A standardized mixture of Chinese herbs, Zemaphyte taken orally as a daily decoction has been shown to be effective in the treatment of atopic eczema. This study showed that Zemaphyte is an efficient antioxidant, being capable of scavenging both superoxide and hydroxyl, and preventing peroxidation of biological membranes. It does not degrade hydrogen peroxide directly, but instead most likely forms a Zemaphyte-hydrogen peroxide complex. The complexed hydrogen peroxide can then be degraded in the presence of catalase to form oxygen and water. It is conceivable that Zemaphyte may contribute to the down-regulation of the activities of cells implicated in atopic eczema through its antioxidant activities.
Garlic and garlic extracts, through their antioxidant activities, have been reported to provide protection against free radical damage in the body. This study investigated antioxidant properties of garlic compounds representing the four main chemical classes, alliin, allyl cysteine, allyl disulfide, and allicin, prepared by chemical synthesis or purification. Alliin scavenged superoxide, while allyl cysteine and allyl disulfide did not react with superoxide. Allicin suppressed the formation of superoxide by the xanthine/xanthine oxidase system, probably via a thiol exchange mechanism. Alliin, allyl cysteine, and allyl disulfide all scavenged hydroxyl radicals; the rate constants calculated based on deoxyribose competitive assay were 1.4-1.7 x 10(10), 2.1-2.2 x 10(9), and 0.7-1.5 x 10(10) M (1) second(1), respectively. Contrary to previous reports, allicin did not exhibit hydroxyl radical scavenging activity in this study. Alliin, allicin, and allyl cysteine did not prevent induced microsomal lipid peroxidation, but both alliin and allyl cysteine were hydroxyl scavengers, and allyl disulfide was a lipid peroxidation terminator. In summary, our findings indicated that allyl disulfide, alliin, allicin, and allyl cysteine exhibit different patterns of antioxidant activities as protective compounds against free radical damage.
This study adopts Differential Scanning Calorimetry (DSC) to analyze the thermal properties of samples (2.5-4.0 mg) from the tip, middle, and base sections of individual paracetamol suppositories, which were sampled carefully using a stainless steel scalpel. The contents of paracetamol present in the samples obtained from these sections were determined from the enthalpies of fusion of paracetamol and expressed as % w/w paracetamol to allow comparison of the amount of paracetamol found in each section. The tip, middle, and base sections contained 10.1+/-0.2%, 10.1+/-0.2%, and 10.3+/-0.2% w/w paracetamol, and are statistically similar (One-way anova; p>0.05). This indicates that the preparation technique adopted produces high quality suppositories in terms of content uniformity. The contents of paracetamol in the 120-mg paracetamol suppositories determined by DSC and UV spectrophotometry were statistically equivalent (Students's t-test; p>0.05), 120.8+/-2.6 mg and 120.8+/-1.5 mg, respectively, making DSC a clear alternative method for the measurement of content of drug in suppositories. The main advantages of the method are that samples of only 2.5-4.0 mg are required and the procedure does not require an extraction process, which allows for the analysis to be completed rapidly. In addition, it is highly sensitive and reproducible, with the lower detection limit at 4.0% w/w paracetamol, which is about 2.5 times lower than the content of paracetamol (10% w/w) present in our 120-mg paracetamol suppositories and commercial paracetamol suppositories, which contained about 125 mg paracetamol. Therefore, this method is particularly suited for determination of content uniformity in individual suppositories in quality control (QC) and in process quality control (PQC).
Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.
The evolution of antibiotic resistance in Staphylococcus aureus showed that there is no long-lasting remedy against this pathogen. The limited number of antibacterial classes and the common occurrence of cross-resistance within and between classes reinforce the urgent need to discover new compounds targeting novel cellular functions not yet targeted by currently used drugs. One of the experimental approaches used to discover novel antibacterials and their in vitro targets is natural product screening. Three known pentacyclic triterpenoids were isolated for the first time from the bark of Callicarpa farinosa Roxb. (Verbenaceae) and identified as α-amyrin [3β-hydroxy-urs-12-en-3-ol], betulinic acid [3β-hydroxy-20(29)-lupaene-28-oic acid], and betulinaldehyde [3β-hydroxy-20(29)-lupen-28-al]. These compounds exhibited antimicrobial activities against reference and clinical strains of methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA), with minimum inhibitory concentration (MIC) ranging from 2 to 512 μg/mL. From the genome-wide transcriptomic analysis to elucidate the antimicrobial effects of these compounds, multiple novel cellular targets in cell division, two-component system, ABC transporters, fatty acid biosynthesis, peptidoglycan biosynthesis, aminoacyl-tRNA synthetases, ribosomes and β-lactam resistance pathways are affected, resulting in destabilization of the bacterial cell membrane, halt in protein synthesis, and inhibition of cell growth that eventually lead to cell death. The novel targets in these essential pathways could be further explored in the development of therapeutic compounds for the treatment of S. aureus infections and help mitigate resistance development due to target alterations.
There has been considerable effort to discover plant-derived antibacterials against methicillin-resistant strains of Staphylococcus aureus (MRSA) which have developed resistance to most existing antibiotics, including the last line of defence, vancomycin. Pentacyclic triterpenoid, a biologically diverse plant-derived natural product, has been reported to show anti-staphylococcal activities. The objective of this study is to evaluate the interaction between three pentacyclic triterpenoid and standard antibiotics (methicillin and vancomycin) against reference strains of Staphylococcus aureus.
Staphylococcus aureus is an important human pathogen in both hospital and the community that has demonstrated resistance to all currently available antibiotics over the last two decades. Multidrug-resistant isolates of methicillin-resistant S. aureus (MRSA) exhibiting decreased susceptibilities to glycopeptides has also emerged, representing a crucial challenge for antimicrobial therapy and infection control. The availability of complete whole-genome nucleotide sequence data of various strains of S. aureus presents an opportunity to explore novel compounds and their targets to address the challenges presented by antimicrobial drug resistance in this organism. Study compounds α-amyrin [3β-hydroxy-urs-12-en-3-ol (AM)], betulinic acid [3β-hydroxy-20(29)-lupaene-28-oic acid (BA)] and betulinaldehyde [3β-hydroxy-20(29)-lupen-28-al (BE)] belong to pentacyclic triterpenoids and were reported to exhibit antimicrobial activities against bacteria and fungi, including S. aureus. The MIC values of these compounds against a reference strain of methicillin-resistant S. aureus (MRSA) (ATCC 43300) ranged from 64 µg/ml to 512 µg/ml. However, the response mechanisms of S. aureus to these compounds are still poorly understood. The transcription profile of reference strain of MRSA treated with sub-inhibitory concentrations of the three compounds was determined using Affymetrix GeneChips. The findings showed that these compounds regulate multiple desirable targets in cell division, two-component system, ABC transporters, fatty acid biosynthesis, peptidoglycan biosynthesis, aminoacyl-tRNA synthetase, ribosome and β-lactam resistance pathways which could be further explored in the development of therapeutic agents for the treatment of S. aureus infections.
Staphylococcus aureus has become a serious concern in hospitals and community due to rapid adaptation to existing antimicrobial agents. Betulinaldehyde [3β-hydroxy-20(29)-lupen-28-al (BE)] belongs to pentacyclic triterpenoids that are based on a 30-carbon skeleton comprising four six-membered rings and one five-membered ring. In a preliminary study, BE exhibited antimicrobial activity against reference strains of methicillin-resistant and methicillin-sensitive S. aureus. However, the response mechanism of S. aureus to this compound is not known. In this study, the global gene expression patterns of both the reference strains in response to sub-inhibitory concentrations of BE were analyzed using DNA microarray to identify gene targets, particularly essential targets in novel pathways, i.e. not targeted by currently used antibiotics, or novel targets in existing pathways. The transcriptome analysis revealed repression of genes in the aminoacyl-tRNA synthetase and ribosome pathways in both the reference strains. Other pathways such as cell division, two-component systems, ABC transporters, fatty acid biosynthesis and peptidoglycan biosynthesis were affected only in the reference strain of methicillin-resistant S. aureus. The findings suggest that BE regulates multiple desirable targets which could be further explored in the development of therapeutic agents for the treatment of S. aureus infections.
Curcumin, the yellow pigment derived from turmeric rhizomes, exhibits antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. We have previously reported in a study that curcumin could induce differentiation in embryonal carcinoma cell (EC). EC cells are the primary constituents of teratocarcinoma tumors, and hence differentiating them to a non-proliferative cell type may be useful in anticancer therapies. Here, we conducted a detailed study using various molecular approaches to characterize this differentiation at the cellular and molecular levels. The cells were treated with 20 µM curcumin, which was the optimal concentration to produce the highest amount of differentiated cells. Changes in protein and RNA expression, membrane dynamics, and migration of these cells after treatment with curcumin were then studied in a time-dependent manner. The differentiated cells were morphologically distinct from the precursor cells, and gene expression profiles were altered in curcumin-treated cells. Curcumin promoted cell motility and cell adhesion. Curcumin also induced changes in membrane fluidity and the lateral mobility of lipids in the plasma membrane. The findings of this study suggest that curcumin might have therapeutic potential in differentiation therapy for the treatment of teratocarcinomas or germ cell tumors (GCTs) such as testicular and ovarian GCTs.
The bark extract of Melicope subunifoliolata (Stapf) T.G. Hartley showed competitive muscarinic receptor binding activity. Six polymethoxyflavones [melibentin (1); melisimplexin (3); 3,3',4',5,7-pentamethoxyflavone (4); meliternatin (5); 3,5,8-trimethoxy-3',4',6,7-bismethylenedioxyflavone (6); and isokanugin (7)] and one furanocoumarin [5-methoxy-8-geranyloxypsoralen (2)] were isolated from the bark extract. Compounds 2 and 6 were isolated for the first time from M. subunifoliolata. The methoxyflavones (compounds 1, 3, 4, 5, 6, and 7) show moderate inhibition in a muscarinic receptor binding assay, while the furanocoumarin (compound 2) is inactive. The potency of the methoxyflavones to inhibit [(3)H]NMS-muscarinic receptor binding is influenced by the position and number of methoxy substitution. The results suggest these compounds are probably muscarinic modulators, agonists or partial agonists/antagonists.
To enhance the stability of the anticancer drug gemcitabine (2'-deoxy-2',2'-difluorocytidine), it was conjugated to poly-l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly-l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48h, and reached equilibrium at 72h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug.
This study reports the in vivo performance of two tribenzyltin carboxylate complexes, tri(4-fluorobenzyl)tin[(N,N-diisopropylcarbamothioyl)sulfanyl]acetate (C1) and tribenzyltin isonicotinate (C9), in their native form as well as in a poly(lactic-co-glycolic acid) (PLGA)-based nanoformulation, to assess their potential to be translated into clinically useful agents. In a 4T1 murine metastatic mammary tumour model, single intravenous administration of C1 (2.7 mg/kg) and C9 (2.1 mg/kg; 2.1 mg/kg C9 is equivalent to 2.7 mg/kg C1) induced greater tumour growth delay than cisplatin and doxorubicin at equivalent doses, while a double-dose regimen demonstrated a much greater tumour growth delay than the single-dose treated groups. To improve the efficacy of the complexes in vivo, C1 and C9 were further integrated into PLGA nanoparticles to yield nanosized PLGA-C1 (183.7 ± 0.8 nm) and PLGA-C9 (163.2 ± 1.2 nm), respectively. Single intravenous administration of PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGA-C9 (2.1 mg C9 equivalent/kg) induced greater tumour growth delay (33% reduction in the area under curve compared to that of free C1 and C9). Multiple-dose administration of PLGA-C1 (5.4 mg C1 equivalent/kg) and PLGA-C9 (4.2 mg C9 equivalent/kg) induced tumour growth suppression at the end of the study (21.7 and 34.6% reduction relative to the size on day 1 for the double-dose regimen; 73.5 and 79.0% reduction relative to the size on day 1 for the triple-dose regimen, respectively). Such tumour growth suppression was not observed in mice receiving multiple-dose regimens of free C1 and C9. Histopathological analysis revealed that metastasis to the lung and liver was inhibited in mice receiving PLGA-C1 and PLGA-C9. The current study has demonstrated the improved in vivo antitumour efficacies of C1 and C9 compared with conventional chemotherapy drugs and the enhancement of the efficacies of these agents via a robust PLGA-based nanoformulation and multiple-drug administration approach.
Radionuclide imaging using (111)In, (99m)Tc and (153)Sm is commonly undertaken for the clinical investigation of gastric emptying, intestinal motility and whole gut transit. However the documented evidence concerning internal radiation dosimetry for such studies is not readily available. This communication documents the internal radiation dosimetry for whole gastrointestinal transit studies using (111)In, (99m)Tc and (153)Sm labeled formulations. The findings were compared to the diagnostic reference levels recommended by the United Kingdom Administration of Radioactive Substances Advisory Committee, for gastrointestinal transit studies.
Oculocutaneous albinism (OCA) is a heterogeneous group of autosomal recessive disorders resulting from mutations of the tyrosinase (TYR) gene and presents with either complete or partial absence of pigment in the skin, hair and eyes due to a defect in an enzyme involved in the production of melanin. In this study, mutations in the TYR gene of 30 unrelated Iranian OCA1 patients and 100 healthy individuals were examined using PCR-sequencing. Additionally, in order to predict the possible effects of new mutations on the structure and function of tyrosinase, these mutations were analyzed by SIFT, PolyPhen and I-Mutant 2 software. Here, two new pathogenic p.C89S and p.H180R mutations were detected in two OCA1 patients. Moreover, the R402Q and S192Y variants, which are common non-pathogenic polymorphisms, were detected in 17.5% and 35% of the patients, respectively. The outcome of this study has extended the genotypic spectrum of OCA1 patients, which paves the way for more efficient carrier detection and genetic counseling.
Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer's disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki=40-110 µM), comparable to that of acetylcholine (Ki=59 µM). Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.
Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM), inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM) and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6) exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.
Phthalocyanines are excellent photosensitizers for photodynamic therapy as they have strong absorbance in the near infra-red region which is most relevant for in vivo activation in deeper tissular regions. However, most phthalocyanines present two major challenges, ie, a strong tendency to aggregate and low water-solubility, limiting their effective usage clinically. In the present study, we evaluated the potential enhancement capability of glycerol substitution on the photodynamic properties of zinc (II) phthalocyanines (ZnPc). Three glycerol substituted ZnPc, 1-3, (tetra peripherally, tetra non-peripherally and mono iodinated tri non-peripherally respectively) were evaluated in terms of their spectroscopic properties, rate of singlet oxygen generation, partition coefficient (log P), intracellular uptake, photo-induced cytotoxicity and vascular occlusion efficiency. Tetrasulfonated ZnPc (ZnPcS4) was included as a reference compound. Here, we showed that 1-3 exhibited 10-100 nm red-shifted absorption peaks with higher molar absorptivity, and at least two-fold greater singlet oxygen generation rates compared to ZnPcS4. Meanwhile, phthalocyanines 1 and 2 showed more hydrophilic log P values than 3 consistent with the number of glycerol attachments but 3 was most readily taken up by cells compared to the rest. Both phthalocyanines 2 and 3 exhibited potent phototoxicity against MCF-7, HCT-116 and HSC-2 cancer cell-lines with IC50 ranging 2.8-3.2 µM and 0.04-0.06 µM respectively, while 1 and ZnPcS4 (up to 100 µM) failed to yield determinable IC50 values. In terms of vascular occlusion efficiency, phthalocyanine 3 showed better effects than 2 by causing total occlusion of vessels with diameter <70 µm of the chorioallantoic membrane. Meanwhile, no detectable vascular occlusion was observed for ZnPcS4 with treatment under similar experimental conditions. These findings provide evidence that glycerol substitution, in particular in structures 2 and 3, is able to improve the photodynamic properties of ZnPc.
This paper describes the use of gamma scintigraphic and magnetic resonance (MR) fusion images for improving the anatomical delineation of orally administered radiotracers used in gastrointestinal (GI) transit investigations.