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Boswellic acids, the main active ingredients of Boswellia serrata, are gaining more and more importance in the treatment of peritumoural oedema and chronic inflammatory diseases. They may be even considered as alternative drugs to corticosteroids in reducing cerebral peritumoural oedema. An important focus for drugs acting in the central nervous system is achieving a high extent of brain penetration. Today there is increasing evidence for the importance of transporters, especially P-glycoprotein (Pgp), for drug disposition and resulting clinical response. Pharmacokinetic studies revealed that the concentrations of the potent keto derivatives, the 11-keto-beta-boswellic acid (KBA) and the acetyl-11-keto-beta-boswellic acid (AKBA), in proportion to boswellic acids lacking a keto group, like the beta-boswellic acid, are much lower in plasma than in the orally administered extract. Moreover the brain/plasma ratio for KBA and AKBA determined in preliminary experiments on rats was only about 0.51 and 0.81, respectively, in spite of their lipophilicity. Until now little is known about the cerebral pharmacokinetics of boswellic acids and how it may be influenced. Since many drugs are known to interact with Pgp at the level of the intestine and the blood-brain barrier the modulatory potencies of the Boswellia serrata extract of H15(R) and the major boswellic acids on the transport activity of Pgp have been determined in two in vitro assays. A human lymphocytic leukaemia cell line (VLB cells) expressing Pgp was chosen as model for human Pgp, and porcine brain capillary endothelial cells (PBCEC cells) were taken as model for the blood-brain barrier using calcein acetoxymethyl ester (calcein-AM) as Pgp substrate. It was found that the Boswellia extract, as well as the keto-boswellic acids inhibit the transport activity of Pgp in the micromolecular range in both cell types. No modulation was observed using those boswellic acids which have no keto group in their structure. The inhibition of Pgp at the blood-brain barrier by Boswellia extract is probably not relevant for the brain availability of other Pgp substrates, because of the low plasma levels determined for KBA and AKBA. However the presented data could not exclude the possibility of drug interactions caused by modulation of Pgp by extracts of Boswellia serrata on the gastrointestinal level.

Two bisanthraquinones named (+)-epicytoskyrin (1) and (+)-1,1'-bislunatin (2) were produced by the endophytic fungus from a tea plant, which is a species closely related to Diaporthe phaseolorum strain sw-93-13. The chemical structures of the metabolites were elucidated on the basis of the physicochemical properties including the circular dichroism (CD) spectrum.

Three new saponins, capilliposide A (1), capilliposide B (2) and capilliposide C (3) were isolated from an ethanol extract of Lysimachia capillipes. Their structures were determined by 1D and 2D NMR (1H-1H COSY, HMBC, HMQC, DEPT and TOCSY) techniques, MS, and hydrolysis. Capilliposide B showed significant cytotoxicity against human A-2780 cells.

Astragaloside-IV (3-O-beta-d-xylopyranosyl-6-O-beta-d-glucopyranosyl-cycloastragenol) is the major active constituent contained in Radix Astragali. This paper describes a rapid, sensitive and specific assay for quantitative determination of astragaloside-IV in rat plasma. After a liquid/liquid extraction (LLE) with n-butanol and high-performance liquid chromatography (HPLC) gradient separation with acetonitrile-NH4Cl solution (0.5 micromol/L) as the mobile phase, the anions adduct [M + Cl]- at m/z 819.4 of astragaloside-IV, and [M + Cl]- at m/z 815.35 of internal standard (IS) digoxin were analyzed by electrospray ionisation-mass spectrometry (LC-ESI-MS) in selected ion monitoring (SIM) mode. Chromatographic separation was achieved in less than 9 min and calibration curve was linear over a concentration range of 2-200 ng/ml. The described assay method was successfully applied to the preclinical pharmacokinetic study of astragaloside-IV. After intragastric administration of astragaloside-IV to rats, Cmax and Tmax of astragaloside-IV were 134.73 +/- 39.86 ng/ml and 1.5 h, respectively, and the elimination half-life (t1/2) was 5.45 +/- 0.39 h.

The biological activity of natural and semisynthetic lupane triterpenoids is discussed in a two-part review. The first part is devoted to the pharmacological properties of natural lupane triterpenoids. Betulinic acid has proven to be the most effective antitumor agent among more than fifty natural lupanes.

A rapid, sensitive and selective HPLC separation with photodiode array detection was developed for the analysis of the novel pentacyclic triterpenoid acetyl-11-keto-alpha-boswellic acid. Complete baseline separation of acetyl-11-keto-alpha-boswellic acid from the corresponding isomer acetyl-11-keto-beta-boswellic acid was achieved on a fluorinated stationary phase. The standard curve was linear from 0.98 nmol/l to 196 nmol/l acetyl-11-keto-alpha-boswellic acid. The compound was isolated from chick embryonic plasma using extraction on diatomaceous earth with an overall average extraction yield of 82%. This method was applied in a kinetic study on the chick chorioallantoic membrane model (CAM) and showed unequivocal separation between acetyl-11-keto-alpha-boswellic acid and acetyl-11-keto-beta-boswellic acid unachievable so far.

Pharmacokinetics of Astragaloside IV (AGS-IV) in rats was studied by high performance liquid chromatography coupled with tandem mass spectrometry. The concentration in plasma was determined after i.v. administration of 1, 2, 4 mg/kg and p.o. administration of 20 mg/kg of AGS-IV. The AUC were linearly correlated to doses. Recoveries of AGS-IV in bile, urine and feces were also analyzed following i.v. dose of 2 mg/kg. Cumulative recovery of AGS-IV in bile reached 30.8% in 24h. Cumulative recovery of AGS-IV in urine and feces was 52.14%, which indicates that about 50% of AGS-IV was metabolized in vivo. The bioavailability of AGS-IV after p.o. administration was found to be 3.66%. These findings provide useful information for the research and development of AGS-IV and other potential agents.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method (LC-MS-MS) had been developed and validated for the quantitation of astragaloside IV (AGS-IV)-an active constituent of Radix Astragali in rat plasma. Assay method was developed by a series of operations described as below. The plasma proteins were precipitated with acetonitrile and digoxin was used as the internal standard (I.S.). The sample solution containing astragaloside IV and the I.S. were obtained and subsequently injected into a LC-MS-MS system following by a gradient elution at a slow flow rate combined with a valve diversion during the liquid chromatography. Chromatographic separation was achieved on a C4 (2.1 mmx10 mm) column with a gradient mobile phase comprised of 90% methanol in water and 10 mM ammonium acetate buffer. The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction-monitoring (MRM) modes were m/z 785.5 (precursor ion) to m/z 143.2 (product ion) for AGS-IV and m/z 781.2 (precursor ion) to m/z 243.3 (product ion) for digoxin (I.S.). The method was validated over a linear range of 1-1000 ng/ml. The low limit of quantitation was 1.0 ng/ml. Results from a 3-day validation study demonstrated that the developed method possessed good precision (CV% values were between 5.9 and 7.6%) and accuracy (96.5-102.1%) across the calibration range. The recoveries were 91 and 90% for astragaloside IV and I.S., and no significant matrix effects were observed. QC samples were stable when kept at room temperature for 4 h, at -20 degrees C for 4 weeks, and after three freeze/thaw cycles.

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.

23-Hydroxybetulinic acid is a newly isolated derivative of betulinic acid. The agent exhibits potential anti-tumor activity and functions in this regard via apoptosis. In support of pharmacokinetic and toxicological evaluations, a new assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of 23-hydroxybetulinic acid. Sample preparation consisted of extraction of the plasma by the addition of methylene chloride followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Molecules of 23-hydroxybetulinic acid and the internal standard limonin were detected using selected ion monitoring at m/z 471 and 469, respectively. The limit of detection of 23-hydroxybetulinic acid was 0.05 pg (0.11 fmol) injected on-column (10 pg/mL, 5 microL injection volume), and the limit of quantitation was 10 pg (21.19 fmol, 2 ng/mL, 5 muL injection volume). 23-Hydroxybetulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were 3.0 and 4.8%, respectively. The utility of the assay was demonstrated by measuring 23-hydroxybetulinicacid in mouse plasma following intragastric administration (IG) in vivo. Pharmacokinetic parameters were calculated using the 3P97 pharmacokinetic software package. A two-compartment, first-order model was selected for pharmacokinetic modeling. The result showed that after IG of 200 mg/kg 23-hydroxybetulinic acid, the plasma concentrations reached peaks at 2 h with C(max) of 3.1 microg/mL. The 200 mg/kg 23-hydroxybetulinic acid suspension IG doses were found to have long elimination half-lives of 25.6 h and low bioavailability of 2.3%. No interference was noted due to endogenous substances. These analytical methods should be of value in future studies related to the development and characterization of 23-hydroxybetulinic acid., (Copyright 2005 John Wiley & Sons, Ltd.)

Astragaloside IV is an important constituent of Radix Astragali, a herbal remedy widely used in traditional Chinese medicine. Radix Astragali is administered orally but little is known about the transport properties and bioavailability of astragaloside IV. In this paper we report studies of the absorption of astragaloside IV in the perfused rat intestinal model, transport and uptake in Caco-2 cell monolayers and in vivo bioavailability in rat after an oral dose. In the perfused rat intestinal model, absorption of astragaloside IV was low from an aqueous solution but was significantly higher from a solution of Radix Astragali. Absorption was not affected by bile duct ligation. Transport through Caco-2 cells gave a very low permeability value (mean P(app) of 6.7+/-1.0 x 10(-8) cm/sec.) and uptake was unaffected by P-glycoprotein inhibitors. The absolute bioavailability of astragaloside IV in rat was 2.2%. The correlation between low permeability in vitro and poor bioavailability in vivo indicates in vitro absorption studies are useful in the evaluation of traditional Chinese medicines.

A 1,2-diglyceride-based multi-step colorimetric assay to measure the pancreatic lipase activity was applied for the determination of the kinetic profiles of the lipase inhibition with a slight modification and the validity verification. With this assay method, our study revealed that platycodin D, one of major constituents of Platycodi Radix, inhibits the pancreatic lipase activity in a competitive type, with the value of KI being 0.18+/-0.02 mM. In addition, PD has affected the values of K(m,app) and Kcat/Km in a dose-dependent manner. The results shed a meaningful light on how PD mediates lipid metabolism in the intestinal tracts. On the other hand, since the revised assay is sensitive, rapid, and does not affect the accuracy to the kinetic properties, it is applicable not only to evaluation of the kinetic properties of the pancreatic lipase, but also to high-throughput screening of pancreatic lipase activity.

Catharanthus roseus cell suspension cultures were used to bioconvert both triptolide (1) and triptonide (2). The same reaction path was followed in both biotransformations. Two biotransformed products were obtained and their structures identified as triptriolide (3) and 12beta,13alpha-dihydroxytriptonide (4), respectively, from 1 and 2. Product 4 is a new compound.

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.

Astragaloside IV (AGS-IV) is an active constituent of Radix Astragali used in many Traditional Chinese Medicines. This paper describes a sensitive and specific assay for the quantitation of AGS-IV in rat plasma. After solid phase extraction (SPE), samples were analyzed by liquid chromatography electrospray ionization mass spectrometry using a reversed-phase C18 column. The assay was linear in the range 1-500 ng/ml with a limit of detection of 0.5 ng/ml. The recovery was 92.5% and within-day and between-day precision were 3.7-6.0 and 2.8-9.8%, respectively. The assay was applied to a pharmacokinetic study in rat after a single oral dose. The drug was rapidly absorbed and subsequently eliminated according to a biphasic concentration-time curve.

Introduction: Boswellia serrata has been used in traditional medicine for treatment of inflammatory diseases since antiquity. However human kinetic studies are lacking for this. Hence to better elucidate its effects in humans and determine its optimal dosing, this study was planned., Material and Methods: Twelve healthy adult men volunteers were given capsule Wok Vel containing 333 mg of Boswellia Serrata Extract, orally, after a seven days washout period. Venous blood samples were drawn through indwelling canula from each volunteer prior to drug administration and at 30, 60, 120, 150, 180, 210, 240, 300, 360, 480, 600, 720, 840 minutes after drug administration. Plasma obtained after centrifuge was analyzed to measure concentration of 11-Keto beta-Boswellic Acid (KBA) by HPLC. Various kinetic parameters were then calculated from the plasma concentrations., Results: The results are expressed as mean +/- Standard Error of Mean. The peak plasma levels (2.72 x 10(-3) +/- 0.18 micromoles/ml) of BSE were reached at 4.5 +/- 0.55 h. The concentration declined with a mean elimination half life of 5.97 +/- 0.95 h. The apparent volume of distribution averaged 142.87 +/- 22.78 L and the plasma clearance was 296.10 +/- 24.09 ml/min. The AUC(0-infinity) was 27.33 x 10(-3) +/- 1.99 micromoles/ml h., Conclusion: Elimination half life of nearly six hours suggests that the drug needs to be given orally at the interval of six hours. The plasma concentration will attain the steady state after approximately 30 hours. BSE is a safe drug and well tolerated on oral administration. No adverse effects were seen with this drug when administered as single dose in 333 mg.

Objective: The aim of this study was to investigate the delivery of Cucurbitacin BE to cervical lymph nodes by peri-cancer submucosal injection of the average diameter 85 nm Cucurbitacin BE polylactic acid nanoparticles (CuBE-PLA-NP)., Methods: Cervical lymph node metastasis model of mice oral cancer was established and CuBE-PLA-NP and CuBE were injected in peri-cancer submucosal of the mice respectively, the concentration in blood, heart, liver, spleen, lung, kidney and cervical lymph node were measured by high performance liquid chromatography at fifteen time points, Targeted cervical lymph nodes were evaluated by targeting index, selectivity index, targeting efficiency, relative targeting efficiency and ratio of maximum concentration., Results: (1) The CuBE concentration in the cervical lymph nodes after CuBE-PLA-NP injection was far higher than that after CuBE injection at every time point, the CuBE duration in the cervical lymph nodes in CuBE-PLA-NP group was far longer than that in CuBE group; (2) The five targeted values of the cervical lymph nodes in CuBE-PLA-NP group was far higher than that in CuBE group; (3) The CuBE concentration in blood, heart, liver, spleen, lung and kidney in CuBE-PLA-NP group was far lower than that in CuBE group., Conclusion: The CuBE-PLA-NP can specifically deliver CuBE to the cervical lymph nodes by peri-cancer submucosal injection, increase CuBE concentration and duration in metastasized cervical lymph nodes, and decrease drug concentration of other organs.

Objective: The aim of this study was to investigate the target delivery of Cucurbitacin BE (CuBE) to cervical lymph nodes by peri-oral-cancer submucosal injection of the average diameter 85 nm Cucurbitacin BE poly-lactic acid nanoparticles (CuBE-PLA-NP) and evaluate its clinical therapy efficacy., Methods: CuBE and CuBE-PLA-NP were respectively injected into peri-oral-cancer submucosa at 2, 12, 24, 48, 72, 96, 120, 144, 168 and 192 hours before operation in 26 patients with oral cancer. The concentrations of CuBE in cervical lymph nodes and blood were measured by high performance liquid chromatography (HPLC) at every time point. Cancer cell degeneration and necrosis in metastasis foci of cervical lymph nodes were observed using light microscope and electron microscope., Results: (1) The CuBE concentrations in cervical lymph nodes after CuBE-PLA-NP injection were far higher than those after CuBE injection at every time point, the duration of CuBE existing in the cervical lymph nodes in CuBE-PLA-NP group was far longer than those in CuBE group, the area under the CuBE concentrations-time curve of the cervical lymph nodes in CuBE-PLA-NP group was 43.67 times as many as that in CuBE group; (2) The CuBE ratio of maximum concentrations in the cervical lymph nodes in CuBE-PLA-NP group was 106.46 times as high as that in CuBE group; (3) The CuBE concentrations in the blood in CuBE-PLA-NP group was far lower than that in CuBE group; (4) Cancer cell necrosis and degeneration in the metastasis foci of cervical lymph nodes were found in CuBE-PLA-NP group, necrosis and degeneration were not found in CuBE group., Conclusion: The peri-oral-cancer submucosa injection of the average diameter of 85 nm CuBE-PLA-NP can specifically delivery CuBE to the cervical lymph nodes, enhance treatment efficiency, and reduce general toxicity.

For the determination of pentacyclic triterpenes of the boswellic acid family in human plasma a novel sensitive method was developed combining serial extraction on diatomaceous earth and graphitized carbon black followed by reversed phase high-performance liquid chromatography (HPLC) and photodiode array detection. The overall average extraction yield of 12 different pentacyclic triterpenic acids was approximately 66%. The calibration graphs were linear with coefficients of correlation for all compounds greater than 0.999. The overall within-day and between-day coefficients of variation (CV) for the 12 pentacyclic triterpenic acids were 5.6 and 6.8%, respectively. This HPLC procedure delivers the analytical sensitivity, precision and accuracy required for clinical pharmacokinetic and therapeutic studies.

The diterpenoid triepoxides are the major active constituents of Tripterygium wilfordii with potent antitumor and immune activities. But the strong toxicity of these compounds has restricted their application to a great extent. In order to find more effective compounds with less toxicity, structural modifications of triptonide (1) by Aspergillus niger (AS 3.739) were investigated and four biotransformed products were obtained. Based on their chemical and spectral data, their structures were elucidated as 5alpha-hydroxytriptonide (2), triptolide (3), 17-hydroxytriptonide (4), and 16-hydroxytriptonide (5), among which 2, 4 and 5 are new compounds. All the three new transformed products showed cytotoxic activities against the majority of the human tumor cell lines tested, however, they are found to possess less cytotoxic activity when compared with 1. Both compounds 4 and 5 showed similar cytotoxic activity and their IC (50) values were 5-15 fold less than 1, while 2 is about 100 times less active than 1.

Eleven serratane-type triterpenoids isolated from the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. jezoensis (Pinaceae) and the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. hondoensis (Mayer) Rehder (Pinaceae) and three synthetic analogs were studied for their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 21-Episerratenediol, serratenediol, diepiserratenediol, 3 beta-hydroxyserrat-14-en-21-one, 3 alpha-methoxy-21 beta-hydroxyserrat-14-en-16-one, 3 beta-methoxyserrat-14-en-21 beta-yl acetate, 3 alpha-methoxyserrat-14-en-21 beta-yl acetate and 3 beta-methoxyserrat-14-en-21 alpha-yl acetate demonstrated strong inhibitory effects on the EBV-EA activation without showing any cytotoxicity, their effects being stronger than that of a representative control, oleanolic acid. Furthermore, 21-episerratenediol exhibited a remarkable inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene as an initiator and TPA as a promoter. The result of the present investigation indicated that 21-episerratenediol might be valuable as a potent cancer chemopreventive agent.

A high-performance liquid chromatographic method for the determination of dehydrotumulosic acid in plasma of rats having been administrated orally with the traditional Chinese medicinal preparation Ling-Gui-Zhu-Gan decoction was developed. Plasma samples taken from rats were acidified with hydrochloric acid and extracted with ethyl acetate. Separation of the main effective constituent dehydrotumulosic acid was accomplished on a C(18) stationary phase and a mobile phase of methanol-acetonitrile-2% glacial acetic acid (13:12:10, v/v), with a UV detector setting at 242 nm. After validation, the method was used for preliminary investigation of the pharmacokinetic profiles of dehydrotumulosic acid administrated in Ling-Gui-Zhu-Gan decoction.

Argentatin B is a naturally occurring tetracyclic triterpene isolated from Parthenium argentatum x P. tomentosa. It was microbiologically transformed to 16, 24-epoxycycloartan-3alpha, 25-diol, (isoargentatin D), by Nocardia corallina var. taoka ATCC 31338, Mycobacterium species NRRL B3683 and Septomyxa affinis ATCC 6737. The later microbe also produced 16, 24-epoxycycloartan-3beta, 25-diol (argentatin D) and 1, 2-didehydroargentatin B, (isoargentatin D). Sodium hydroxide converted argentatin B to argentatin D and isoargentatin D. Hydrochloric acid treatment gave cycloartan-25-ol-3, 24-dione. Cerium sulfate/sulfuric acid/aqueous methanol induced scission of the isopropanol moiety and provided an isomeric mixture of 24-methoxy-25-27-trinorargentatin B. Oxidation of this isomeric mixture with pyridinium chlorochromate, selectively, attacked the isomer with the equatorial proton at position-24 to give the corresponding lactone, 24-oxo-25-27-trinorargentatin B. The produced compounds were characterized by spectroscopic methods.

Ginsenosides, the glycosides of Panax ginseng, are metabolized (deglycosylated) by intestinal bacteria after oral administration. 20(S)-Protopanaxatriol (M4) is the main bacterial metabolite of protopanaxatriol-type ginsenosides and mediates their antitumor effects. To clarify the mechanism of the M4-mediated antitumor effect, the antitumor activity and metabolism of M4 was examined, using the C57BL/6 mice implanted with B16-BL6 melanoma. The chronic oral administration of M4 inhibited the growth of B16-BL6 melanoma at the implanted site. Analyses using TLC, HPLC, MS and NMR suggest that orally administered M4 was absorbed from the small intestine into the mesenteric lymphatics followed by the rapid esterification of M4 with fatty acids and its accumulation in the tissues including the liver and lung. The administration of M4 prior to the intravenous injection of B16-BL6 cells abrogated the enhanced lung metastasis in the mice pretreated with 2-chloroadenosine more effectively than in those pretreated with anti-asialo GM1. The esterified M4 (EM4) did not directly affect tumor growth in vitro, whereas it stimulated splenic NK cells to become cytotoxic to tumor cells. These results indicate that the antitumor activity of M4 is based on the NK cell-mediated tumor lysis enhanced by EM4.

A sensitive, specific, accurate, fast and reproducible GC/MS-method for the quantitative determination of 11-keto-boswellic acid in human plasma using 18alpha-glycyrrhetinic acid as the internal standard was developed and validated. 11-Keto-boswellic acid and the internal standard were separated from acidified plasma by liquid/liquid extraction. The extracted samples were methylated and analyzed by GC/MS in the negative ion chemical ionization mode (NICI) and selected ion monitoring (SIM). The total run time was 8 min between injections. The assay described in this paper demonstrates a validated lower limit of quantification of 0.0100 microg/ml using 1 ml of plasma. The calibration curves are linear in the measured range between 10.0 and 2000 ng/ml plasma. The overall precision (expressed as CV) and accuracy (expressed as bias) for all concentrations of quality controls and standards is better than 15%. No indications were found for possible instabilities of 11-keto-boswellic acid in plasma, in whole blood, in the extraction solvent or after repeated thawing/freezing cycles. The recovery of the extraction method is calculated as 84%. The assay was applied successfully to determine the plasma level of 11-keto-boswellic acid in a clinical pilot study.

The biotransformation of a mixture of argentatin A (20%) 1 and incanilin (80%) 2 by Gibberella suabinetti ATCC 20193 and Septomyxa affinis ATCC 6737 demonstrated the conversion of incanilin to 16beta-hydroxylanosta-2, 8, 23-triene, while argentatin A did not react. The acetate of this triterpenoid mixture was biotransformed by Septomyxa affinis ATCC 6737 to give five metabolites. Argentatin A acetate was transformed to 3beta, 16beta,30-trihydroxycycloart-20, 24-diene, 20R, 24R-epoxy-16beta, 25-dihydroxy-3, 4-seco-cycloart-4(28)-en-3-oic acid acetate and 20R, 24R-epoxy-16beta, 25-dihydroxy-3, 4-seco-cycloart-4(28)-en-3-oic acid. Incanilin acetate was converted to 16beta-hydroxylanosta-2, 8, 23-triene and 20R, 24R-epoxy-16beta, 25-dihydroxy-3, 4-seco-lanost-1, 4(28), 8-trien-3-oic acid acetate. The structural elucidations of these metabolites were achieved by different spectroscopic methods.

The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpenoid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1, 14 and ursolic acid (20) were isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2alpha,13-dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl 3beta,7beta,21beta-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3beta,28-dihydroxyurs-12-ene (uvaol) (23) and 3beta,28-bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the micro-organism, however.

The leaves of Gymnema inodorum (GI) have been known to be effective for some diseases including diabetes mellitus, rheumatic arthritis and gout. The crude saponin mixtures extracted from GI leaves inhibited glucose absorption in the isolated intestinal tract and suppressed the increased blood glucose in rats. In this study, we examined the relationship between chemical structure and pharmacological activity of the four components from GI leave extracts (GiA-1, GiA-2, GiA-5 and GiA-7). These components were the derivatives of (3beta,4alpha,16beta)-16,23,28-trihydroxyolean-12-en-3-yl-beta-D-glucopyranosiduroic acid. GiA-2, GiA-5 and GiA-7 that have suppressive effects on the high K+-induced contraction, an increase in deltaPD and the increased blood glucose level in the glucose tolerance test have -H at the 21st position and -CH2OH at 4beta of aglycon. On the other hand, GiA-1 that does not have any effects on the three parameters mentioned above has -H at the 21st position and -CH3 at 4beta of aglycon. In conclusion, it is suggested that the inhibitory effect of triterpenoids in Gymnema leaves on glucose absorption from the intestinal tract relies on -CH2OH at 4beta.

When ginsenoside Rb1 and Rb2 were anaerobically incubated with human intestinal microflora, these ginsenosides were metabolized to 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) and 20(S)-protopanaxadiol. Several kinds of intestinal bacteria hydrolyzed these ginsenosides. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp., which more potently hydrolyzed gentiobiose than sophorose, metabolized ginsenoside Rb1 to compound K via ginsenoside Rd rather than gypenoside XVII. However, Fusobacterium K-60, which more potently hydrolyzed sophorose than gentiobiose, metabolized to compound K via gypenoside XVII. Ginsenoside Rb2 was also metabolized to compound K via ginsenoside Rd or compound O by human intestinal microflora. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp. metabolized ginsenoside Rb2 to compound K via ginsenoside Rd rather than compound O. Fusobacterium K-60 metabolized ginsenoside Rb2 to compound K via compound O.

Azadirachtin-A on exposure to UV-light (254 nm) as a thin film on glass surface gave a isomerised (Z)-2-methylbut-2-enoate product. Half-life of azadirachtin-A as thin film under UV light was found to be 48 min. Azadirachtin-A was irradiated along with saturated and unsaturated fatty acids, and fatty oils under ultra-violet light as thin film. Saturated fatty acid increased the rate of photodegradation of azadirachtin-A, whereas unsaturated fatty acids such as oleic, linoleic and elaidic acid reduced the rate of degradation. Castor, linseed and olive oil accelerated the rate of degradation, whereas neem oil showed no or little change in the rate of degradation of azadirachtin-A. None of these fatty acids and fatty oils were effective in controlling the rate of degradation of azadirachtin-A under UV-light as thin film.

After intravenous administration of AS 2-006A, 20, 50, and 90 mg/kg, to rats, the pharmacokinetic parameters, terminal half-life (69.8-86. 6 min), mean residence time (56.2-75.2 min), apparent volume of distribution at steady state (809-1040 mL/kg), and total body clearance (11.4-11.9 mL/min/kg), were dose-independent. After topical application of 0.5 or 1% AS 2-006A ointment, 300 mg, to abraded rat skin, the absorbed amounts were dose (0.5 and 1%) and time (30, 60, 120, 240, 360 and 480 min)-independent; the value was approximately 20%. The tissue-to-plasma ratios of AS 2-006A were greater than unity in all rat tissues studied, except in the muscle and large intestine. AS 2-006A was stable for up to 24 h incubation in rat plasma, and human plasma and urine; however, it seemed not to be stable in rat urine; the disappearance rate constant was 0.0218/h. AS 2-006A reached equilibrium fast between plasma and blood cells, and the equilibrium plasma/blood cells partition ratios were independent of the initial rabbit blood concentrations of AS 2-006A, 10, 20, and 50 microg/mL; the mean values were in the range of 2.38-2.75 for three rabbit blood. The protein binding of AS 2-006A to rat plasma was high, as the drug was under detection limit in the filtrate at the plasma concentrations of the drug, ranging from 7.21 to 228 microg/mL., (Copyright 2000 John Wiley & Sons, Ltd.)

From the use of silicones within O/W/O multiple emulsions, we can expect, two principal advantages: (1) the silicones with the lowest molecular weight decrease the oily touch; (2) due to the large range of viscosity, this excipient should influence the skin distribution of actives after topical application. The purpose of our work is to highlight these advantages. Multiple emulsions were formulated with several dimethicones and with drug model. Firstly, the effects of different dimethicones incorporated within multiple emulsions were studied, through in vitro penetration results. Secondly, we investigated the residual film on the skin by Differential Scanning Calorimetry (DSC) and by Fourier Transform Infrared (FTIR) to determine its structure. Correlations were established between the silicone structure and the distribution of drugs in different skin levels or between the silicone structure and the percutaneous penetration. The incorporation of silicones within O/W/O multiple emulsions seems to be an efficient means of modulating the penetration and the distribution of drugs in the skin.

Betulinic acid is a naturally occurring pentacyclic triterpenoid. Betulinic acid has recently been selected by the National Cancer Institute for addition into the RAID (Rapid Access to Intervention in Development) programme. The agent exhibits potential anti-tumour activity and functions in this regard via apoptosis. The objective of the present study was to determine the pharmacokinetics of betulinic acid in CD-1 mice. Serum samples were obtained at designed times after a single 250 or 500 mg/kg intraperitoneal (IP) dose of betulinic acid. Tissue samples (skin, heart, liver, spleen, kidney, lung, brain, colon, caecum, ovary, uterus, thymus, lymph node, bladder, perirenal fat, mammary gland and small intestine) were collected after betulinic acid administration (500 mg/kg). Betulinic acid was extracted with methylene chloride and quantitatively analysed by HPLC/MS. Oleanolic acid and madecassic acid were used as internal standards. Pharmacokinetic parameters were calculated using the WinNonlin pharmacokinetic software package. A two-compartment, first-order model was selected for pharmacokinetic modelling. The results showed that after IP 250 and 500 mg/kg betulinic acid, the serum concentrations reached peaks at 0.15 and 0.23 h, respectively. The 250 and 500 mg/kg above betulinic acid IP doses were found to have elimination half-lives of 11.5 and 11.8 h and total clearances of 13.6 and 13.5 L/kg/h, respectively. The pharmacokinetic parameters observed for IP betulinic acid 500 mg/kg in the skin of mice were as follows: k(a) (h(-1)) 0.257, k(10) (h(-1)) 0.234, t(1/2(alpha)) (h) 2.63, t(1/2(beta)) (h) 20.2, V (L/kg) 0.61, AUC (microg/h/mL) 3504, T(max) (h) 3.90 and C(max) (microg/mL) 300.9. The distribution of betulinic acid in tissues at 24 h post-IP administration in a descending order was as follows: perirenal fat, ovary, spleen, mammary gland, uterus, bladder, lymph node, liver, small intestine, caecum, lung, thymus, colon, kidney, skin, heart and brain., (Copyright 1999 John Wiley & Sons, Ltd.)

Betulinic acid has been coupled with a series of amino acids at C-28 carboxylic acid position and the toxicity of the derivatives has been evaluated against cultured human melanoma (MEL-2) and human epidermoid carcinoma of the mouth (KB) cell lines. A number of amino acid conjugates of betulinic acid showed improved water solubility as well as selective cytotoxicity. This investigation demonstrates that amino acid conjugates of betulinic acid can produce potentially important derivatives, which may be developed as antitumor agents.

A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O-isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found.

Triptonide, isolated from Tripterygium wilfordii Hook., was found to show significant antiinflammatory, immunosuppression and antitumor activities. A RP-HPLC method was applied to determine the plasma concentration of triptonide at different times in rats. Concentration-time curves after i.v., 0.7, 1.4 and 2.8 mg.kg-1 of triptonide were fitted to a two-compartment open model with T1/2 alpha of 0.167-0.195 h and T1/2 beta of 4.95-6.49 h. The area under curves (AUCs) were linearly related to the dosages (gamma = 0.9894). Systematic clearances (CLs) were independent of dosages. Mean residence time (MRT) of the three doses was 3.26-5.14 h by noncompartmental (the statistical moment method) analyses. The tissue distribution of triptonide in rats appeared to be wide throughout the body. The triptonide levels were high in the lung and liver, moderate in the heart, kidney, spleen and muscle and low in the testis, intestine and brain. Data of the urine and bile excretion indicated that only a small percent of unchanged triptonide was excreted. Plasma protein binding of triptonide rate was about 75%.

The comparative steady-state bioavailability of asiatic acid was studied in 12 healthy male and female volunteers following oral administration of approximately equimolar doses of either asiatic acid (12 mg) or the glycoside derivative of asiatic acid, asiaticoside (24 mg). Both asiatic acid and asiaticoside are constituents of the marketed dermatological product Madecassol. Asiaticoside is converted in vivo to asiatic acid by hydrolytic cleavage of the sugar moiety. Steady-state AUC0-12h values for asiatic acid on either regimen were similar (614 +/- 250 ng.h/ml following asiatic acid compared to 606 +/- 316 ng.h/ml following asiaticoside) indicating comparable bioavailability for asiatic acid with the two ingredients at approximately equimolar doses. Since asiatic acid is considered to be the most therapeutically active ingredient of Madecassol, the current data suggest that the therapeutic effects of asiaticoside may be mediated through conversion to asiatic acid.

The uptake and metabolism of ginsenoside Rh2 (Rh2) by B16 melanoma cells were studied. In a medium containing 2% fetal calf serum, the uptake of Rh2 reached a maximum of 3 nmol/10(6) cells at 3-6 h after Rh2 (12.5 microM) was added, but gradually decreased to 0.8 nmol/10(6) cells. In these cells, protopanaxadiol (PPD), which is an aglycon of Rh2, increased inversely with the decrease in Rh2 as a result of deglycosylation by the cells. When PPD (8 microM) was added to the medium, the uptake reached a plateau of 2.4 nmol/10(6) cells, within 0.5 h. The association constant of Rh2 (1.74 +/- 1.08 x 10(6) M-1) for bovine serum albumin (BSA) was significantly higher than that of PPD (9.90 +/- 1.10 x 10(4) M-1). In a serum-free medium, both Rh2 and PPD were incorporated within 1.5 h. The uptake rate constant of Rh2 (1.20 +/- 0.20 h-1) was not significantly different from that of PPD (1.02 +/- 0.15 h-1), but the release rate constant of PPD (2.12 +/- 0.38 h-1) was significantly lower than that of Rh2 (3.03 +/- 0.57 h-1). These differences in affinity for BSA and the release rate constants were thought to be the cause of the difference in uptake kinetics between these drugs. The effects of Rh2 and PPD on the cells were identical, and there was no difference in the lag periods before the appearance of their effects, despite their differing rates of uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

A new HPLC assay method was used to investigate the pharmacokinetics of asiatic acid after oral administration of the total triterpenic fraction of Centella asiatica in single doses (30 or 60 mg) and after a 7-day treatment (30 or 60 mg twice daily). Twelve healthy volunteers received each treatment following a randomized cross-over design with trials separated by a 3-week interval. The time of peak plasma concentration was not affected by dosage difference or by treatment scheme. Differences in peak plasma concentration and area under the concentration vs. time curve from 0 to 24 h (AUC0-24) calculated after 30 or 60 mg administration (single dose) were accounted for by the different dose regimen. However, after chronic treatment with both 30 and 60 mg, peak plasma concentrations, AUC0-24 and half-life were significantly higher than those observed after the corresponding single dose administration. This phenomenon could be explained by a metabolic interaction between asiatic acid and asiaticoside, which is transformed into asiatic acid in vivo.