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Although it has been known that pomegranate (Punica granatum L.) contains several steroid hormones, concrete experimental proofs about that have not been published until now. In order to identify and evaluate the contents of steroid hormones including estrone in pomegranate, we analyzed pomegranate seed, fruit juice and commercial preparations. We developed a reproducible and sensitive method for separation and identification of steroid hormones in pomegranate samples using both high performance liquid chromatography (HPLC)–PDA and gas chromatography (GC)–MS. In case of HPLC, an isocratic elution method using 35% aqueous acetonitrile solution at 1.0 ml/min with photodiode-array (PDA) detection at 225 nm and 254 nm was found to optimally separate and identify the steroid hormones from the pomegranate samples with a run time of less than 30 min. The pomegranate samples were comparatively analyzed to the HPLC results by GC/FID or GC/MS detection on a HP-1 (30 m length, 0.32 mm I.D.) with helium as carrier gas under the oven temperature control as follows: start 220 °C for 5 min, raising 5 °C per min, final 280 °C for 10 min. The HPLC and GC methods were successfully applied to the identification of steroid hormones in pomegranate samples. Our results suggested that there were no steroid estrogens including estrone, estradiol and testosterone in pomegranate seed, fruit juice and preparations. Consequently, we assumed that the previously reported analysts of pomegranate were misunderstood their analytical results according to either the estrogen-like effects or similarity of peak retention time and Rf values in experiments.

In order to evaluate the improvement in the treatment of chronic arthritis, we investigated chondroitin sulfate depolymerization product (low molecular weight chondroitin sulfate, LMWCS) and intact chondroitin sulfate (CS) in vitro and in vivo. LMWCS was prepared by a chemical depolymerization process induced by hydrogen peroxide in the presence of copper salts. LMWCS (300 mg/kg) and CS (1200 mg/kg) were orally administered to DBA/1J mice once daily for 14 d prior to initial immunization with type II collagen. Their elastase activities and the production of cytokines in sera were examined on type II collagen-induced arthritis in DBA/1J mice. We also compared the paracellular transport of LMWCS and CS across Caco-2 cell monolayers and examined the inhibitory effects on elastase activities. LMWCS inhibited elastase activity slightly, but CS did not show inhibition. Hind paw edema was significantly decreased by LMWCS treatment. Levels of anti-type II collagen antibody and tumor necrosis factor-alpha (TNF-alpha) in sera were also reduced by LMWCS treatment but not in case of CS, although no significant difference was observed between LMWCS and CS on interleukin-6 (IL-6) induction. The LMWCS preparation showed preventive effects on the type II collagen-induced arthritis in DBA/1J mice and better permeability through Caco-2 cells.

Three new ( 1–3 ) and two known ( 4–5 ) triterpene glycosides were isolated from the leaves of Acanthopanax japonicus (Araliaceae) and elucidated structurally by mass, 1D, and 2D NMR spectroscopy. All the compounds possessed a nor -oleanene triterpene skeleton as the aglycone. The structures of 1–5 were established as 28- O -α- l - rhamno-pyranosyl-(1→4)-β- d -glucopyranosyl-(1→6)-β- d -glucopyranosyl ester of 3β-hydroxy- 30- nor -olean-12,20(29)-diene-23,28-dioic acid, designated as acanjaposide A, 3β- hydroxy-23-oxo-30- nor -olean-12,20(29)-diene-28-oic acid, named acanjaposide B, 3β,20α-dihydroxy-23-oxo-30- nor -olean-12-en-28-oic acid, named acanjaposide C, and nipponoside E, a known saponin, respectively.

The metabolic pathway of chiisanoside isolated from leaves of Acanthopanax divaricatus var. albeofructus (Araliaceae) by human intestinal bacteria and by the protein fraction of leaves of this plant were investigated, and the cytotoxic and anti-rotaviral activities of chiisanoside and its metabolite, chiisanogenin, were assayed. Chiisanogenin was produced as a main metabolite, when chiisanoside were incubated for 15 h with human intestinal bacteria. This metabolic pathway proceeded more potently with the protein fraction than with human intestinal bacteria. The in vitro cytotoxicity of chiisanogenin was superior to that of chiisanoside. H+/K+ ATPase was more potently inhibited by chiisanogenin than by chiisanoside. However, the anti-rotaviral activity of chiisanoside was more potent than that of chiisanogenin.

Two new lupane-triterpene glycosides named acankoreosides C and D, were isolated from the leaves of Acanthopanax koreanum. Based on spectroscopic data, the chemical structures were determined as 3-O-β- d -glucopyranosyl 3α,11α-dihydroxylup-20(29)-en-28-oic acid 28-O-α- l -rhamnopyranosyl-(1→4)-β- d -glucopyranosyl-(1→6)-β- d -glucopyranosyl ester and 3α,11α-dihydroxylup-23-al-20(29)-en-28-oic acid 28-O-α- l -rhamnopyranosyl-(1→4)-β- d -glucopyranosyl-(1→6)-β- d -glucopyranosyl ester, respectively.

A new lupane triterpene glycoside, acantrifoside A, was isolated from the leaves of Acanthopanax trifoliatus and A. koreanum . Based on spectroscopic data, the compound was identified as 3α,11α-dihydroxylup-20(29)-en-28-oic acid 28- O -α- l -rhamnopyranosyl-(1→4)-β- d -glucopyranosyl-(1→6)-β- d -glucopyranosyl ester.

Two new lupane-triterpene glycosides, acankoreoside A (I) and B (2), were isolated from the leaves of Acanthopanax koreanum NAKAI (Araliaceae). Based on spectroscopic data, the chemical structures of 1 and 2 were determined as 3α-hydroxy-lup-20(29)-en-23,28-dioic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester and 3α,11α,23-trihydroxy-lup-20(29)-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, respectively.

Two androstane alkaloids were isolated from the musk of Moschus moschiferus. The structures were elucidated to be 3alpha-ureido-androst-4-en-17-one (1) and 3alpha-ureido-androst-4-en-17beta-ol (2) by two-dimensional NMR analysis (HMQC, 1H-1H COSY, HMBC, and NOESY).

Two 3,4-seco-lupane triterpenoids (1, 2) were isolated from the leaves of Acanthopanax divaricatus var. albeofructus (Araliaceae). Based on the spectroscopic data, the chemical structures of 1 and 2 were characterized as 24-hydroxychiisanogenin and 22alpha-hydroxychiisanogenin, respectively. 1 was a new compound and 2 was isolated for the first time from the natural source, although it had been obtained as an enzymatically hydrolyzed artifact from 22alpha-hydroxychiisanoside.

Two new lupane-triterpene glycosides, acankoreoside A (I) and B (2), were isolated from the leaves of Acanthopanax koreanum NAKAI (Araliaceae). Based on spectroscopic data, the chemical structures of 1 and 2 were determined as 3α-hydroxy-lup-20(29)-en-23,28-dioic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester and 3α,11α,23-trihydroxy-lup-20(29)-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, respectively.

We performed the quantitative analysis of chondroitin sulfate (CS) obtained from raw materials and various pharmaceutical preparations. To quantify CS content in raw materials and in an ophthalmic solution, each test sample and the authentic CS were first digested by chondroitinase ABC. The CS disaccharides produced were analyzed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC) and CS content was quantified by calculating the total peak areas of the disaccharides derived from a CS calibration curve. In the case of soft capsules, CS was first extracted with hexane followed by phenol-chloroform to remove oil and protein ingredients. The extracted CS samples were depolymerized by chondroitinase ABC and CS content was determined. Quantitative analysis of the disaccharides derived from raw materials and an ophthalmic solution showed the CS contents (%) were 39.5+/-0.1 to 105.6+/-0.1 and 103.3+/-1.2, respectively. In case of CS analysis in soft capsules and liquid preparations, the overall recovery (%) of the spiked CS was 96.79+/-0.53-103.54+/-1.78 and 97.10+/-1.82 to 103.17+/-2.34, respectively. In conclusion, the quantitative analysis of the disaccharides produced by enzymatic digestion can be used in the direct quantitation of CS containing pharmaceutical formulations.

A novel lupane-triterpene glycoside, called wujiapioside B (1), was isolated from the leaves of Acanthopanax gracilistylus (Araliaceae) together with three known lupane-triterpene glycosides, acankoreoside C (2), acantrifoside A (3) and 3-epibetulinic acid 28-O-alpha-L-rhamnopyranosyl-(1--4)-beta-D-glucopyranosyl-(1--6)-beta-D-glucopyranosyl ester (4). Based on spectroscopic data, the chemical structure of 1 was determined as 3alpha,23-dihydroxy-lup-20(29)-en-28-oic acid 28-O-alpha-L-rhamnopyranosyl-(1--4)-beta-D-glucopyranosyl-(1--6)-beta-D-glucopyranosyl ester. Compounds 2-3 were obtained for the first time from this plant and compound 4 has not been isolated from Acanthopanax genus yet.

We report the development of enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of a unique musk protein (MP-1) in musk samples. Musk defatted with ethyl acetate/methanol (9:1, v/v) was dipped in cold water and ammonium sulfate was added to the supernatant up to 85% saturation. The resulting precipitate was applied to a Bio-Gel P-100 chromatography. The fraction eluted at the void region was collected and it was consecutively purified by affinity chromatography on a DEAE Affi-Gel Blue and on anion-exchange columns containing DEAE-Sepharose CL-6B. This protein was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions with an apparent molecular weight of 35000 Da and was called as musk protein-1 (MP-1). Polyclonal antibodies of MP-1 were produced by injecting it into a rabbit. These antibodies were reactive to the aqueous extract of musk and the pure antigen. The ELISA could be applied to detect nano gram quantities of the antigen in musk samples. This method made it possible to distinguish musk samples from different origins.

Two 3,4-seco-lupane triterpenoids (1, 2) were isolated from the leaves of Acanthopanax divaricatus var. albeofructus (Araliaceae). Based on the spectroscopic data, the chemical structures of 1 and 2 were characterized as 24-hydroxychiisanogenin and 22alpha-hydroxychiisanogenin, respectively. 1 was a new compound and 2 was isolated for the first time from the natural source, although it had been obtained as an enzymatically hydrolyzed artifact from 22alpha-hydroxychiisanoside.

Two androstane alkaloids were isolated from the musk of Moschus moschiferus. The structures were elucidated to be 3alpha-ureido-androst-4-en-17-one (1) and 3alpha-ureido-androst-4-en-17beta-ol (2) by two-dimensional NMR analysis (HMQC, 1H-1H COSY, HMBC, and NOESY).