Phytomedicine

This study was undertaken to evaluate a possible effect of the extracts PY102 of Pygeum africanum (Hook), and UR 102 of Urtica dioica L. as well as their combination PHL-00801 (Prostatonin®) on the enzymes 5 α-reductase (5 α-RE) and aromatase (AR): Inhibition of 5 α-RE: Pygeum africanum extract PY 102, and Urtica dioica extract UR 102, inhibited the 5 α-RE activity in a concentration dependent manner. Whereas UR102 extract was only able to influence the enzyme activity at high concentrations (≥ 12mg/ml) and its ED(50) being calculated as 14.7mg/ml, the PY102 extract showed a much higher activity starting with low concentrations (0.1 mg/ml) its ED(50) being calculated as 0.78 mg/ml. When compared with the effects of UR 102, the combination of both extracts, PHL-00801 (Prostatonin®), led to a similar inhibition of the enzyme (ED(50) 14.15 mg/ml). Inhibition of AR: The PY 102 extract showed a concentration dependent and strong activity (ED(50) = 0.98 mg/ml). The activity of the UR 102 extract was also concentration dependent (ED(50) = 3.58 mg/ml). The combination of both extracts, PHL-00801 (Prostatonin®) showed a synergistic action and significantly (p = 0.05) increased the AR-inhibitory activity in concentrations as low as 0.1 mg/ml (ED(50) 0.24 mg/ml). These observations are an explanation for the beneficial effects of PHL-00801 (Prostatonin®) observed in the clinical studies on BPH.

A 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-generating system was used to evaluate the antioxidant properties of Korean medicinal plants that have been used widely as folk medicines for several disorders, as well as compounds isolated from them. Among the Rosaceae, Rosa rugosa and Rosa davurica showed strong DPPH radical-scavenging activity. The most effective medicinal plant from families other than Rosaceae was Cedrela sinensis, followed in order by Nelumbo nucifera, Eucommia ulmoides, Zanthoxylum piperitum, Cudrania tricuspidata and Houttuynia cordata. These results serve as a good index of the free radical-scavenging activities of Korean medicinal plants. Furthermore, the polyphenols isolated from these plants, procyanidin B-3, (+)-catechin, gallic acid, methyl gallate, quercetin, quercetin-3-O-beta-D-glucoside, quercetin-3-O-beta-galactoside, quercetin-3-O-rutinose and kaempferol, exerted strong DPPH radical-scavenging activity. These results suggest that the Korean medicinal plants and the polyphenols isolated from them that exhibited effective radical-scavenging activity may be promising agents for scavenging free radicals and treating diseases associated with excess free radicals.

A dichloromethane extract from the leaves of Lithraea molleoides (Anacardiaceae), an argentine medicinal plant, showed cytotoxicity on human hepatocellular carcinoma cell line. Bioassay guided fractionation of this extract led to the isolation of a new active 5-alkyl resorcinol: 1,3-dihydroxy-5-(tridec-4',7'-dienyl)benzene. Chemical structure was established based on spectroscopic data (UV, IR, MS, 1H-NMR, 13C-NMR, COSY). This compound presented cytotoxic activity on 3 human tumoral cell lines: hepatocellular carcinoma cell line-Hep G2 (IC50 +/- SD of 68 +/- 2 microM), mucoepidermoid pulmonary carcinoma cell line-H292 (IC50 +/- SD of 63 +/- 5 microM) and mammary gland adenocarcinoma cell line -MCF7 (IC50 +/- SD of 147 +/- 5).

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chicken. However the increasing resistance of these parasites to currently used anticoccidial drugs has stimulated the search for new methods of control. As part of this effort we investigated the root bark of Berberis lycium (barberry) as a potential source of compounds with anticoccidial activity. In the present study anticoccidial activity of different solvent extracts of the root bark of B. lycium and berberine was evaluated in vivo using broiler chicken. Results of the study demonstrated equipotent efficacy of pure berberine in comparison to that of standard drug amprolium on the basis of reduction in coccidian oocyst output, body weight gain of chicken and feed conversion ratio. Among the extracts crude methanolic extract showed highest anticoccidial activity tested at 300mg/kg body weight which could be due to the presence of alcohol-soluble active ingredients in root bark of B. lycium. Toxicological studies revealed that B. lycium extracts as well as berberine were not lethal up to dosage of 2000mg/kg body weight. LD(50) was not determined as mortalities were not recorded in any of the five groups of chicken. From the present study it can be concluded that root bark of B. lycium has the immense potential to contribute to the control of coccidian parasites of chicken. Our results corroborate the use of berberine for treatment of severe diarrhoea, amoebiasis and intestinal infections and could justify its use in folk medicine for treatment of haemorrhagic dysentery.

Previously, it was isolated from the fruiting bodies of the gilled mushroom Pholiota spumosa (Basidiomycetes, Strophariaceae), putrescine-1,4-dicinnamide, a phenylpropanoid derivative conjugated with polyamine putrescine never isolated before as a natural compound. Recently, polyamine analogs that are similar in structure to the natural polyamines but that cannot mimic their functions that are essential for cellular growth and differentiation, have shown antitumor activity in several types of human cancer cells. Therefore, we have now investigated the response of DU-145 cells, a well characterized androgen-independent human prostate cancer (PCA) cell line, to this phenylpropanoid derivative. The results presented here demonstrate that putrescine-1,4-dicinnamide, as suggested for polyamine analogs synthesized artificially, inhibits the cell growth of cancer cells inducing apoptosis cell death, mediated, at least in part, by the activation of caspase cascades, that at higher doses shift to necrosis, through the increase of reactive oxygen species (ROS) generation.

Curcuma comosa Roxb. is ginger-family plant used to relieve menopausal symptoms. Previous work showed that C. comosa extracts protect mice from ovariectomy-induced osteopenia with minimal effects on reproductive organs, and identified the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD) as the major active component of C. comosa rhizomes. At 1-10μM, DPHD increased differentiation in transformed mouse osteoblasts, but the effect of DPHD on normal bone cells was unknown. We examined the concentration dependency and mechanism of action of DPHD relative to 17β-estradiol in nontransformed human osteoblasts (h-OB). The h-OB were 10-100 fold more sensitive to DPHD than transformed osteoblasts: DPHD increased h-OB proliferation at 10nM and, at 100nM, activated MAP kinase signaling within 30min. In long-term differentiation assays, responses of h-OB to DPHD were significant at 10nM, and optimal response in most cases was at 100nM. At 7-21 days, DPHD accelerated osteoblast differentiation, indicated by alkaline phosphatase activity and osteoblast-specific mRNA production. Effects of DPHD were eliminated by the estrogen receptor antagonist ICI182780. During differentiation, DPHD promoted early expression of osteoblast transcription factors, RUNX2 and osterix. Subsequently, DPHD accelerated production of bone structural genes, including COL1A1 and osteocalcin comparably to 17β-estradiol. In h-OB, DPHD increased the osteoprotegerin to RANKL ratio and supported mineralization more efficiently than 10nM 17β-estradiol. We conclude that DPHD promotes human osteoblast function in vitro effectively at nanomolar concentrations, making it a promising compound to protect bone in menopausal women.

The aim of the present study was to investigate the chemical composition of the essential oil of the fruits of Eucalyptus globulus and to examine the potential application of the fruit oil against multidrug-resistant bacteria. GLC/MS analysis in the fruit oil showed that aromadendrene was the main compound followed by 1,8-cineole and globulol. The three most abundant components of the fruit oil were also tested individually against microorganisms. In addition, the synergistic effects of combinations of the major constituents (aromadendrene and 1,8-cineole) of the fruit oil were also investigated. All Gram-positive bacteria were susceptible to the fruit oil with different degrees of susceptibility as determined by microdilution method. The oil exerted a marked inhibition against multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) Enterococcus faecalis. The results indicated that aromadendrene might be responsible for the antimicrobial properties, whereas 1,8-cineole and globulol exhibited low activities. The checkerboard assay demonstrated that combinations of 1,8-cineole and aromadendrene reduce the MIC in most cases in an additive way, whereas the time-kill assay indicates a synergistic effect.

A number of clinically useful drugs were tested for their ability to inhibit the inflammatory edema induced by 1,8-cineole (cineole), a terpenoid oxide, in the hindpaw of rats. Paw edema was measured by plethysmography following subplantar injection of cineole (20 μl/paw). The edema inducing effect of cineol was rapid in its onset (early phase, 0.5-1 h); attained slowly its peak level at 2 h and thereafter remained relatively constant up to 5 h post-injection (late phase, 2-5 h). Rats pretreatad with the antiallergic drugs cyproheptadine, ketotifen and cromoglycate demonstrated significant inhibition at both the early and the late phases of paw edema, while the rats that received nonsteroidal anti-inflammatory compounds phenylbutazone, indomethacin, and piroxicam and steroidal anti-inflammatory drug dexamethasone exhibited significant edema inhibition only at the late phase, suggesting that the anti-allergic drugs are more effective in this model of acute inflammation. However, all the drugs tested were found to inhibit the late phase edema effect of 1,8-cineole. The data indicate that 1,8-cineole-induced acute inflammation is a useful model to screen compounds that possess anti-allergic and anti-inflammatory potential.

Vasorelaxant effects of essential oil of Alpinia zerumbet (EOAZ) and its main constituent, 1,8-cineole (CIN) were studied. In rat isolated aorta preparations with intact endothelium, EOAZ (0.01-3000 microg/ml) induced significant but incomplete relaxation of the phenylephrine-induced contraction, an effect that was abolished by removal of vascular endothelium. However, at the same concentrations (0.01-3000 microg/ml corresponding to 0.0000647-19.5 mM), CIN induced a complete vasorelaxant effects (IC(50)=663.2+/-63.8 microg/ml) that were significantly reduced in endothelium-denuded rings (IC(50)=1620.6+/-35.7 microg/ml). Neither EOAZ nor CIN affected the basal tonus of isolated aorta. Vasorelaxant effects of both EOAZ and CIN remained unaffected by the addition of tetraethylamonium chloride (500 microM) or indomethacin (10 microM) into the bath, but were significantly reduced by N(G)-nitro-L-arginine methyl ester (100 microM). It is concluded that EOAZ induces a potent vasorelaxant effect that could not be fully attributed to the actions of the main constituent CIN, and appears totally dependent on the integrity of a functional vascular endothelium. The data is novel and corroborate the popular use of A. zerumbet for the treatment of hypertension.

As part of our ongoing search for flavonoids that are bioactive in humans, it was determined that FRS 1000, a beverage containing flavonoids extracted from onion peel, showed unexpected improvement of male sexual function. An in vitro enzyme assay clearly showed that FRS 1000 has a strong phosphodiesterase 5A (PDE 5A) inhibitory activity, which is considered to be important for treatment of erectile dysfunction. Detailed assays of each major ingredient indicated that the antioxidative flavonoid quercetin was responsible for the activity. Results also suggested that PDE 5A inhibition is not directly related to the free radical scavenging activity of flavonoids.

Hot flashes are a disorder of thermoregulation due to the lack of estrogens and are the most common and characteristic climacteric complaint. Hormone replacement therapy is the gold standard treatment but now its use is limited due to several side effects. Need therefore arises to search for non-estrogenic alternatives. It is well established that extracts of Cimicifuga racemosa (CR) ease climacteric complaints but solid animal experimental data supporting such effects are not available. The availability of sensitive transponders which record subcutaneous temperature continuously enables nowadays experiments in rats to establish whether they have hot flashes following ovariectomy (Seidlova-Wuttke et al. 2003) and if so, whether they can be influenced by the extract of CR BNO 1055. Intact Sprague-Dawley rats (n=16) were acclimatized and their subcutaneous body temperature was measured in 5 min intervals and mean values from 3h recordings were calculated. Thereafter, the rats were ovx and fed either with soy free (sf) or CR BNO 1055 (25 mg/animal/day) food. Temperature was recorded again after acute and sub-acute application of CR. In individual intact animals temperature was stable over the 3h recording period. Following ovx temperature pulses appeared with peaks occurring every 20-40 min. These fluctuations were not seen in CR BNO 1055 treated animals resulting in significantly higher mean temperatures in ovx in comparison to intact or ovx CR BNO treated rats. This reduction of hot flashes by BNO 1055 outlasted the experimental period of 3 weeks. These results suggest that the ovx rats and the new temperature-sensitive device may be useful for the study of hot flashes. Furthermore the results prove that the CR BNO 1055 exerts hot flash reducing effects.

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do not bind to either of the known estrogen receptors. Thus, the anti-tumor effect of CR me be mediated by mechanisms not involving these receptors. Polycyclic aromatic hydrocarbons are toxic environmental pollutants, which indirectly act as anti-estrogens by activating the aryl hydrocarbon receptor (AhR). The AhR is widely expressed in mammalian tissues and tumors. A recent screening study demonstrated activation of the AhR by a variety of herbal extracts, among others, CR. Since activation of the AhR causes inhibition of growth of prostate cancer cells, we addressed the question, whether CR may not only inhibit growth of breast cancer--but also of prostate cancer cells. In the AhR ligand assay, the CR extract BNO 1055 reduced tracer binding to 71% of the control demonstrating interaction of constituents of this extract with the receptor. Under basal as well as under estradiol- and dihydrotestosterone stimulated conditions, the CR extract dose dependently inhibited proliferation of LNCaP cells. A significant reduction of cell growth was observed at a concentration as low as 50 ng/ml. Thus, it is demonstrated for the first time that CR compounds potently inhibit the growth of human prostate cancer cells in vitro. This anti-proliferative effect may be mediated via the AhR.

In order to investigate the pharmacodynamic basis of the previously-established anticonvulsant properties of linalool, we examined the effects of this compound on behavioral and neurochemical aspects of glutamate expression in experimental seizure models. Specifically, linalool effects were investigated to determine its inhibition of (i) L-[3H]glutamate binding at CNS (central nervous system membranes), (ii) N-methyl-D-aspartate (NMDA)-induced convulsions, (iii) quinolinic acid (QUIN)-induced convulsions, and the behavioral and neurochemical correlates of PTZ-kindling. The data indicate that linalool modulates glutamate activation expression in vitro (competitive antagonism of L-[3H]glutamate binding) and in vivo (delayed NMDA convulsions and blockage of QUIN convulsions). Linalool partially inhibited and significantly delayed the behavioral expression of PTZ-kindling, but did not modify the PTZ-kindling-induced increase in L-[3H]glutamate binding.

Acetyl-11-keto-β-boswellic acid (AKAB) from Boswellia serrata and B. carterii acts directly on purified 5-lipoxygenase of human blood leukocytes at a selective site for pentacyclic triterpenes that is different from the arachidonate substrate binding site. The pentacyclic triterpene ring is crucial for binding to the enzyme, whereas functional groups (11-keto function in addition to a hydrophilic group on C 4 of ring A) are essential for the 5-lipoxygenase activity.

Acetyl-11-keto-β-boswellic acid (AKBA) from Boswellia serrata Roxb. and italics Boswellia carterii Birdw. is the first selective, direct, non-competitive and non-redox-type inhibitor of 5-lipoxygenase, the key enzyme for leukotriene biosynthesis (Safayhi et al., 1992). Previously, we showed that AKBA interacts with the 5-lipoxygenase via a pentacyclic triterpene selective effector site (Safayhi et al., 1995). In order to study the impact of AKBA's functional groups on enzyme inhibition, natural and synthetic analogues of this boswellic acid were tested for 5-lipoxygenase inhibition in intact rat neutrophils (Sailer et al., 1996 a). The results reveal that the carboxylic group of AKBA combined with the 11-keto-group is essential for enzyme inhibition, whereas the acetoxy-group on position C-3 α increases the affinity of AKBA to its effector site. Furthermore, other experiments demonstrated that minor structural modifications could cause a total loss of binding affinity and/or inhibitory activity of these compounds.

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. 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. 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. 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.

Saireito (TJ-114) is a traditional Japanese herbal medicine that has been used for treating edema and inflammation in diseases such as nephritic disease. This study investigates the effect of TJ-114 on postoperative edema and inflammation after total hip arthroplasty (THA). Patients who underwent cementless THA were randomly divided into two groups: Group A consisted of 8 hips of 8 patients who were treated with TJ-114 at a dose of 9 g/day 2 days before surgery and for 2 weeks after surgery; Group B consisted of 9 hips of 9 patients who did not take TJ-114. Although no significant difference was observed between the two groups for lower extremity edema, it was found that swelling of the proximal leg in Group A was less than that in Group B. Furthermore, 3 weeks after surgery, every measuring point in the lower extremity showed that TJ-114 tended to decrease postoperative swelling compared to measurements of swelling of patients who did not take TJ-114. Serum C-reactive protein (CRP) levels of 6 out of 8 patients in Group A decreased and became negative 2 weeks after surgery; however, there were no patients in Group B whose CRP levels became negative after 2 weeks. In conclusion, TJ-114 is safe and useful for the prevention and early recovery of postoperative leg edema after THA with an association of rapid CRP reduction.

Various assay methods have been developed to evaluate the effectiveness of substances against human immunodeficiency virus (HIV). One of them is the Syncytia formation inhibition assay, which is based on the inhibition of the interaction between the HIV-1 envelope protein gp 120 and the cellular membrane protein CD4. A variation of this assay using recombinant virus vPE 16 and CD4(+) HeLa cell was developed to find anti-HIV compounds in natural products that inhibit gp 120-CD4 binding. VPE 16 expresses glycoprotein gp 160, which is glycosylated then processed into gp 120 and gp 41 on its envelope. A total of 50 plant extracts were screened with this system. Extracts from Calicarpa japonica and Sedum sarmentosum were among those that showed strong inhibition of the gp 120-CD4 interaction.

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.