The role of the PCM in reducing oxidative stress induced by radical initiated photoencapsulation of chondrocytes in poly(ethylene glycol) hydrogels.

OBJECTIVE: The objectives for this study were to determine whether radical initiated photopolymerizations typically employed for cell encapsulations lead to oxidative stress incurred by chondrocytes and whether the development of a pericellular matrix (PCM) decreases this oxidative stress and has longer-term benefits on chondrocyte function. METHODS: Freshly isolated bovine chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels devoid of a PCM or with a PCM, confirmed by immunocytochemistry (IC), and cultured for up to 2 weeks. Reactive oxygen species (ROS) production and damage to cell membrane by lipid peroxidation were accomplished using carboxy-2,7-difluorodihydrofluorescein diacetate (carboxy-H(2)DFFDA) and by malondialdehyde (MDA) content, respectively. Gene expression and proteoglycan synthesis were analyzed using reverse transcription (RT)-quantitative PCR (qPCR) and (35)SO(4) incorporation, respectively. RESULTS: The photopolymerization reaction, which alone generates radicals and extracellular ROS, led to oxidative stress in chondrocytes evidenced by increased intracellular ROS and lipid peroxidation. The presence of a PCM decreased intracellular ROS and abrogated membrane lipid peroxidation, improved aggrecan, collagen II and collagen VI expression, and enhanced proteoglycan synthesis. CONCLUSIONS: The development of the PCM prior to photoencapsulation in PEG hydrogels reduces oxidative stress and improves chondrocyte anabolic activity. Our data suggest this reduction occurs by decreased ROS diffusion into the cell and decreased membrane damage. Our findings suggest that minimizing oxidative stress, such as through the presence of a PCM, may have long-term beneficial effects on tissue elaboration when employing photopolymerizations to encapsulate chondrocytes for cartilage tissue engineering applications.

Pharmacokinetics of 23-epi-26-deoxyactein in women after oral administration of a standardized extract of black cohosh.

Dietary supplements containing black cohosh are alternatives to conventional hormone replacement therapy in menopause. This study investigates the maximum tolerated dose of a 75% ethanol extract of black cohosh and determines the pharmacokinetics of one of its most abundant triterpene glycosides, 23-epi-26-deoxyactein. Single doses of black cohosh extract containing 1.4, 2.8, or 5.6 mg of 23-epi-26-deoxyactein were administered to 15 healthy, menopausal women. Serial blood samples and 24-h urine samples were obtained; blood chemistry, hormonal levels, and 23-epi-26-deoxyactein levels were determined. No acute toxicity or estrogenic hormone effects were observed. Pharmacokinetic analyses of 23-epi-26-deoxyactein in sera indicated that the maximum concentration and area under the curve increased proportionately with dosage, and that the half-life was ~2 h for all dosages. Less than 0.01% of the 23-epi-26-deoxyactein was recovered in urine 24 h after administration. No phase I or phase II metabolites were observed either in clinical specimens or in vitro.

Activation of the mu-opiate receptor by Vitex agnus-castus methanol extracts: implication for its use in PMS.

The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to opiate receptors; however, functional activity was not determined. We tested two different VAC extracts in receptor binding and functional assays. Our objectives were: (1) to confirm the opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the mu-opiate receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the mu-opiate receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human mu-opiate receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p<0.001), it argued against significant interference by fatty acids. In an assay to determine receptor activation, VAC-M1 and VAC-M2 stimulated [35S]GTPgammaS binding by 41 and 61% (p<0.001), respectively. These results suggested for the first time that VAC acted as an agonist at the mu-opiate receptor, supporting its beneficial action in PMS.

The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties.

As the population ages, there is an ever-increasing need for therapeutic agents that can be used safely and efficaciously to manage symptoms related to postmenopausal estrogen deficiency. Endogenous estrogens, e.g., 17beta-estradiol, of exogenous mammalian origin, e.g., horses, have long been used to manage such symptoms. There are more than 20 different classes of phytochemicals that have demonstrated affinity for human estrogen receptors in vitro. Some studies on exogenous estrogenic substances of botanical origin (phytoestrogens), such as standardized formulations of plant extracts with in vitro and in vivo estrogenic activity from soy (Glycine max Merill.) and red clover (Trifolium pratense L.), suggest clinical efficacy. Few clinical data for phytoestrogens other than isoflavonoids are available. In an exhaustive review of the literature through 2003, only two clinical trials were identified that were designed to evaluate the effect of hops (Humulus lupulus L.) on symptoms related to menopause. Folkloric, chemical, and biological literature relating primarily to the use of hops for their estrogenic activity, and two human clinical trials, are reviewed.

Chemical and biological characterization and clinical evaluation of botanical dietary supplements: a phase I red clover extract as a model.

Botanical dietary supplements, as compared with nutritional supplements or single-component pharmaceutical drugs, are typically less-refined preparations derived from bulk plant material and, as such, require a modified approach to their development, production, and evaluation. An integrated, multidisciplinary team of scientific and clinical investigators is required in order to develop high quality phytomedicines and rigorously evaluate their safety and efficacy. Research on botanicals involves unique challenges as plant source materials frequently vary in chemical content and may contain unwanted pesticides, heavy metals, contaminant plant species, or other adulterants. Ideally, a botanical formulation should be standardized, both chemically and biologically, by a combination of analytical techniques and bioassays. This combination approach provides multiple measures by which reproducible quality and efficacy of botanical supplements may be achieved, and is particularly useful for botanical products for which the active compound(s) have not yet been identified. Safety and toxicity should be evaluated during the supplement development process in both in vitro and in vivo systems. A number of liquid chromatography-mass spectrometry methods can aid in the assessment of purity, bioavailability, toxicity, metabolism, and molecular target profiling of botanical extracts. Clinical investigators must appreciate the complexity of multi-component phytomedicines and adjust trial protocols accordingly. This review highlights practical considerations of value to basic science and clinical investigators engaged in the study of botanical supplements. Lessons and examples are drawn from the authors' experience in designing and developing a red clover (Trifolium pratense L.) standardized extract for evaluation in Phase I and Phase II clinical trials.

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.

Antimycobacterial evaluation of Peruvian plants.

We present the results of an antimycobacterial screening of 270 Peruvian plant samples representing 216 species from 171 genera in 63 families. Dichloromethane extracts were tested at a concentration of 50 microg/ml for inhibition of Mycobacterium tuberculosis in radiometric culture. Slightly more than half of the samples tested showed inhibition of M. tuberculosis at this concentration.

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.

A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women's health.

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.

Macharistol, a new cytotoxic cinnamylphenol from the stems of Machaerium aristulatum.

A new cinnamylphenol, macharistol (1), along with a known pterocarpan, (+)-medicarpin (2), were isolated as cytotoxic constituents from the stems of Machaerium aristulatum. In addition, a known pterocarpan, (+)-maackiain (3), and a known isoflavone, formononetin (4), were identified as inactive constituents. Compound 1 was evaluated in the in vivo hollow fiber assay with KB, Col-2, and hTERT-RPE1 cells and found to be inactive at the highest dose (25 mg/kg body weight) tested.