Geum Soog Kim

Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu.

ETHNOPHARMACOLOGICAL RELEVANCE Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu. MATERIAL AND METHODS Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells. RESULTS A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) ((1)H, (13)C, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfey׳s method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70μM. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-κB activation by blocking IκBα degradation and NF-κB phosphorylation in LPS-stimulated RAW264.7 cells. CONCLUSIONS These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-κB activation associated with IκBα degradation and NF-κB phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases.

Comparison of Growth Characteristics and Ginsenosides Content of 6-Year-Old Ginseng (Panax ginseng C. A. Meyer) by Drainage Class in Paddy Field

**Ginseng Research Division, Department of Herbal Crop Research, NIHHS, RDA, Eumseong 369-873, Korea.**Horticultural and Herbal Crop Environment Division, NIHHS, RDA, Suwon 440-706, Korea.ABSTRACT : To develop the practical cultivation for paddy field, we investigated the properties of paddy soil, growth char-acteristics and ginsenoside content of 6-year-old ginseng, Cheonpung variety between poor drainage class (PDC) and imper-fect drainage class (IDC). Groundwater level in PDC showed monthly small changes of 20~30㎝, while IDC showedmonthly great changes of 28~71 ㎝ depending on rainfall. Soil moisture content in PDC and IDC was 17.2%, 22.5%, respec-tively. Air temperature in IDC was lower than 0.3 ℃, while soil temperature was higher than 0.8 ℃ compare to PDC, respec-tively. Main soil color of PDC was grayish olive, while IDC was brownish olive. PDC showed yellowish mottles only atunderground of 20~40㎝, while IDC showed that at underground of 30~90㎝. IDC showed lower pH, EC, potassium, cal-cium and magnesium content, but higher organic matter, phosphate, and iron content than that of PDC, respectively. All ofEC, organic matter, potassium, calcium, and magnesium content were decreased, but iron content was increased at the sub-soil layers of PDC. All of EC, organic matter, phosphorus, and potassium content were decreased, but calcium and magne-sium content were increased at the subsoil layers of IDC. Root yield in IDC was more increased by 33% than that of PDC.The moisture content and rusty ratio of ginseng root in IDC were lower than that of PDC. Ginsenoside content in IDC washigher than that of PDC because the ratio of lateral and fine root showing relatively high content of ginsenoside was higherin IDC than that of PDC.Key Words : Panax ginseng, Poor Drainage Class, Imperfect Drainage Class, Paddy Soil, Root Yield, Ginsenoside

A direct protein kinase B-targeted anti-inflammatory activity of cordycepin from artificially cultured fruit body of Cordyceps militaris.

Background: Cordyceps militaris is one of well-known medicinal mushrooms with anti-inflammatory, anti-cancer, anti-diabetic, and anti-obesity activities. Objective: The objective of the following study is to isolate chemical components from the ethanol extract (Cm-EE) from Cordyceps militaris and to evaluate their anti-inflammatory activities. Materials and Methods: Column chromatographic separation was performed and anti-inflammatory roles of these compounds were also examined by using NO production and protein kinase B (AKT) activity assays. Results: From Cm-EE, 13 constituents, including trehalose (1), cordycepin (2), 6-hydroxyethyladenosine (3), nicotinic amide (4), butyric acid (5), β-dimorphecolic acid (6), α-dimorphecolic acid (7), palmitic acid (8), linoleic acid (9), cordycepeptide A (10), 4-(2-hydroxy-3-((9E,12E)-octadeca-9,12-dienoyloxy)propoxy)-2-(trimethylammonio)butanoate (11), 4-(2-hydroxy-3-(palmitoyloxy)propoxy)-2-(trimethylammonio)butanoate (12), and linoleic acid methyl ester (13) were isolated. Of these components, compound 2 displayed a significant inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells. Furthermore, this compound strongly and directly suppressed the kinase activity of AKT, an essential signalling enzyme in LPS-induced NO production, by interacting with its ATP binding site. Conclusion: C. militaris could have anti-inflammatory activity mediated by cordycepin-induced suppression of AKT.

Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells

Abstract Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1–6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated β-galactosidase (SA-β-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases.

Investigation of Shikonin Pigments and Antioxidant activity of the Roots from Lithospermum erythrorhizon according to the Different Growth Stages and Areas of Cultivation

This study was conducted to evaluate the quality variation of Lithospermum radix on the pigment contents and antioxidant activities according to different growth stages and areas of cultivation. Acetylshikonin contents showed the tendency to decrease gradually from the middle of July (0.28%) to the end of August (0.05%) and then the content was increased again to the end of October (0.25%). Shikonin content was detected as small amount of about 0.009% during the period. The weight of plants was increased from the end of September to the end of October and showed the highest value as 19.8 g on October 25. ROS scavenging activity was the highest in the early of October as IC50 value of 0.11 ㎍/mL. Lithosper- mum radix of September showed lower ROS scavenging activities than those of other growth stages as IC50 value of 1.02 and 0.49 ㎍/mL on September 9 and September 27, respectively. Among 17 areas cultivated Lithospermum radix, 10 areas (59%) showed 0.05-0.10% of acetylshikonin contents and 3 areas (18%) were measured to contain 0.16-0.26% of acetylshikonin.

The Neuroprotective Effect of White Ginseng (Panax ginseng C. A. Meyer) on the Trimethyltin (TMT)-Induced Memory Deficit Rats

The present study examined the effects of Korean white ginseng (WG, Panax ginseng C. A. Meyer) on the learning and memory function and the neural activity in rats with trimethyltin (TMT)-induced memory deficits. The rats were administered with saline or WG (WG 100 or 300 ㎎/㎏, p.o.) daily for 21 days. The cognitive improving efficacy of WG on the amnesic rats, which was induced by TMT, was investigated by assessing the Morris water maze test and by perform- ing immunohistochemistries on choline acetyltransferase (ChAT), acetylcholinesterase (AchE), cAMP responsive element binding protein (CREB) and brain derived neurotrophic factor (BDNF). The rats treated with TMT injection (control group) showed impaired learning and memory of the tasks, but the rats treated with TMT injection and WG administration produced significant improvement of the escape latency to find the platform in the Morris water maze at the 2nd and 4th days compared to that of the control group. In the retention test, the WG 100 and WG 300 groups showed significantly increased crossing number around the platform compared to that of the control group (p < 0.001). Consistently with the behavioral data, result of immunohistochemistry analysis showed that WG 100 ㎎/㎏ significantly alleviated the loss of BDNF-ir neurons in the hippocampus compared to that of the control group (p < 0.01). Also, treatment with WG has a trend to be increased the cholinergic neurons in the hippocampal CA1 and CA3 areas as compared to that of the control group. These results suggest that WG may be useful for improving the cognitive function via regulation of neurotrophic activity.

Growth Characteristics and Ginsenoside Contents of 6-Year-Old Ginseng (Panax ginseng C. A. Meyer) by Shade Materials in Paddy Field

This study was carried out to select optimal shade materials among four-layered polyethylene (PE) net (FLPN), aluminium-coated PE sheet (APSS), and blue PE sheet (BPSS) in condition of paddy field cultivated 6-year-old gin- seng. The order of light-penetrated ratio and air temperature by shade materials was BPSS > APSS > FLPN. Light-pene- trated ratio of BPSS before two fold shade was more 3 times and 2 times than that of FLPN and APSS, respectively. Air temperature of BPSS was also higher 1.6 o C and 1.4 o C than that of FLPN and APSS, respectively. BPSS showed good cul- tural environment because all of light-penetrated ratio and air temperature were become higher in spring and fall season but lower in summer season by additional shade with two-layered PE net. Survived-leaf ratio was highest in BPSS and low- est in FLPN causing a little water leak on a rainy day. Rusty-root ratio was also highest in FLPN because soil moisture con- tent was increased by water leak. The order of root yield was BPSS > APSS > FLPN, and the cause of highest yield in BPSS was higher light-penetrated ratio during spring and fall season, higher survived-leaf ratio, and lower rusty-root ratio than that of APSS and FLPN. BPSS showed highest total ginsenoside content because of high light-penetrated ratio, blue light effect, and the difference in dry matter partitioning ratio such as low taproot ratio, and high lateral root ratio.

Effects of Supercritical Fluid Extract, Shikonin and Acetylshikonin from Lithospermum erythrorhizon on Chondrocytes and MIA-Induced Osteoarthritis in Rats

*Department of Herbal Crop Research, NIHHS, RDA, Eumseong 369-873, Korea.**YD Life Science Research Institute, YD Life Science Co. Ltd., Seoul 139-743, Korea.ABSTRACT : This study investigates the effect of supercritical fluid extract (CMPB803-C) of Lithospermum erythrorhizon,shikonin and acetylshikonin isolated from Lithospermum erythrorhizon on IL-1β-induced chondrocytes and monosodiumiodoacetate (MIA)-induced osteoarthritis in rat. Shikonin (50μM) and acetylshikonin (3μM) treatment reduced signifi-cantly the mRNA expression and enzyme activity of matrix metalloproteinase (MMP)-1, −3 and −13 in IL-1β-inducedSW1353 chondrosarcoma cells. The chondro-protective effects of CMPB803-C and acetylshikonin were than analyzed in arat OA model using a single intra-articular injection of MIA (1㎎) in the right knee joint. CMPB803-C (200㎎/㎏) or ace-tylshikonin (5㎎/㎏) was orally administered daily for two weeks starting after 1 week of MIA injection. In the histologicalobservation, CMPB803-C and acetylshikonin clearly improved OA lesions being comparable to or better that control group.Our results demonstrated that CMPB803-C and acetylshikonin as active compound of Lithospermum erythrorhizon have astrong chondro-protective effect in OA rats, which likely attributes to its anti-inflammatory activity and inhibition ofMMPs production. Key Words : Lithospermum erythrorhizon , Shikonin, Acetylshikonin, Osteoarthritis, MIA, MMP

Evaluation on Extraction Conditions and HPLC Analysis Method for Bioactive Compounds of Astragali Radix

This study has been conducted to establish the optimal extraction process and HPLC analysis method for the determination of marker compounds as a part of the materials standardization for the development of health functional food materials from Astragali radix. Five extraction conditions including the shaking extraction at room temperature and the reflux extraction at 85°C with 30%, 50% and 95% ethanol were evaluated. Reflux extraction with 50% ethanol showed the highest extraction yield as 27.27 ± 2.27%, while the extraction under reflux with 95% ethanol showed significantly the lowest yield of 10.55 ± 0.24%. The quantitative determination methods of calycosin-7-O-β-D-glucoside and calycosin as marker compounds of Astragali radix extracts were optimized by HPLC analysis using a Thermo Hypersil column (4.6 × 250 mm, 5 μm) with the gradient elution of water and acetonitrile as the mobile phase at the flow rate of 0.8 mL min and a detection wavelength of 230 nm. The HPLC/UV method was applied successfully to the quantification of two marker compounds in Astragali radix extracts after validation of the method with the linearity, accuracy and precision. The contents of calycosin-7-O-β-D-glucoside and calycosin in 50% ethanol extracts by reflux extraction were significantly higher as 1,700.3 ± 30.4 and 443.6 ± 8.4 μg g-1, respectively, comparing with those in other extracts. The results indicate that the reflux extraction with 50% ethanol at 85°C is optimal for the extraction of Astragali radix, and the established HPLC method are very useful for the evaluation of marker compounds in Astragali radix extracts to develop the health functional material from Astragali radix.

Monitoring of Biological Hazards in Herbal Crops from Korean Market

Background: The public has increasing concerns about herbal crops owing to insufficient information on biological hazards such as foodborne pathogens. Therefore, the objective of this study is the development of a herbal crop quality control system through monitoring with biological hazard analysis. Today, it is estimated that millions of people become ill every year from food contamination. The public demands agricultural products of stable and consistent quality. Governments have the responsibility of establishing the standards, legislation and enforcement programs necessary to control food quality and safety. However, research on the biosafety of herbal crop products is still insufficient. Therefore, the implementation of monitoring systems with high standards is critical for public safety. Methods and Results: In this study, we collected 52 samples of herbal crop products, and conducted both quantitative and qualitative biological hazard analysis. With biological hazard analysis, aerobic bacteria, Staphylococcus aureus, Salmonella spp., Escherichia coli, Coliforms, and Listeria spp. could be detected. Conclusions: Herbal crops were found to be contaminated with aerobic bacteria at . Staphylococcus aureus, Salmonella spp., Escherichia coli, Coliforms, and Listeria spp. were not detected in any of the samples. This research suggests that continuous monitoring of biological hazards is required to improve the quality of herbal crops.