Purification of phenoloxidase from Haliotis discus hannai and its anti-inflammatory activity in vitro.

Haliotis discus hannai, a food with a high protein content, is widely consumed in Asian countries. It is known to have antioxidant, anticancer, and antibacterial effects. Since the biological significance of H. discus hannai hemolymph has not been widely studied, the objective of the present study was to purify phenoloxidase (PO) and investigate its immunological effects on human colonic epithelial cells. PO was purified through ammonium sulfate precipitation and one step column chromatography. The molecular weight of the protein was about 270 kDa. When PO was mixed with Gram-negative bacteria-derived lipopolysaccharide (LPS) at various ratios (10:1-1:10, w/w), the amount of residual LPS was reduced. PO at concentrations up to 200 µg/mL was not cytotoxic to HT-29 cells. The inflammatory response induced by LPS in HT-29 cells was regulated when the concentration of PO was increased. With increasing concentration of PO, production levels of pro-inflammatory cytokines, cytokines associated with hyperimmune responses such as IL4, IL-5, and INF-γ, and prostaglandin 2 (PGE2) were regulated. It was thought that simultaneous treatment with PO and LPS anti-inflammatory effects in HT-29 cells showed by regulating the ERK1/2-mediated NF-κB pathway. Results of this study suggest that H. discus hannai hemolymph is involved in the regulation of Gram-negative bacteria-related inflammatory immune responses in human colonic epithelial cells.

Echinatin induces reactive oxygen species-mediated apoptosis via JNK/p38 MAPK signaling pathway in colorectal cancer cells.

Colorectal cancer (CRC) is a very common and deadly cancer worldwide, and oxaliplatin is used as first-line chemotherapy. However, resistance usually develops, limiting treatment. Echinatin (Ech) is the main component of licorice and exhibits various therapeutic effects on inflammation-mediated diseases and cancer, ischemia/reperfusion, and liver injuries. The present study elucidated the underlying molecular mechanism of Ech-induced apoptosis in both oxaliplatin-sensitive (HT116 and HT29) and -resistant (HCT116-OxR and HT29-OxR) CRC cells. To evaluate the antiproliferative activities of Ech, we performed MTT and soft agar assays. Ech reduced viability, colony size, and numbers of CRC cells. The underlying molecular mechanisms were explored by various flow cytometry analyses. Ech-induced annexin-V stained cells, reactive oxygen species (ROS) generation, cell cycle arrest, JNK/p38 MAPK activation, endoplasmic reticulum (ER) stress, mitochondrial membrane potential depolarization, and multi-caspase activity. In addition apoptosis-, cell cycle-, and ER stress-related protein levels were confirmed by western blotting. Moreover, we verified ROS-mediated cell death by treatment with inhibitors such as N-acetyl-L-cysteine, SP600125, and SB203580. Taken together, Ech exhibits anticancer activity in oxaliplatin-sensitive and -resistant CRCs by inducing ROS-mediated apoptosis through the JNK/p38 MAPK signaling pathway. This is the first study to show that Ech has the potential to treat drug-resistant CRC, providing new directions for therapeutic strategies targeting drug-resistant CRC.

Drug Development from Natural Products Based on the Pathogenic Mechanism of Asthma.

Asthma is a chronic inflammatory disease of the pulmonary system associated with many wheeze-to-sleep apnea complications that may lead to death. In 2019, approximately 262 million patients suffered from asthma, and 455 thousand died from the disease worldwide. It is a more severe health problem in children and older adults, and as the aging of society intensifies, the problem will continue to worsen. Asthma inducers can be classified as indoor and outdoor allergens and can cause asthma due to their repeated invasion. There are several theories about asthma occurrence, such as the imbalance between Th1 and Th2, inflammation in the pulmonary system, and the abnormal apoptosis/cell proliferation of cells related to asthma. Although there are many medications for asthma, as it is an incurable disease, the purpose of the drugs is only to suppress the symptoms. The current drugs can be divided into relievers and controllers; however, as they have many adverse effects, such as immune suppression, growth retardation, promotion of cataracts, hyperactivity, and convulsions, developing new asthma drugs is necessary. Although natural products can have adverse effects, the development of asthma drugs from natural products may be beneficial, as some have anti-asthmatic effects such as immune modulation, anti-inflammation, and/or apoptosis modulation.

Dual inhibition of EGFR and MET by Echinatin retards cell growth and induces apoptosis of lung cancer cells sensitive or resistant to gefitinib.

Patients with non-small-cell lung cancer (NSCLC) containing epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to tyrosine kinase inhibitor gefitinib; however, the treatment is less effective over time. Gefitinib resistance mechanisms include MET gene amplification. A therapeutic strategy targeting MET as well as EGFR can overcome resistance to gefitinib. In the present study we identified Echinatin (Ecn), a characteristic chalcone in licorice, which inhibited both EGFR and MET and strongly altered NSCLC cell growth. The antitumor efficacy of Ecn against gefitinib-sensitive or -resistant NSCLC cells with EGFR mutations and MET amplification was confirmed by suppressing cell proliferation and anchorage-independent colony growth. During the targeting of EGFR and MET, Ecn significantly blocked the kinase activity, which was validated with competitive ATP binding. Inhibition of EGFR and MET by Ecn decreases the phosphorylation of downstream target proteins ERBB3, AKT and ERK compared with total protein expression or control. Ecn induced the G2/M cell cycle arrest, and apoptosis via the intrinsic pathway of caspase-dependent activation. Ecn induced ROS production and GRP78, CHOP, DR5 and DR4 expression as well as depolarized the mitochondria membrane potential. Therefore, our results suggest that Ecn is a promising therapeutic agent in NSCLC therapy.

Industrial attributes of ß-glucanase produced by Bacillus sp. CSB55 and its potential application as bio-industrial catalyst.

The ß-glucanase produced from Bacillus sp. CSB55 not only depicts the potent industrial characteristics but also relates as bio-industrial catalyst supporting the spontaneous formation of the products, high hydrolytic efficiency, and feasibility of the enzymatic reaction. A homogeneous ß-glucanase (GluB55) was purified via various purification processes resulting in 11.69% yield and 14.24-fold purity. Biochemical characterization of the purified enzyme revealed the molecular mass of approximately 40 kDa, which was verified by zymography. The optimum activity of GluB55 was determined at pH 7.2 and 55 °C. GluB55 could highly hydrolyze carboxymethylcellulose and was stable over a wide range of pH, retaining more than 70% residual activity at pH 5.8-11.0 and carried 100% thermostability as high as 60 °C. In addition, it showed 68% residual activity at 70 °C. The N-terminal amino acid sequence of GluB55 was Ala-Asn-Pro-Glu-Leu-Val-Asn-X-Gln-Ala-X-X-Ala-X-Gln-Gly. The enzyme activity was stimulated by Co2+ (158.6%), Zn2+ (211.1%), Mn2+ (264.4%), and Ba2+ (211.4%). Enzyme kinetics showed Km and Vmax values of 0.022 mg mL-1 and 994.56 ± 3.72 U mg-1, respectively. Q10 was calculated to be 1.12. ∆H, ∆G, and ∆S were low revealing that the formation of the transition phase and conversion to the product is very well organized. The lower the free energy change (∆G), the more feasible is the reaction.

Deoxypodophyllotoxin, a Lignan from Anthriscus sylvestris, Induces Apoptosis and Cell Cycle Arrest by Inhibiting the EGFR Signaling Pathways in Esophageal Squamous Cell Carcinoma Cells.

Deoxypodophyllotoxin (DPT) derived from Anthriscus sylvestris (L.) Hoffm has attracted considerable interest in recent years because of its anti-inflammatory, antitumor, and antiviral activity. However, the mechanisms underlying DPT mediated antitumor activity have yet to be fully elucidated in esophageal squamous cell carcinoma (ESCC). We show here that DPT inhibited the kinase activity of epidermal growth factor receptor (EGFR) directly, as well as phosphorylation of its downstream signaling kinases, AKT, GSK-3ß, and ERK. We confirmed a direct interaction between DPT and EGFR by pull-down assay using DPT-beads. DPT treatment suppressed ESCC cell viability and colony formation in a time- and dose-dependent manner, as shown by MTT analysis and soft agar assay. DPT also down-regulated cyclin B1 and cdc2 expression to induce G2/M phase arrest of the cell cycle and upregulated p21 and p27 expression. DPT treatment of ESCC cells triggered the release of cytochrome c via loss of mitochondrial membrane potential, thereby inducing apoptosis by upregulation of related proteins. In addition, treatment of KYSE 30 and KYSE 450 cells with DPT increased endoplasmic reticulum stress, reactive oxygen species generation, and multi-caspase activation. Consequently, our results suggest that DPT has the potential to become a new anticancer therapeutic by inhibiting EGFR mediated AKT/ERK signaling pathway in ESCC.

Picropodophyllotoxin, an Epimer of Podophyllotoxin, Causes Apoptosis of Human Esophageal Squamous Cell Carcinoma Cells Through ROS-Mediated JNK/P38 MAPK Pathways.

Esophageal squamous cell carcinoma (ESCC), a major histologic type of esophageal cancer, is one of the frequent causes of cancer-related death worldwide. Picropodophyllotoxin (PPT) is the main component of Podophyllum hexandrum root with antitumor activity via apoptosis-mediated mechanisms in several cancer cells. However, the underlying mechanism of the PPT effects in apoptosis induction in cancer remains ambiguous. Hence, in this study, we evaluate the anti-cancer effects of PPT in apoptotic signaling pathway-related mechanisms in ESCC cells. First, to verify the effect of PPT on ESCC cell viability, we employed an MTT assay. PPT inhibited the viability of ESCC cells in time- and dose-dependent manners. PPT induced G2/M phase cell cycle arrest and annexin V-stained cell apoptosis through the activation of the c-Jun N-terminal kinase (JNK)/p38 pathways. Furthermore, the treatment of KYSE 30 and KYSE 450 ESCC cells with PPT induced apoptosis involving the regulation of endoplasmic reticulum stress- and apoptosis-related proteins by reactive oxygen species (ROS) generation, the loss of mitochondrial membrane potential, and multi-caspase activation. In conclusion, our results indicate that the apoptotic effect of PPT on ESCC cells has the potential to become a new anti-cancer drug by increasing ROS levels and inducing the JNK/p38 signaling pathways.

Socheongryongtang suppresses COPD-related changes in the pulmonary system through both cytokines and chemokines in a LPS COPD model.

Context: Socheongryongtang is a traditional Korean medical prescription used to treat pulmonary diseases.Objective: This study investigated the therapeutic mechanism of socheongryongtang for pulmonary diseases.Materials and methods: Seventy BALB/c mice were used: control, 0.8 mg/kg/study LPS intranasal instillation, 1 mg/kg/day Spiriva oral administration for five days, two socheongryongtang groups (150 or 1500 mg/kg/day orally treatment for five days). To illuminate the anti-COPD mechanism, several factors were evaluated such as WBC and differential counts in BALF and IgE in serum, morphological changes, and changes of COPD-related cytokines (TNF-α, IFN-γ, TGF-ß) and chemokines (CXCL1, CCL-2, CCR2) in the lung. In order to confirm the statistical significance, all results were compared under p < 0.01 and p < 0.05.Results: LPS induced a high level of WBC, neutrophils and eosinophils in our in vivo study. Additionally, COPD related cytokines and chemokines such as TNF-α, IFN-γ, TGF-ß, CXCL1, CCL-2 and CCR2 were induced by LPS. Compared to the LPS treatment group, socheongryongtang significantly controlled the level of WBC, neutrophils and eosinophils as well as the level of IgE. It effectively down-regulated the morphological changes, such as fibrosis near bronchoalveolar spaces, small airway destruction (emphysema), etc. It also inhibited the levels of COPD-related cytokines (TNF-α, IFN-γ, TGF-ß) and chemokines (CXCL1, CCL-2, CCR2) compared to the LPS treatment group. In particular, socheongryongtang significantly down-regulated the levels of TNF-α, IFN-γ, and CCR2.Conclusions: Socheongryongtang controlled COPD, but as it has been used as a prescription for respiratory disease, we should additionally evaluate the therapeutic effects against various pulmonary diseases.

Licochalcone D directly targets JAK2 to induced apoptosis in human oral squamous cell carcinoma.

Licochalcone (LC) families have been reported to have a wide range of biological function such as antioxidant, antibacterial, antiviral, and anticancer effects. Although various beneficial effects of LCD were revealed, its anticancer effect in human oral squamous cancer has not been identified. To examine the signaling pathway of LCD's anticancer effect, we determined whether LCD has physical interaction with Janus kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) signaling, which is critical in promoting cancer cell survival and proliferation. Our results demonstrated that LCD inhibited the kinase activity of JAK2, soft agar colony formation, and the proliferation of HN22 and HSC4 cells. LCD also induced mitochondrial apoptotic events such as altered mitochondrial membrane potential and reactive oxygen species production. LCD increased the expression of apoptosis-associated proteins in oral squamous cell carcinoma (OSCC) cells. Finally, the xenograft study showed that LCD significantly inhibited HN22 tumor growth. Immunohistochemical data supported that LCD suppressed p-JAK2 and p-STAT3 expression and induced cleaved-caspase-3 expression. These results indicate that the anticancer effect of LCD is due to the direct targeting of JAK2 kinase. Therefore, LCD can be used for therapeutic application against OSCC.

Correction: Kim, Y. K. et al. Tussilagone Inhibits the Inflammatory Response and Improves Survival in CLP-Induced Septic Mice. Int. J. Mol. Sci. 2017, 18, 2744.

We wish to make the following corrections to this paper [...].

Identification and Extraction Optimization of Active Constituents in Citrus junos Seib ex TANAKA Peel and Its Biological Evaluation.

Citrus junos Seib ex TANAKA possesses various biological effects. It has been used in oriental remedies for blood circulation and the common cold. Recently, biological effects of C. junos peel have been reported. However, optimization of the biological properties of C. junos peel preparations has yet to be reported on. We developed a high-performance liquid chromatography (HPLC) method for quantification of the active constituents in C. junos peel. Hot water and ethanolic extracts of C. junos peel were prepared and their chemical profiles and biological activities were evaluated. The 80% ethanolic extract demonstrated the greatest antioxidant activity and phenolic content, while the 100% ethanolic extract had the greatest xanthine oxidase inhibitory activity. Elastase inhibition activity was superior in aqueous and 20% ethanolic extracts. The contents of two flavonoids were highest in the 100% ethanolic extract. We postulated that the antioxidant and anti-aging effects of C. junos peel extract could be attributed to phenolics such as flavonoids. Our results suggest that the flavonoid-rich extract of C. junos may be utilized for the treatment and prevention of metabolic disease and hyperuricemia while the water-soluble extract of C. junos could be used as a source for its anti-aging properties.

Metabolomic Approach for Discrimination of Cultivation Age and Ripening Stage in Ginseng Berry Using Gas Chromatography-Mass Spectrometry.

The purpose of this study was to analyze metabolic differences of ginseng berries according to cultivation age and ripening stage using gas chromatography-mass spectrometry (GC-MS)-based metabolomics method. Ginseng berries were harvested every week during five different ripening stages of three-year-old and four-year-old ginseng. Using identified metabolites, a random forest machine learning approach was applied to obtain predictive models for the classification of cultivation age or ripening stage. Principal component analysis (PCA) score plot showed a clear separation by ripening stage, indicating that continuous metabolic changes occurred until the fifth ripening stage. Three-year-old ginseng berries had higher levels of valine, glutamic acid, and tryptophan, but lower levels of lactic acid and galactose than four-year-old ginseng berries at fully ripened stage. Metabolic pathways affected by different cultivation age were involved in amino acid metabolism pathways. A random forest machine learning approach extracted some important metabolites for predicting cultivation age or ripening stage with low error rate. This study demonstrates that different cultivation ages or ripening stages of ginseng berry can be successfully discriminated using a GC-MS-based metabolomic approach together with random forest analysis.

Retrochalcone Echinatin Triggers Apoptosis of Esophageal Squamous Cell Carcinoma via ROS- and ER Stress-Mediated Signaling Pathways.

Esophageal squamous cell carcinoma (ESCC) is a poor prognostic cancer with a low five-year survival rate. Echinatin (Ech) is a retrochalone from licorice. It has been used as a herbal medicine due to its anti-inflammatory and anti-oxidative effects. However, its anticancer activity or underlying mechanism has not been elucidated yet. Thus, the objective of this study was to investigate the anti-tumor activity of Ech on ESCC by inducing ROS and ER stress dependent apoptosis. Ech inhibited ESCC cell growth in anchorage-dependent and independent analysis. Treatment with Ech induced G2/M phase of cell cycle and apoptosis of ESCC cells. It also regulated their related protein markers including p21, p27, cyclin B1, and cdc2. Ech also led to phosphorylation of JNK and p38. Regarding ROS and ER stress formation associated with apoptosis, we found that Ech increased ROS production, whereas its increase was diminished by NAC treatment. In addition, ER stress proteins were induced by treatment with Ech. Moreover, Ech enhanced MMP dysfunction and caspases activity. Furthermore, it regulated related biomarkers. Taken together, our results suggest that Ech can induce apoptosis in human ESCC cells via ROS/ER stress generation and p38 MAPK/JNK activation.

Effects of Harvest Time on Phytochemical Constituents and Biological Activities of Panax ginseng Berry Extracts.

Ginseng (Panax ginseng) has long been used as a traditional medicine for the prevention and treatment of various diseases. Generally, the harvest time and age of ginseng have been regarded as important factors determining the efficacy of ginseng. However, most studies have mainly focused on the root of ginseng, while studies on other parts of ginseng such as its berry have been relatively limited. Thus, the aim of this study iss to determine effects of harvest time on yields, phenolics/ginsenosides contents, and the antioxidant/anti-elastase activities of ethanol extracts of three- and four-year-old ginseng berry. In both three- and fourfour-year-old ginseng berry extracts, antioxidant and anti-elastase activities tended to increase as berries ripen from the first week to the last week of July. Liquid chromatography-tandem mass spectrometry analysis has revealed that contents of ginsenosides except Rg1 tend to be the highest in fourfour-year-old ginseng berries harvested in early July. These results indicate that biological activities and ginsenoside profiles of ginseng berry extracts depend on their age and harvest time in July, suggesting the importance of harvest time in the development of functional foods and medicinal products containing ginseng berry extracts. To the best of our knowledge, this is the first report on the influence of harvest time on the biological activity and ginsenoside contents of ginseng berry extracts.

Tyrosinase Inhibition Antioxidant Effect and Cytotoxicity Studies of the Extracts of Cudrania tricuspidata Fruit Standardized in Chlorogenic Acid.

In the present study, various extracts of C. tricuspidata fruit were prepared with varying ethanol contents and evaluated for their biomarker and biological properties. The 80% ethanolic extract showed the best tyrosinase inhibitory activity, while the 100% ethanolic extract showed the best total phenolics and flavonoids contents. The HPLC method was applied to analyze the chlorogenic acid in C. tricuspidata fruit extracts. The results suggest that the observed antioxidant and tyrosinase inhibitory activity of C. tricuspidata fruit extract could partially be attributed to the presence of marker compounds in the extract. In this study, we present an analytical method for standardization and optimization of C. tricuspidata fruit preparations. Further investigations are warranted to confirm the in vivo pharmacological activity of C. tricuspidata fruit extract and its active constituents and assess the safe use of the plant for the potential development of the extract as a skin depigmentation agent.

Licochalcone C induced apoptosis in human oral squamous cell carcinoma cells by regulation of the JAK2/STAT3 signaling pathway.

Oral cancer is of an aggressive malignancy that arises on oral cavity and lip, 90% of cancers histologically originated in the squamous cells. Licochalcone (LC)C has been known as natural phenolic chalconoid substances, and its origin is the root of Glycyrrhiza glabra or Glycyrrhiza inflata. LCC inhibited oral squamous cell carcinoma (OSCC) cell viability, mitochondrial function, and anchorage-independent growth in a dose-dependent manner. To investigate the ability of LCC to target Janus kinase 2 (JAK2), we performed pull-down binding assay, kinase assay, and docking simulation. The molecular docking studies were performed between JAK2 and the potent inhibitor LCC. It was shown that LCC tightly interacted with ATP-binding site of JAK2. In addition, LCC inhibited the JAK2/signal transducer and activator of transcription 3 pathway, upregulated p21, and downregulated Bcl-2, Mcl-1, and Survivin, while it disrupted mitochondrial membrane potential and subsequently caused cytochrome c release with activation of multi-caspase, eventually leading to apoptosis in HN22 and HSC4 cells. LCC elevated the protein levels of Bax, cleaved Bid and PARP, and increased Apaf-1, and this effect was reversed by LCC treatment. Our results demonstrated that treatment of OSCC cells with LCC induced the death receptor (DR)4 and DR5 expression level with the generation of reactive oxygen species and the upregulation of CHOP protein expression. Taken together, these results could provide the basis for clinical application as a new therapeutic strategy in the treatment of oral cancer.

Phytochemical Constituents and the Evaluation Biological Effect of Cinnamomum yabunikkei H.Ohba Leaf.

Cinnamomum yabunikkei H.Ohba leaf is known as a traditional medicinal material in Korea. However, no scientific identification of the components or efficacy of C.yabunikkei H.Ohba leaf has been reported. In the present study, we prepared various solvent extracts of C.yabunikkei H.Ohba leaf to understand its basic properties and evaluated the antioxidant, xanthine oxidase inhibitory, and elastase inhibitory activities of hexane, ethyl acetate, acetone, methanol, ethanol, and water extracts for the first time. The antioxidant properties were evaluated based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, reducing power, and total phenolic contents. The hot water extract showed the highest DPPH radical scavenging activity and total phenolic contents, and the reducing power was the highest in the water extract. The hexane extract showed an excellent elastase inhibitory effect compared to control (phosphoramidone) and the highest xanthine oxidase inhibitory activity. These results present basic information for the possible uses of the hot water and hexane extracts from C. yabunikkei leaf for the treatment of diseases caused by oxidative imbalance. In the present study, individual extracts exhibited different effects. Therefore, it is hypothesized that the applicability of C. yabunikkei will depend on the extraction method and nature of the extract. The hot water and hexane extracts could be used as antioxidants, and as anti-gout and anti-wrinkle materials respectively. Several biologically active substances present in hexane extract of C. yabunikkei have been analyzed by GCMS and demonstrated to possess antioxidant and xanthine oxidase inhibitory activity. To the best of our knowledge, this is the first study that reports the chemical profiling and biological effects of various C. yabunikkei leaf extracts, suggesting their potential use in food therapy, cosmetics or alternative medicine.

Optimization of the Extraction Conditions and Biological Evaluation of Dendropanax morbifera H. Lev as an Anti-Hyperuricemic Source.

Dendropanax morbifera H. Levis a medicinal plant native to South Korea, East Asia, and South America. Among some 75 species, one species grows in Korea. In previous studies, D. morbifera extracts with anti-oxidant, anti-inflammatory, anti-complementary and anti-cancer activities were reported. The present study aims to investigate optimization of extraction and evaluation of anti-hyperuricemic effects of D. morbifera leaf and the phytochemicals contained therein. Ethanol and hexane extract were found to display the best xanthine oxidase inhibition among six types of solvent and water extract. The antioxidant effect of the ethanol extract was superior to that of the hexane extract. The DPPH radical scavenging effect of the ethanol and hexane extracts were 81.52 ± 1.57% and 2.69 ± 0.16. The reducing power of the ethanol and hexane extracts were 9.71 ± 0.15 and 0.89 ± 0.01 mg/g equivalent of gallic acid. Total phenols of the ethanol and hexane extracts were 6.53 ± 0.16 and 0.63 ± 0.001 mg/g equivalent of gallic acid. In addition, we compared the two marker compounds from D. morbifera, chlorogenic acid and rutin, which were determined in the ethanol extract at 0.80 ± 0.03% and 0.52 ± 0.01%, respectively. We found that the ethanol extracts showed better xanthine oxidase inhibition than hexane extracts. Especially, ethanol extracts showed higher antioxidant activity than hexane extracts. Based on these results, we selected the ethanol extract as an effective xanthine oxidase inhibitor and confirmed whether ethanol extracts showed xanthine oxidase inhibition in animal experiments. The in vivo mouse study demonstrated that ethanol extract of D. morbifera leaf at the dose of 300 mg/kg could inhibit blood/hepatic xanthine oxidase activity and this result shows that the xanthine oxidase inhibitory activity in vitro is reproduced in vivo. The present study showed that ethanol extract was optimal xanthine oxidase inhibitor which can be applied to prevent diseases related to hyperuricemia.

Development and Validation of a HPLC-UV Method for Extraction Optimization and Biological Evaluation of Hot-Water and Ethanolic Extracts of Dendropanax morbifera Leaves.

Dendropanax morbifera Leveille (Araliaceae) has been used in traditional oriental remedies for cancer, inflammation, diabetes, and thrombosis. However, a validated analytical method, standardization, and optimization of extraction conditions with respect to biological activity have not been reported. In this study, a simple and validated HPLC method for identifying and quantifying active substances in D. morbifera was developed. Hot water and ethanolic D. morbifera leaf extracts from different production regions were prepared and evaluated with regard to their chemical compositions and biological activities. The contents of active compounds such as rutin and chlorogenic acid were determined in four samples collected from different regions. The 80% ethanolic extract showed the best antioxidant activity, phenolic content, reducing power, and xanthine oxidase (XO) inhibitory activity. The validated HPLC method confirmed the presence of chlorogenic acid and rutin in D. morbifera leaf extracts. The antioxidant and XO inhibitory activity of D. morbifera extract could be attributed to the marker compounds. Collectively, these results suggest that D. morbifera leaves could be beneficial for the treatment or prevention of hyperuricemia-related disease, and the validated HPLC method could be a useful tool for the quality control of food or drug formulations containing D. morbifera.

Molecular Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by InDel Markers of Chloroplast DNA.

The tuber of Cynanchum wilfordii (Baekshuoh Radix in Korean) is an important medicinal herb in Korea and China; however, it is difficult to differentiate C. wilfordii from a related medicinal herb, C. auriculatum (Baishouwu Radix in Chinese). We sought to develop a molecular method that could be used to distinguish between the tubers of C. wilfordii and C. auriculatum. We aligned the chloroplast genome sequences (available in the NCBI database) of the two species and identified three species-specific insertion and deletion (InDel) sites in the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 intergenic spacer (IGS) regions. To confirm the presence of these three InDels and validate their use as markers, we designed three primer pairs to amplify the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 IGS regions. Polymerase chain reaction (PCR) amplification of the trnQ-psbK IGS region yielded a 249 bp fragment for C. wilfordii, and 419 bp fragment for C. auriculatum, whereas the rps2-rpoC2 IGS primers produced a 629 bp fragment from C. wilfordii and a 282 bp fragment from C. auriculatum. In the psaJ-rpl33 IGS region, allele fragments of 342 and 360 bp in length were amplified from C. wilfordii, whereas 249 and 250 bp fragment were amplified from C. auriculatum. We propose these three InDel markers as a valuable, simple, and efficient tool for identifying these medicinal herbs and will thus reduce adulteration of these herbal materials in commercial markets.