Chondroprotective Effects of a Standardized Extract (KBH-JP-040) from Kalopanax pictus, Hericium erinaceus, and Astragalus membranaceus in Experimentally Induced In Vitro and In Vivo Osteoarthritis Models.

The aim of this study was to investigate the chondroprotective effect of a standardized extract (KBH-JP-040) of the Korean traditional herbs Kalopanax pictus Castor-Aralia, Hericium erinaceus (Bull.) Persoon, and Astragalus membranaceus Schischkin on in vivo and in vitro osteoarthritis (OA) models. Cultured rat chondrocytes were pre-treated with KBH-JP-040 (50, 100 and 200 µg/mL) for 1 h, then recombinant human IL-1α (rhIL-1α) for 24 h. For the in vivo model, rabbits (n = 60) were equally divided into experimental groups: normal control (NC), a collagenase-induced OA group, and OA groups treated with KBH-JP-040 (75, 100, and 150 mg/kg body weight) and celecoxib (Cx, 100 mg/kg) orally for 28 days. Treatment with KBH-JP-040 significantly attenuated inflammatory cytokines and matrix metalloproteinases (MMPs), suppressed the expression of IκBα, NF-κB, and JNK/p38 mitogen-activated protein (MAP) kinase, and upregulated aggrecan and collagen type-II expression in rhIL-1α-stimulated chondrocytes. Furthermore, the serum and synovial levels of inflammatory cytokines of rabbits also decreased in the treatment groups when compared with the OA group. Improved magnetic resonance imaging and histopathological findings further confirmed the therapeutic efficacy of KBH-JP-040 against OA. In conclusion, these results indicate that KBH-JP-040 possesses chondroprotective effects, suppressing inflammation and MMPs, and downregulating IκBα, NF-κB, and JNK/p38 MAP kinase-signaling pathways. This might be a potential therapeutic candidate for OA treatment.

Kalopanacis Cortex extract-capped gold nanoparticles activate NRF2 signaling and ameliorate damage in human neuronal SH-SY5Y cells exposed to oxygen-glucose deprivation and reoxygenation.

Recently, environment-friendly synthesis of gold nanoparticles (GNPs) has been extensively explored by biologists and chemists. However, significant research is still required to determine whether "eco-friendly" GNPs are beneficial to human health and to elucidate the molecular mechanisms of their effects on human cells. We used human neuronal SH-SY5Y cells to show that treatment with Kalopanacis Cortex extract-capped GNPs (KC-GNs), prepared via an eco-friendly, fast, one-pot synthetic route, protected neuronal cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced damage. To prepare GNPs, Kalopanacis Cortex was used without any chemical reducing and stabilizing agents. Ultraviolet-visible spectroscopy showed maximum absorbance at 526 nm owing to KC-GN surface plasmon resonance. Hydrodynamic size (54.02±2.19 nm) and zeta potential (-20.3±0.04 mV) were determined by dynamic light scattering. The average diameter (41.07±3.05 nm) was determined by high-resolution transmission electron microscopy. Energy-dispersive X-ray diffraction spectroscopy and X-ray diffraction confirmed the presence of assembled GNPs. Fourier transform infrared analysis suggested that functional groups such as O-H, C-C, and C-N participated in KC-GN formation. Cell viability assays indicated that KC-GNs restored the viability of OGD/R-treated SH-SY5Y cells. Flow cytometry demonstrated that KC-GNs inhibited the OGD/R-induced reactive oxygen species production and mitochondrial membrane potential disruption. KC-GNs also inhibited the apoptosis of OGD/R-exposed cells. Western blot analysis indicated that the OGD/R-induced cellular apoptosis and simultaneous increases in the expression of cleaved caspase-3, p53, p21, and B-cell lymphoma 2-associated X protein were reversed by KC-GNs. The KC-GN-mediated protection against OGD/R-induced neurotoxicity was diminished by NRF2 and heme oxygenase-1 gene knockdowns. Collectively, these results suggested that KC-GNs exerted strong neuroprotective effects on human neuronal cells, which might be attributed to the attenuation of OGD/R-induced neuronal cell injury through the NRF2 signaling pathway.

Ethanol extract of Kalopanax septemlobus leaf induces caspase-dependent apoptosis associated with activation of AMPK in human hepatocellular carcinoma cells.

The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.

Biological synthesis of manganese dioxide nanoparticles by Kalopanax pictus plant extract.

Manganese dioxide (MnO2) nanoparticles were synthesised by the reduction of potassium permanganate (KMnO4) using Kalopanax pictus leaf extract at room temperature. A transparent dark-brown colour appeared after the addition of K. pictus leaf extract to the solution of permanganate. The time course of the reduction of KMnO4and synthesis of MnO2 nanoparticles was monitored by means of UV-Vis spectra. The reduction of KMnO4occurred after addition of plant extract with disappearance of KMnO4specific peaks and emergence of peak specific for MnO2nanoparticles. MnO2nanoparticles showed absorption maxima at 404 nm. The electron dispersive X-ray spectroscopy analyses confirmed the presence of Mn and O in the sample. X-ray photoelectron spectroscopy revealed characteristic binding energies for MnO2nanoparticles. Transmission electron microscopy micrographs revealed presence of uniformly dispersed spherical shaped particles with average size of 19.2 nm. The selected area electron diffraction patterns revealed the crystalline nature of MnO2nanoparticles. Fourier transform-infrared spectroscopy spectra of pure MnO2show the occurrence of O-Mn-O vibrational mode at around 518 cm⁻¹. The phyto-synthesised MnO2nanoparticles showed degradation ability of dyes (congo red and safranin O) similar to chemically synthesised MnO2nanoparticles. This study shows simple and eco-friendly synthesis of MnO2nanoparticles by plant extract and their utilisation for dye degradation for the first time.

Potential of Kalopanax septemlobus leaf extract in synthesis of silver nanoparticles for selective inhibition of specific bacterial strain in mixed culture.

Silver nanoparticles (AgNPs) were synthesised using Kalopanax septemlobus plant leaf extracts. UV-visible spectrophotometric, Fourier-transform infrared, electron dispersive X-ray spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed synthesis of AgNPs. TEM micrographs revealed presence of well-dispersed AgNPs predominantly of small size and different shapes with an average particle size of 30.8 nm. Antimicrobial susceptibility tests of AgNP treatments revealed variability in sensitivity of bacteria Bacillus cereus and Saccharophagus degradans under study. Minimum inhibitory concentration (MIC) values of the AgNPs for B. cereus and S. degradans were found to be 30 and 10 µg/mL, respectively. The mixed culture of B. cereus and S. degradans treated with AgNPs at 10 µg/mL showed increase in growth with time, suggesting survival of bacteria in liquid media. The plating of mixed culture before AgNP treatment showed presence of both bacteria, but 24-h-old mixed culture treated with AgNPs at the concentration of 10 µg/mL showed presence of B. cereus colonies. SEM micrographs revealed damage to S. degradans cells but no effect on B. cereus cells after AgNP treatment. Confocal microscopic observations of AgNP-treated mixed cultures by Nile blue A staining indicated intact polyhydroxyalkanoates producing flourescent cells of B. cereus but damage and deformities in S. degradans cells. This study suggests that AgNPs can selectively inhibit growth of S. degradans and retain B. cereus at MIC of S. degradans. This report is a case study for selective inhibition of one bacteria and growth of the other in a culture using plant-synthesized silver nanoparticles.

Quantification and identification of bioactive metabolites from Kalopanacis Cortex by HPLC with evaporative light scattering detection and ESI quadrupole TOF MS.

A method based on HPLC coupled with an evaporative light scattering detection and ESI quadrupole TOF MS was established for the quantification and identification of phenolics and triterpene saponins in Kalopanacis Cortex using a gradient elution of acetonitrile with 0.1% formic acid and water with 0.1% formic acid on an RP C18 column (4.6 × 250 mm, 5 µm). Diverse validation parameters, such as the linearity, LOD and LOQ, accuracy, precision, repeatability, and stability, were successfully obtained. Additionally, the efficiencies of different extraction methods were compared. The developed method was applied for the quantitative analysis of twelve representative metabolites in 61 Kalopanacis Cortex samples. The quantitation results showed that coniferin, kalopanaxsaponin C, septemlosides II, III, C, and D exhibited distinct regional patterns in Kalopanacis Cortex samples. These six compounds including one new triterpene saponin show potential as marker compounds for evaluating the quality of Kalopanacis Cortex and the geographical variation in its chemical composition.

Involvement of the p38 MAPK and ERK signaling pathway in the anti-melanogenic effect of methyl 3,5-dicaffeoyl quinate in B16F10 mouse melanoma cells.

Methyl 3,5-dicaffeoyl quinate (MDQ), an active compound present in Kalopanax pictus, Salicornia herbacea L., Aster oharai and Solidago virga-aurea var. gigantean, is a dicaffeoylquinic acid derivative esterified by methanol. Recent studies have revealed that MDQ possesses multiple pharmacological activities, such as antitumor, antioxidative and cytoprotective activities. To date, there has been no attempt to test the action of MDQ in melanocytes. In this study, we investigated the effect of MDQ on melanogenesis in B16F10 mouse melanoma cells. MDQ inhibited melanin production and tyrosinase activity in B16F10 mouse melanoma cells without a direct inhibitory effect on mushroom tyrosinase activity. Furthermore, we also found that MDQ decreased protein expression levels of microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 melanin cells. Meanwhile, phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was significantly reduced after 6h MDQ treatment, and this expression recovered at 48 h. The phosphorylation of extracellular signal-regulated kinase (ERK) was significantly enhanced at 12-48 h, whereas no effect was observed in the phosphorylation of Akt. In addition, MDQ treatment did not significantly alter the expression levels of total p38 MAPK, ERK, and Akt. Thus, it seems that inhibition of phospho-p38 MAPK and activation of phospho-ERK may lead to the suppression of melanogenesis in MDQ-treated B16F10 mouse melanoma cells.

Kalopanaxsaponins A and B isolated from Kalopanax pictus ameliorate memory deficits in mice.

The stem-bark of Kalopanax pictus (KP, family Araliaceae), which contains triterpenoid saponins, has been shown to exhibit anticarcinogenic, antiinflammatory, antirheumatoid and antidiabetic activities. In a preliminary study, a KP methanol extract demonstrated acetylcholinesterase activity in vitro and memory enhancement in scopolamine-treated mice. Therefore, we isolated acetylcholinesterase inhibitors, kalopanaxsaponins A and B, from a KP butanol (BuOH) fraction, measured acetylcholinesterase activity in vitro, and investigated their memory-enhancing effects in a passive avoidance test, Y-maze test and Morris water maze test. These constituents inhibited acetylcholinesterase activity and significantly reversed scopolamine-induced deficits. They also increased brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding (p-CREB) protein expression but reduced TNF-α increased by scopolamine. Based on these findings, kalopanaxsaponins A and B may ameliorate memory deficits by inhibiting acetylcholinesterase activity and inducing BDNF and p-CREB expression.

Effect of triterpenes and triterpene saponins from the stem bark of Kalopanax pictus on the transactivational activities of three PPAR subtypes.

Kalopanax pictus (Araliaceae) is a deciduous tree that grows in East Asian countries. Its stem bark and leaves have been used in traditional medicine to treat rheumatic arthritis, neurotic pain, and diabetes mellitus. A phytochemical study on a methanol extract of the stem bark of K. pictus resulted in the isolation of three new compounds, 6ß,16α-dihydroxy-hederagenin 3-O-ß-D-glucuronopyranoside (1), 3-O-ß-D-glucuronopyranosyl-28-O-ß-D-glucopyranosyl-6ß,16α-dihydroxy-oleanolic acid (2), and 3-O-ß-D-galactopyranosyl(1→3)-α-L-arabinopyranosyl hederagenin 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester (3), along with eight known compounds (4-11). Their structures were established on the basis of chemical and spectroscopic methods (IR, 1D and 2D NMR, and HRESITOFMS). Compounds 1-6 and 8-10 upregulated PPARs transcriptional activity in a dose-dependent manner in HepG2 cells, with EC(50) values in the range 0.20-15.5 µM. Moreover, the specific PPAR transactivational effects of compounds 1-6 and 8-10 on separate PPAR subtypes, PPARα, -γ, and -ß(δ) were further investigated. Compounds 4, 5, 8, and 10 showed significant PPARα transactivational activity, with EC(50) values of 7.8, 8.0, 10.3, and 17.3 µM, respectively. Compounds 2, 4, 6, and 8-10 exhibited PPARγ dose-dependent transactivational activity, with EC(50) values of 14.7, 15.5, 14.8, 10.9, 17.1, and 16.3 µM, whereas compounds 8 and 10 significantly upregulated PPARß(δ) transcriptional activity, with EC(50) values of 15.7 and 17.7 µM, respectively.

[Studies on the chemical constituents from the roots of Kalopanax septemlobus].

OBJECTIVE: To investigate the chemical constituents of Kalopanax septemlobus. METHODS: Chromatographic techniques including silica gel, gel, semi-preparative HPLC and PTLC as well as recrystallization were employed in the isolation and purification, and the structures were elucidated by spectral analysis and physical and chemical properties. RESULTS: 6 compounds were identified as liriodendrin (1), (-) -syringarenol (2), trans-coniferyl aldehyde (3), trans-caffeic acid (4), beta-daucosterol (5), beta-sitosterol (6). CONCLUSION: Compounds 2 -5 are obtained from this genus for the first time.

Anti-inflammatory triterpenoid saponins from the stem bark of Kalopanax pictus.

Five new compounds, 16,23,29-trihydroxy-3-oxo-olean-12-en-28-oic acid (1), 4,23,29-trihydroxy-3,4-seco-olean-12-en-3-oate-28-oic acid (2), 3ß,6ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-glucopyranoside (3), 3-O-[2,3-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (4), and 3-O-[3,4-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (5), as well as 10 known compounds (6-15), were isolated from the stem bark of Kalopanax pictus. Compounds 1-5 and 7-14 inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values ranging from 0.6 to 16.4 µM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed on the basis of decreases in COX-2 and iNOS gene expression in HepG2 cells. The structure-activity relationship of the compounds with respect to anti-inflammatory activity is also discussed.

Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography.

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, high-performance liquid chromatography (HPLC) was used for fast screening of G-quadruplex ligands from the crude extract of Kalopanax septemlobus (Thunb.) Koidz, a traditional Chinese medicine. Four potent G-quadruplex ligands were firstly selected through HPLC by comparing the peak profiles and absorption intensity of the crude sample before and after interaction with G-quadruplex DNA. Then the target compounds were isolated and purified by high-speed countercurrent chromatography (HSCCC) for further confirmation of their stabilities of G-quadruplex by temperature-dependent circular dichroism (CD). Four compounds were isolated and identified as 2,4-dihydroxybenzoic acid (I), chlorogenic acid (II), caffeic acid (III) and 5-feruloylquinic acid (IV) each by MS and NMR. Finally, compound I, II, III were each proved to be potent G-quadruplex ligands by decreasing the peak intensity in HPLC chromatogram after complexation with G-quadruplex, which stabilize G-quadruplex by 7±2 °C, 10±2 °C, and 3±2 °C respectively, based on CD analyses. However, compound IV showed no G-quadruplex stability. The decrease of peak absorption intensity in HPLC chromatogram is the most important signal to find G-quadruplex ligands. This provides a very promising strategy for fast screening G-quadruplex ligands from natural plant extracts.

Kalopanax pictus as feed additive controls bacterial and parasitic infections in kelp grouper, Epinephelus bruneus.

Feeding kelp grouper, Epinephelus bruneus (26.1 ± 1.4), with 0%, 0.1%, 1.0%, and 2.0% Kalopanax pictus extract-supplementation diets, for 30 days reduced mortality on being challenged intraperitoneally 100 µl with bacterium Vibrio alginolyticus (2.1 × 10(7) cfu ml(-1)) and ciliate parasite Philasterides dicentrarchi (2.3 × 10(7) ciliates ml(-1)). The red blood cells (RBC), white blood cells (WBC), haemoglobin, haematocrit, lymphocytes, and monocytes levels significantly increased in kelp grouper fed with all doses of K. pictus-supplementation diets and challenged with bacterium and parasite when compared to control. However, the levels of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), neutrophils, and thrombocytes did not significantly. The phagocytic activity, complement activity, and antiprotease activity did not significantly change in kelp grouper fed with 0.1% K. pictus-supplementation diets and challenged with bacterium and parasite. The respiratory activity, lysozyme activity, bactericidal activity, total protein level, and myeloperoxidase levels significantly increased in kelp grouper fed with all the doses of K. pictus-supplementation diet and challenged with bacterium and parasite. However, α2-macroglobulin level significantly increased with 1.0% diet, but not with 0.1% and 2.0% diets. Therefore this study suggests that 1.0% and 2.0% K. pictus-supplementation diets positively protected and enhanced the immune system in kelp grouper E. bruneus against V. alginolyticus and P. dicentrarchi infection.

Multiple component quantitative analysis for the pattern recognition and quality evaluation of Kalopanacis Cortex using HPLC.

A quantitative and pattern recognition analyses were conducted for quality evaluation of Kalopanacis Cortex (KC) using HPLC. For quantitative analysis, four bioactive compounds, liriodendrin, pinoresinol O-ß-D-glucopyranoside, acanthoside B and kalopanaxin B, were determined. The analysis method was optimized and validated using ODS column with mobile phase of methanol and aqueous phosphoric acid. The validation gave acceptable linearities (r > 0.9995), recoveries (98.4% to 101.9%) and precisions (RSD < 2.20). The limit of detection of compounds ranged from 0.4 to 0.9 µg/mL. Among the four compounds, liriodendrin was recommended as a marker compound for the quality control of KC. The pattern analysis was successfully carried out by analyzing thirty two samples from four species, and the authentic KC samples were completely discriminated from other inauthentic species by linear discriminant analysis. The results indicated that the method was suitable for the quantitative analysis of liriodendrin and the quality evaluation of KC.

Induction of cell cycle arrest and apoptosis by the ethyl acetate fraction of Kalopanax pictus leaves in human colon cancer cells.

Kalopanax pictus is a deciduous tree used in traditional medicine; its leaves are also consumed as a vegetable. In this study, the ethyl acetate fraction of K. pictus leaves (EFK) was tested in vitro for anticancer activity against four cell lines: human colon cancer (HT-29) cells, human stomach cancer (NCI-N87) cells, human breast cancer (MDA-MB231) cells, and mouse melanoma (B16F1) cells. Results indicated that EFK showed the most potent tumor selective growth inhibitory activity against HT-29 cells with less cytotoxic effect on normal cell lines. Cytotoxicity of EFK on HT-29 cells was associated mainly with cell chromatin condensation, DNA fragmentation, and loss of membrane phospholipid asymmetry with appearance of G2/M phase arrest. Cell death induced by EFK displayed features characteristic of apoptosis, and was associated with generation of reactive oxygen species (ROS) and increase of Bax/Bcl-2 ratio. These findings suggest that K. pictus leaves have anticancer properties and may be valuable for application in pharmaceutical industry.

[Studies on extraction process of the main saponin constituents from the stem bark of Kalopanax septemlobus in Guangxi].

Using orthogonal experiment design, the total saponin constituents were obtained by refluxing extraction with alcohol and separated by macroporous adsorption resin and n-Butyl alcohol from the stem bark of Kalopanax septemlobus. According to the purity analysis and the yield, the extraction process was optimized. The results showed that the main saponin constituents were gained with a yield of 1.32% by using macroporous adsorption resin but 1.05% by using n-Butyl alcohol. The former was more efficient than the latter on both yield and color. The optimal process with isolation by macroporous adsorption resin is cheap, simple and practical.

A new triterpene hexaglycoside from the bark of Kalopanax septemlobus (Thunb.) Koidz.

The new triterpene glycoside 3-O-beta-D-xylopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosylhederagenin 28-O-beta-D-gluco-pyranosyl-(1-->6)-beta-D-glucopyranoside, named septemoside A (1), and the known 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-alpha-L-arabinopyranoside-28-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester of hederagenin (2), were isolated from the bark of Kalopanax septemlobus. The structure elucidation of the compounds was based on spectroscopic evidence, including HRESIMS, 1D and 2D-NMR analysis.

[Effects of Cunninghamia lanceolata-broadleaved tree species mixed leaf litters on active soil organic matter].

With incubation test, this paper studied the effects of Cunninghamia lanceolata leaf litter and its mixture with the litters of main broadleaved tree species in subtropical China, such as Alnus cremastogyne, Kalopanax septemlobus and Michelia macclurei on active soil organic matter. The results showed that adding leaf litters into soil could significantly increase soil microbial biomass C and N, respiration rate and dissolved organic C, and mixed leaf litters were more effective than C. lanceolata leaf litter in increasing soil dissolved organic C. By the end of the incubation, the increment of soil microbial biomass C and N, respiration rate, and dissolved organic C in treatments C. lanceolata leaf litter and C. lanceolata-broadleaved tree species mixed leaf litters was 49% and 63%, 35% and 75%, 65% and 100%, and 66% and 108%, respectively, compared with control. The addition of leaf litters had no significant effects on soil microbial quotient and microbial biomass C/N ratio.

Isolation of ginsenoside Rb1 from Kalopanax pictus by eastern blotting using anti-ginsenoside Rb1 monoclonal antibody.

Araliaceous species containing ginsenosides, the major active component in Panax species, were surveyed using ELISA and eastern blotting with anti-ginsenoside Rb1 monoclonal antibody. Immunoassay-guided fractionation of the methanol-soluble extracts of Araliaceous species led to the isolation of a known compound in the bark of Kalopanax pictus Nakai. The known compound was identified as ginsenoside Rb1 by spectroscopic methods and by comparison with an authentic sample. The result provided evidence that a combination of two immunoassays using monoclonal antibodies against ginsenosides is a powerful means of surveying new ginsenoside resources.

Activity-guided isolation of saponins from Kalopanax pictus with anti-inflammatory activity.

By bioassay-guided separation, a known saponin, kalopanaxsaponin A (1) and a new saponin, pictoside A (2) were isolated from the stem bark of Kalopanax pictus as anti-inflammatory components when evaluated by vascular permeability test. Another novel saponin, pictoside B (3) was also isolated but was inactive in the test system used. The structures of pictosides A and B were elucidated as caulophyllogenin 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside (2) and pictogenin (3beta,6beta,16alpha,23-tetrahydroxyolean-12-ene-28-oic acid) 3-O-alpha-L-arabinopyranoside (3), respectively, by spectral analysis and by chemical degradation. Kalopanaxsaponin A and pictoside A showed significant anti-inflammatory activity at the oral doses of 50 mg/kg.