Huo-Luo-Xiao-Ling (HLXL)-Dan, a Traditional Chinese Medicine, for patients with osteoarthritis of the knee: a multi-site, randomized, double-blind, placebo-controlled phase II clinical trial.

OBJECTIVE: To examine the efficacy and safety of Huo-Luo-Xiao-Ling (HLXL)-Dan, a Traditional Chinese Medicine (TCM), in patients with knee osteoarthritis (OA). DESIGN: A multi-site, randomized, double-blind, placebo-controlled phase II dose-escalation clinical trial was conducted. Eligible patients who fulfilled American College of Rheumatology criteria were randomized to receive either HLXL or placebo. Clinical assessments included measurement of knee pain and function with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), patient global assessment (PGA), and knee pain scores every 2 weeks. A Data and Safety Monitoring Board (DSMB) was established to review the data for ensuring the quality of the trial. RESULTS: In the first stage, 28 participants were randomized to receive either low-dose HLXL-Dan (2400 mg/day) or placebo for 6 weeks. The results showed no statistical difference between the two groups. The study was then re-designed following the recommendation of DSMB. Ninety-two patients were enrolled in the second stage and were randomized to receive either high-dose HLXL-Dan (4000 mg/day for week 1-2, and 5600 mg/day for week 3-8) or placebo for 8 weeks. All outcome assessments showed significant improvements for both groups after 8 weeks but no significant between-group differences. The change (mean ± SD) of WOMAC pain and WOMAC function scores of HLXL and placebo group after 8 weeks were -1.2 ± 1.7 vs -1.4 ± 1.5, and -1.1 ± 1.6 vs -1.3 ± 1.5 respectively. No serious adverse events were reported. CONCLUSION: Although safe to use, an 8-week treatment of HLXL-Dan was not superior to placebo for reduction in pain or functional improvement in patients with knee OA. CLINICAL TRIAL REGISTRATION NUMBER: Clinicaltrials.gov (NCT00755326).

Role of neuronal nitric oxide synthase in colonic distension-induced hyperalgesia in distal colon of neonatal maternal separated male rats.

BACKGROUND: Nitric oxide (NO) is implicated in the pathogenesis of irritable bowel syndrome (IBS) but the underlying mechanism is unclear. Thus, the aim of the present study is to examine the role of NO synthase (NOS) expression in the distal colon of neonatal maternal separation (NMS) model rats employed in IBS studies. METHODS: Male neonates of Sprague-Dawley rats were randomly assigned into NMS and normal control (N) groups. Rats of NMS group were subjected to 3 h daily maternal separation on postnatal day 2-21. Rats were administrated non-selective NOS inhibitor l-NAME (100 mg kg(-1) ), selective neuronal NOS (nNOS) inhibitor 7-NINA (10mgkg(-1) ), selective inducible NOS (iNOS) inhibitor, endothelial NOS (eNOS) inhibitor (10mgkg(-1) ) or Vehicle (Veh; distilled water) intraperitoneally 1h prior to the experiment for the test and control groups, respectively. KEY RESULTS: The amount of NO was significantly higher in the NMS Veh rats compared with unseparated N rats. Western-blotting and real-time quantitative PCR studies showed that protein and mRNA expression of nNOS were higher in the NMS group than that in the N rats; whereas no significant change in iNOS and eNOS was found in either groups. Neonatal maternal separation Veh rats showed low pain threshold and increased electromyogram (EMG) activity in response to colonic distension stimuli. l-NAME and 7-Nitroindazole monosodium salt (7-NINA) increased pain threshold pressure and attenuated EMG activity in the NMS rats. In addition, l-NAME and 7-NINA substantially reduced oxidative marker malondialdehyde level in NMS rats. CONCLUSIONS & INFERENCES: Neonatal maternal separation increased the NO generation by nNOS upregulation that interact with reactive oxygen species contributing to the visceral hypersensitivity in IBS.

Ethnobotany/ethnopharmacology and mass bioprospecting: issues on intellectual property and benefit-sharing.

Ethnobotany/ethnopharmacology has contributed to the discovery of many important plant-derived drugs. Field explorations to seek and document indigenous/traditional medical knowledge (IMK/TMK), and/or the biodiversity with which the IMK/TMK is attached, and its conversion into a commercialized product is known as bioprospecting or biodiversity prospecting. When performed in a large-scale operation, the effort is referred to as mass bioprospecting. Experiences from the mass bioprospecting efforts undertaken by the United States National Cancer Institute, the National Cooperative Drug Discovery Groups (NCDDG) and the International Cooperative Biodiversity Groups (ICBG) programs demonstrate that mass bioprospecting is a complex process, involving expertise from diverse areas of human endeavors, but central to it is the Memorandum of Agreement (MOA) that recognizes issues on genetic access, prior informed consent, intellectual property and the sharing of benefits that may arise as a result of the effort. Future mass bioprospecting endeavors must take heed of the lessons learned from past and present experiences in the planning for a successful mass bioprospecting venture.

The UIC ICBG (University of Illinois at Chicago International Cooperative Biodiversity Group) Memorandum of Agreement: a model of benefit-sharing arrangement in natural products drug discovery and development.

The Convention on Biodiversity mandates a new approach to the discovery of natural product drugs, one that incorporates concepts of national ownership of genetic resources, intellectual property rights in traditional knowledge, and sharing of economic benefits with countries that are the source of new natural products. The International Cooperative Biodiversity Group (ICBG) program was established to support experimentation in implementation of the Convention through development and execution of international agreements for bioprospecting. The agreement of one such ICBG program, between the University of Illinois at Chicago and institutions in Vietnam and Laos, is presented here. The core elements contained in the single, five-way Memorandum of Agreement are the arrangements for intellectual property rights, treatment of informed consent, and plans for benefit-sharing (including the sharing of short- and long-term royalty benefits, capacity building, and community reciprocity). Program participants were able to develop a practical and flexible agreement that satisfies the wishes of all institutions that are parties to it.

Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry).

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.

In vitro cytotoxicity activity of Diosquinone, a naphthoquinone epoxide.

Diosquinone [1], a naphthoquinone epoxide previously isolated from the root bark of Diospyros mespiliformis (Hostch) and D. tricolor [Ebenaceae] is been assessed for cytotoxicity activity against ten cancer cell lines by standard NIH method. The ethno-pharmacological claim of this plant and the previously observed good antibacterial activity of this compound among the others isolated from this plant suggest its probable cytotoxicity activity. Diosquinone was observed to be very active against most of the cancer cell lines. It shows very good activity against all the cell lines tested with ED50 value ranging between 0.18 microg/ml. against Human Glioblastoma (U373) to 4.5 microg/ml. against Hormone dependent human prostrate cancer( LNCaP).

Evaluation of commercial Ginkgo biloba dietary supplements for the presence of colchicine by high-performance liquid chromatography.

A high-performance liquid chromatographic method with photodiode array detection was developed for the detection of the presence of colchicine in commercial ginkgo products. The method is based on the baseline separation of constituents in ginkgo samples plus reference colchicine. The minimal detectable concentration of colchicine is 1.0 ng on column in the current assay. By analysis of retention time and UV profile of suspect peaks in the sample with those of reference colchicine, none of the nine commercial ginkgo products analyzed contained colchicine.

Aromatase inhibitors from Broussonetia papyrifera.

Bioassay-guided fractionation of an ethyl acetate-soluble extract from the whole plants of Broussonetia papyrifera, using an in vitro aromatase inhibition assay, led to the isolation of five new active compounds, 5,7,2',4'-tetrahydroxy-3-geranylflavone (1), isogemichalcone C (8), 3'-[gamma-hydroxymethyl-(E)-gamma-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11'-O-coumarate (9), demethylmoracin I (10), and (2S)-2',4'-dihydroxy-2' '-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h]flavanone (11), and 10 known (12-21) compounds which were also found to be active. Of these compounds, the most potent were 9 (IC(50) 0.5 microM), 11 (IC(50) 0.1 microM), isolicoflavonol (12, IC(50) 0.1 microM), and (2S)-abyssinone II (13, IC(50) 0.4 microM). Additionally, six new compounds, 5,7,3',4'-tetrahydroxy-6-geranylflavonol (2), 5,7,3',4'-tetrahydroxy-3-methoxy-6-geranylflavone (3), (2S)-7,4'-dihydroxy-3'-prenylflavan (4), 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propane (5), 1-(2,4-dihydroxy-3-prenylphenyl)-3-(4-hydroxyphenyl)propane (6), and 1-(4-hydroxy-2-methoxyphenyl)-3-(4-hydroxy-3-prenylphenyl)propane (7), were isolated and characterized, but proved to be inactive as aromatase inhibitors, as were an additional 21 known compounds. The structures of the new compounds (1-11) were elucidated by spectroscopic methods. Structure-activity relationships in the aromatase assay were determined for the benzofurans, biphenylpropanoids, coumarins, and various types of flavonoids (chalcones, flavans, flavanones, and flavones) obtained among a total of 42 constituents of B. papyrifera.

Taxane diterpenoids from the bark of Taxus yunnanensis.

Further investigation on the alcohol extract of the barks of Taxus yunnanensis led to the isolation of four taxoids, namely, 7beta-xylosyl-taxol D, taxuyunnanines P, Q and R, along with the known taxuyuntin G (2). Four are rearranged taxoids with an 11(15-->1)-abeotaxoid skeleton and an opened oxetane ring moiety. Structures were determined by spectroscopic and chemical means.

A novel cyclooxygenase-inhibitory stilbenolignan from the seeds of Aiphanes aculeata.

[structure: see text] Aiphanol (1), a novel stilbenolignan, along with isorhapontigenin (2), piceatannol (3), and luteolin, were isolated by bioassay-guided fractionation from the seeds of Aiphanes aculeata Willd. (Arecaceae). The structure of compound 1 was elucidated by spectroscopic methods. Compound 1 is based on an unprecedented stilbenolignan skeleton in which a stilbene moiety is linked with a phenylpropane unit through a dioxane bridge. Compounds 1 and 2 exhibited significant inhibitory activities against cyclooxygenases-1 and -2.

Antimalarial compounds from Rhaphidophora decursiva.

Bioassay-directed fractionation led to the isolation of 14 compounds, six of which possess antimalarial activity, from the dried leaves and stems of Rhaphidophora decursiva. Polysyphorin (1) and rhaphidecurperoxin (6) showed strong activities against Plasmodium falciparum. Rhaphidecursinol A (2), rhaphidecursinol B (3), grandisin (4), and epigrandisin (5) were less active against the same organism. Among the isolates, rhaphidecursinol A (2) and rhaphidecursinol B (3) were determined to be new neolignans, and rhaphidecurperoxin (6) is a new benzoperoxide. Known compounds isolated include polysyphorin (1), grandisin (4), epigrandisin (5), (+)-medioresinol, (-)-pinoresinol, (-)-syringaresinol, (+)-glaberide I, (+)-dehydrovomifoliol, (-)-liliolide, (-)-hydroxydihydrobovolide, and N-butylbenzamide, of which compound 1 appears worthy of further evaluation as an antimalarial agent. Structure elucidation and identification were accomplished by spectroscopic means including 1D and 2D NMR analyses.

Cyclooxygenase-inhibitory and antioxidant constituents of the aerial parts of Antirhea acutata.

Two new compounds, (6S)-hydroxy-29-nor-3,4-seco-cycloart-4(30),24-dien-3-oic acid (1) and 8-[1-(3,4-dihydroxyphenyl)-3-methoxy-3-oxopropyl]epicatechin (3), were isolated by bioassay-guided fractionation from the aerial parts of Antirhea acutata (DC.) Urb. (Rubiaceae). Compound 1 showed moderate inhibitory activities in cyclooxygenase-1 and -2 assays (IC(50) 43.7 and 4.7 microM, respectively), while compound 3 was active in 1,1-diphenyl-2-picrylhydrazyl free-radical and cytochrome c reduction antioxidant assays (IC(50) 29.1 and 16.3 microM, respectively). Additionally, one further new compound was isolated, (3S,24S)-25-trihydroxy-9,19-cycloartane-29-oic acid (2), but this was inactive in the bioassay systems used. Compound 1 is based on the unprecedented 29-nor-3,4-seco-cycloartane skeleton.

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography.

The pregnane steroid, (E)-aglawone, along with four known triterpenes, and a known sterol mixture were isolated from the bark of Aglaia lawii (Wight) Saldanha ex Ramamoorty (Meliaceae). The structural determination/identification was accomplished by a combination of 1D- and 2D-NMR spectroscopic techniques. The relative stereochemistry of the known triterpene, 20S,24S-epoxydammarane-3alpha,25-diol acetate, was also unequivocally determined for the first time by X-ray crystallography. The isolates were not active against various human cancer cell lines.

Use of high-performance liquid chromatography-tandem mass spectrometry to distinguish Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L. (North American ginseng).

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to distinguish Asian ginseng (Panax ginseng C. A. Meyer) and North American ginseng (Panax quinquefolius L.). The method is based on the baseline chromatographic separation of ginsenoside Rf and 24(R)-pseudoginsenoside F11, two potential chemical markers present in ginseng root methanolic extracts, and their unambiguous on-line identification using tandem mass spectrometry. Consistent with the literature, 24(R)-pseudoginsenoside F11 was detected in abundance in North American ginseng roots in excess of 0.1% (w/w) of the dried root. In contrast to some reports, 24(R)-pseudoginsenoside F11 was also identified in Asian ginseng roots at trace levels using LC-MS-MS but at less than 0.0001% (w/w). Besides showing identical tandem mass spectra to authentic 24(R)-pseudoginsenoside F11, the corresponding compound in Asian ginseng root coeluted with standard under different HPLC conditions, thus confirming this compound as 24(R)-pseudoginsenoside F11. Another ginsenoside often used to distinguish Asian and North American ginseng, ginsenoside Rf, was found in abundance in Asian ginseng roots at more than 0.021% (w/w). In Asian ginseng roots, the ratio of ginsenoside Rf to 24(R)-pseudoginsenoside F11 exceeded 700:1. The limit of detection of ginsenoside Rf or 24(R)-pseudoginsenoside F11 was 120 pg injected on-column, and the limit of quantification was 240 pg on-column. In summary, LC-MS-MS analysis of ginseng products for the presence and ratio of ginsenoside Rf and 24(R)-pseudoginsenoside F11 may be used for the unambiguous identification of Asian and North American ginsengs.

Rearranged taxanes from the bark of Taxus yunnanensis.

Five new 11(15-->1)-abeo-taxane diterpenoids, taxuyunnanines K-O (1-5), were isolated from an ethanol extract of the bark of Taxus yunnanensis, and their structures were determined using MS and NMR techniques. Compounds 1/2 and 4/5 are rearranged taxane diterpenoids possessing an opened oxetane ring moiety at C4(20). Compounds 4/5 are rearranged taxoids lacking an oxygenated functionality at C-4.

Isolation and characterization of cytotoxic saponin chloromaloside A from Chlorophytum malayense.

A cytotoxic steroidal glycoside was isolated from Chlorophytum malayense Ridley and its structure was characterized as a known compound, neohecogenin 3-O-beta-D-glucopyranosyl(1-->2)-[beta-D-xylopyranosyl(1-->3)]-beta-D- glucopyranosyl(1-->4)-beta-D-galactopyranoside (chloromaloside A). The structural identification was performed using 2D-NMR and LC/MS/MS analysis. The previous, erroneously assigned 1H-NMR spectral data were revised whereas the published 13C-NMR spectral assignments were confirmed. This compound showed in vitro cytotoxicity against several human cancer cell lines.

Antibacterial activity of diospyrin, isodiospyrin and bisisodiospyrin from the root of Diospyros piscatoria (Gurke) (Ebenaceae).

Two dimeric naphthoquinones, diospyrin and isodiospyrin, isolated from the root of Diospyros piscatoria (Gurke), a common ingredient in several folk medicines, have been shown to have a broad spectrum of antibacterial activity. The minimum inhibitory concentrations (MICs) of diospyrin against Streptococcus pyogenes ATCC 12344 and Streptococcus pneumoniae ATCC 33400 ranged from 1.56 to 50 microg/mL. While those against Salmonella choleraesuis serotype typhi (S. typhi), ATCC 6539 and Mycobacterium chelonae ATCC 19977 were between 25 and 100 microg/mL. Isodiospyrin was more active than its racemic isomer diospyrin. The MICs against Gram-positive bacteria ranged from 0.78 to 50 microg/mL. While those against Pseudomonas aeruginosa ATCC 15443 and S. typhi ranged from 50 to 100 microg/mL. The MIC for M. chelonae was between 6.25 and 25 microg/mL. MICs were found to increase with the concentration of cells used for the inoculum. The MICs for Bacillus subtilis ATCC 6633 increased up to the highest concentration of cells tested. The same phenomenon was observed on M. chelonae, but with better effect in the latter. The kinetics of bacteria studies against both B. subtilis and M. chelonae increases with increasing concentration of isodiospyrin tested. Two tetrameric forms of plumbagin were isolated. The naphthoquinone bisisodiospyrin, gave MIC values between 300 and 400 micro g/mL. The second, as yet unidentified tetramer, was not active at 500 micro g/mL.

Activity-guided isolation of antioxidative constituents of Cotinus coggygria.

Six constituents (1-6) were isolated from EtOAc-soluble partitions of two separate collections of the whole plants of Cotinus coggygria, namely, disulfuretin ¿2,2'-[1,2-bis(3,4-dihydroxyphenyl)-1, 2-ethanedylidene]bis[6-hydroxy-3(2H)-benzofuranone] (1)¿, sulfuretin (2), sulfurein (3), gallic acid (4), methyl gallate (5), and pentagalloyl glucose (6). The structure of the novel biaurone 1 was determined by spectral and chemical methods. Compounds 1-6 were found to be potent antioxidants in a 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging assay.

Activity-guided isolation of steroidal alkaloid antiestrogen-binding site inhibitors from Pachysandra procumbens.

Four novel steroidal alkaloids, (+)-(20S)-20-(dimethylamino)-3-(3'alpha-isopropyl)-lactam-5alpha-+ ++preg n-2-en-4-one (1), (+)-(20S)-20-(dimethylamino)-16alpha-hydroxy-3-(3'alpha-isopropyl) -la ctam-5alpha-pregn-2-en-4-one (2), (+)-(20S)-3-(benzoylamino)-20-(dimethylamino)-5alpha-pregn-2-en-++ +4beta -yl acetate (3), and (+)-(20S)-2alpha-hydroxy-20-(dimethylamino)-3beta-phthalimido-5 alpha- pregnan-4beta-yl acetate (4), as well as five known compounds, (-)-pachyaximine A (5), (+)-spiropachysine (6), (+)-axillaridine A (7), (+)-epipachysamine D (8), and (+)-pachysamine B (9), were isolated from Pachysandra procumbens, using a bioassay-guided fractionation based on inhibition of 3H-tamoxifen binding at the antiestrogen binding site (AEBS). Compounds 1-7 and 9 demonstrated significant activity as AEBS-inhibitory agents, and compounds 3, 5 and 9 were found to potentiate significantly the antiestrogenic effect mediated by tamoxifen in cultured Ishikawa cells. The structure elucidation of compounds 1-4 was carried out by spectral data interpretation.