension (concentration, 1×106 conidia/mL) and compared with a control group inoculated with sterilized water. The inoculated Apios tubers were placed in a plastic box maintained in conditions of high humidity at 25°C. Five days after inoculation, the typical symptoms were observed on inoculated tubers, and no symptoms were observed in the control one. P. expansum was again isolated from artificially inoculated tubers to complete Koch's assumption. This is the first report of P. expansum causing tuber rot in A. americana in South Korea. As the cultivated area of Apios is increasing in Korea, it will be necessary to develop effective storage methods and management strategies for the control of storage diseases such as blue mold.

Purification, cDNA cloning and characterization of Kunitz-type protease inhibitors from Apios americana tubers.

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10-7 M and 6 × 10-6 M, respectively.Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-ß-D-thio-galactopyranoside; Ki: inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p-nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p-nitroanilide; PFLNA: Pyr-Phe-Leu-p-nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala-p-nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.

Anti-Inflammatory Effect of Lupinalbin A Isolated from Apios americana on Lipopolysaccharide-Treated RAW264.7 Cells.

Apios americana, a leguminous plant, is used as food in some countries. Although the biological activities of Apios extract have been reported, there have been no reports about the anti-inflammatory mechanism of lupinalbin A on the RAW264.7 cells. In this study, we investigated the anti-inflammatory effect of A. americana lupinalbin A on lipopolysaccharide (LPS)-treated RAW264.7 cells. Lupinalbin A significantly inhibited nitric oxide production and inducible nitric oxide synthase expression in LPS-treated RAW264.7 cells. The expression of cytokines, including interleukin-6, tumor necrosis factor-α, and chemokine of monocyte chemoattractant protein, was reduced under lupinalbin A exposure in LPS-treated RAW264.7 cells. In addition, lupinalbin A significantly decreased LPS-induced interferon (IFN)-ß production and STAT1 protein levels in RAW264.7 cells. Taken together, these results suggest that A. americana lupinalbin A exerts anti-inflammatory effects via the inhibition of pro-inflammatory cytokines and blocking of IFN-ß/STAT1 pathway activation.

Chemical Constituents from Apios americana and Their Inhibitory Activity on Tyrosinase.

The goal of this study was to identify phytochemicals with inhibitory activity against tyrosinase. Nine compounds 1-9 were isolated from the tubers of Apios americana. This is the first report of aromadendrin 5-methyl ether (1) being isolated from the Apios species. Among them, compounds 2 and 8 showed inhibitory activity toward tyrosinase. Based on a Dixon plot, the potential Ki values of competitive inhibitors 2 and 8 were calculated as 10.3 ± 0.8 µM and 44.2 ± 1.7 µM, respectively. An IC50 value of 13.2 ± 1.0 µM was calculated for the slow-binding inhibitor 2 after preincubation with tyrosinase. Additionally, the predicted binding sites between the receptor and ligand, as well as secondary structure changes, in the presence of 2 were examined by molecular simulation.

Soluble Epoxide Hydrolase Inhibitory Activity of Components Isolated from Apios americana Medik.

A new compound 1, 5-methoxy-2,5,7,4'-tetrahydroxy-coumaronochromone, along with seven known compounds (2-8), were isolated from Apios americana using open column chromatography. Their structures were established based on an analysis of 1D and 2D NMR, and MS spectra. Among these, two compounds 1 and 2 showed inhibitory activity on soluble epoxide hydrolase (sEH) at a concentration below 50 µM. The respective competitive (1) and mixed (2) inhibitors were revealed to have Ki values of 21.0 ± 0.8 and 14.5 ± 1.5 µM, based on the Dixon plot. The potential inhibitor (2) was visually presented in a predicted binding pose in the receptor by molecular docking. Additionally, molecular dynamics were performed for a detailed understanding of their complex by Gromacs 4.6.5 package.

Biological activities of unique isoflavones prepared from Apios americana Medik.

Four unique isoflavone aglycones (barpisoflavone A (1), 2'-hydroxygenistein (2), 5-methylgenistein (3), and gerontoisoflavone A (4)) whose structures were related to genistein were prepared from the tuber of Apios americana Medik. We examined the estrogen receptor and androgen receptor binding activities, estrogen agonistic activities, antioxidant activities, and α-glucosidase inhibitory activities of 1-4. The results obtained showed that 2 possessed potent and 1, 3, and 4 possessed moderate estrogen partial agonistic activities, 1 and 2 possessed moderate antioxidant activities, and 2 and 3 possessed moderate α-glucosidase inhibitory activities.

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers.

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.

Investigation of isoflavone constituents from tuber of Apios americana Medik and its protective effect against oxidative damage on RIN-m5F cells.

In our previous study, AI-3, a mixture of isoflavones, was obtained from Apios Americana Medik tuber and showed strong protective ability on oxidative damaged RIN-m5F cells. This study aimed to identify the main compounds of AI-3 and elucidate their activities and underlying mechanism. In results, eleven compounds were purified from AI-3. Among them, Compound 8 (2'-Hydroxy, 5-methoxy genistein-7-O-glucoside, HMG) was the most effective compound against H2O2-induced injury in RIN-m5F cells (stronger than positive control α-LA). Further RNA-seq analysis found that compared with H2O2 group, 388 differentially expressed genes (DEGs) were identified in HMG group. The enrichment analyses revealed fluid shear stress and atherosclerosis pathway and hepatocellular carcinoma pathway enriched the most DEGs, in which HOX-1, GST, NQO1, SQSTM, TrxR1 were significantly up-regulated. The finding indicated Keap1-Nrf2-ARE signaling pathway may play essential role in the protective effect of HMG on oxidative damaged RIN-m5F cells.

Anti-diabetic effect of the lupinalbin A compound isolated from Apios americana: In vitro analysis and molecular docking study.

Dipeptidyl peptidase 4 (DPP4) and α-glucosidase inhibitors have been developed as anti-diabetic agents for the treatment of diabetes mellitus. In the present study, the anti-diabetic effects of the lupinalbin A compound isolated from Apios americana was investigated by measuring its inhibitory activity against DPP4 and α-glucosidase. To detect the inhibitory effect of lupinalbin A, DPP4 and α-glucosidase assays were performed in vitro. Molecular docking analysis was performed using AutoDock 4.2. The IC50 values of lupinalbin A against DPP4 and α-glucosidase were 45.2 and 53.4 µM, respectively. Analysis of the enzyme kinetics revealed that lupinalbin A interacted with the active site of DPP4 in a competitive manner, with an inhibition constant (Ki) value of 35.1±2.0 µM, whereas the lupinalbin A interaction with α-glucosidase was non-competitive, with a Ki value of 45.0 µM. Molecular docking analysis revealed a binding pose between the DPP4 enzyme and lupinalbin A. Taken together, these data suggest lupinalbin A is more effective against DPP4 than α-glucosidase, with regard to its anti-diabetic effects.

Apios americana Medik flowers polysaccharide (AFP) alleviate Cyclophosphamide-induced immunosuppression in ICR mice.

Immunosuppression refers to the suppression of the immune response. The immune function of immunocompromised people is not enough to resist bacterial, viral, fungal and other infections, leading to a series of diseases. A large number of experimental data show that polysaccharide compounds are immune modulators, which can enhance the body immunity with little toxic. Meanwhile, it can reduce the side effects of commonly used immunosuppressants, such as cytotoxicity, decreased ability of the body to fight infection, and inhibition of the reproduction of bone marrow hematopoietic cells. It can be used as oral or injectable drugs. In this study, a purified polysaccharide was primarily extracted from the flowers of Apios americana Medik (AAM), which can improve the immunosuppression induced by cyclophosphamide (CTX). The immunoenhancement effect of AFP was evaluated by measuring the body weight, immune organ index, cytokine secretion and antibody generated levels of CTX-induced mice. Our results showed that AFP could significantly improve the above immune indexes, which indicated AFP could alleviate immunosuppression induced by CTX. The study provided a theoretical basis for the promotion, development and application of AAM as a newly introduced food material.

Improvement in post-partum uterine involution in rats treated with Apios americana.

OBJECTIVE: Apios americana, a plant used as a staple ingredient of native American diets, has various properties, including anti-cancer, anti-hyperglycemic, hypotensive, and anti-inflammatory activity. In Japan, Apios is used as a post-natal medication. After parturition, women undergo a period of recovery as they return to pre-pregnancy conditions. However, few health products that aid post-partum recovery are on the market. We explored whether Apios can accelerate the post-partum recovery process, in particular the involution of the uterus. METHODS: Female rats kept in individual cages were mated with two male rats, with the exception of the control group (female rats without mating, on basal diet; n=6). After delivery, rats were divided into five groups based on their diet: basal diet (model; n=6); basal diet+oral intake at 5.4 g/kg of Chanfukang granules (a Chinese patent medicine preparation for post-partum lochia) (positive; n=6); basal diet containing 10% Apios powder (low; n=6); basal diet containing 20% Apios powder (medium; n=6); basal diet containing 40% Apios powder (high; n=6). Five days later, uteri and spleens were weighed. Uterus and spleen indices for each rat were calculated by dividing visceral weight by the total weight. Hormone and cytokine concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Histological analysis of uteri was completed using hematoxylin and eosin (H&E) staining. Expression of matrix metalloproteinases and inhibitors in uteri was measured by western blotting. RESULTS: Our results showed that Apios treatment reduced the post-partum uterus index and regulated the hormone concentrations. Moreover, we found that the process of uterine involution was accelerated, based on morphological changes in the uterus. In addition, our results indicated that Apios alleviated the inflammatory response induced by the involution process. Transforming growth factor ß was also found to be regulated by Apios. There were significant downregulation of matrix metalloproteinases and upregulation of their inhibitors by Apios, which suggested that Apios increased the rate of the collagen clearance process. CONCLUSIONS: These results, based on experimental observations at the molecular and protein levels, verified our hypothesis that Apios can improve uterine involution, and demonstrated the potential application of Apios in post-partum care.

Apios americana Medik flowers polysaccharide (AFP-2) attenuates H2O2 induced neurotoxicity in PC12 cells.

Oxidative stress is a critical cause for the progression of neurodegenerative diseases, indicating that screening natural antioxidant and pharmacological targeting of mechanism need more attention. Natural plant polysaccharides are the primary group of natural antioxidants showing mighty antioxidant activity. Therefore, a polysaccharide (AFP-2) from flowers of Apios americana Medik was isolated, purified and characterized. Then, a H2O2-induced PC12 cells oxidative stress model was established to investigate the neuroprotective role of AFP-2 and to clarify the potential mechanism of action against H2O2-induced neurotoxicity. It was found that AFP-2 reduced ROS production and mitochondrial damage caused by hydrogen peroxide in PC12 cells. In addition, data from transmission electron microscope and western blot displayed that AFP-2 significantly activated autophagy in PC12 cells via Akt-mTOR pathway. These results together qualify AFP-2 as an interesting natural polysaccharide worth further investigation as a neuroprotective agent.

Apios americana Medikus tuber polysaccharide exerts anti-inflammatory effects by activating autophagy.

The tubers of Apios americana Medikus possess high nutritional value and have been used as food in many countries for a long time. However, few researches have focused on the tuber polysaccharides. In the present study, a purified polysaccharide (ATP-1) was isolated with the average molecular weights of 12.16 kDa. ATP-1 significantly suppressed the release of nitric oxide (NO) and inflammatory cytokines from LPS-induced RAW 264.7 cells, as well as oxidative stress and mitochondrial dysfunction. Meanwhile, ATP-1 reduced oxidative damage via the NF-κB, MAPKs and Nrf2-Keap1 signaling pathways in RAW264.7 macrophages. Furthermore, autophagy was activated by HMGB1-Beclin1, Sirt1-FoxO1 and Akt-mTOR signaling pathways, leading to a relief of oxidative stress, mitochondrial dysfunction, inflammation and an expression enhancement of autophagy-related proteins, such as LC3, Beclin1, Atg4, Atg5, and Atg7. In summary, our results suggested that ATP-1 might help to activate the anti-inflammation system，resulting in prevention of LPS-induced damage in RAW264.7 cells.

Apios americana Medik flowers extract protects PC12 cells against H2O2 induced neurotoxicity via regulating autophagy.

Since flavonoids are antioxidant compounds, they could beneficially affect neurodegenerative diseases where reactive oxygen species are involved. In this study, we firstly isolated and identified fourteen compounds from the flowers of Apios americana Medik. Then, we tested whether Apios americana Medik flowers water extract (AFWE) exerts a protective effect on H2O2 induced PC12 cells injure. As expected, pretreatment with AFWE inhibited cytotoxicity and DNA condensation in H2O2induced PC12 cells. Exposure of PC12 cells to H2O2 resulted in reactive oxygen species accumulation and mitochondrial dysfunction, while AFWE alleviated these damages. AFWE obviously reversed the alternations as H2O2 increased Caspase-3 and decreased the ratio of Bcl-2/Bax expressions. Furthermore, autophagy in PC12 cells was further activated by AFWE, which was beneficial to resisting adversity. These results manifest that AFWE prevents H2O2 induced damage via regulating autophagy.

Effects of C-Glycosides from Apios americana Leaves against Oxidative Stress during Hyperglycemia through Regulating Mitogen-Activated Protein Kinases and Nuclear Factor Erythroid 2-Related Factor 2.

Main components of Apios americana leaves extract (ALE) were flavonoid C-glycosides, including vitexin (46.7%), schaftoside (18.9%), and orientin (4.32%). In vitro, ALE restored glucose consumption, glucose uptake, and glycogen content in glucose-induced hepatic cells. Exposure of HepG2 cells to high glucose resulted in reactive oxygen species and O2- accumulation, while ALE alleviated these increases by 47 ± 0.68 and 68 ± 0.74%, respectively. Glucose increased c-Jun N-terminal kinase (JNK) and decreased extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 phosphorylation, while ALE reduced p-JNK and p-p38 but not p-ERK1/2, accompanied by nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1, and NAD(P)H quinine oxidoreductase 1 downregulation. In vivo, the lifespan of Caenorhabditis elegans was more violently shortened by paraquat under hyperglycemia, while ALE protected this damage in N2 worms (2.6 times extension) but not in daf-16 mutants. Furthermore, p38/PMK-1 and Nrf2/SKN-1 expressions in worms were suppressed by glucose, which were reversed by ALE treatment. These results suggest that ALE prevents glucose-induced damage via regulating specific mitogen-activated protein kinases and Nrf2 pathways.