Enhancement of the release of azelaic acid through the synthetic membranes by inclusion complex formation with hydroxypropyl-beta-cyclodextrin

The aim of this study was to investigate the release rates of azelaic acid and azelaic acid-hydroxypropyl-beta-cyclodextrin (HPbetaCD) inclusion complex through three types of synthetic membranes, namely cellophane, silicone and elastomer membranes. Solid inclusion complexes of azelaic acid-HPbetaCD at the molar ratio of 1:1 were prepared by coevaporation and freeze-drying methods, subsequently characterized by differential scanning calorimetry, X-ray diffractometry and dissolution studies. Solid inclusion complex obtained by coevaporation method which exhibited the inclusion of azelaic acid in the HPbetaCD cavity and gave the highest dissolution rate of azelaic acid was selected for the release study. Release studies of azelaic acid and this complex through the synthetic membranes were conducted using vertical Franz diffusion cells at 30 degrees C for 6 days. The release rates of azelaic acid through the synthetic membranes were enhanced by the formation of inclusion complex with HPbetaCD at the molar ratio of 1:1, with the increasing fluxes of about 41, 81 and 28 times of the uncomplexed system in cellophane, silicone and elastomer membranes, respectively. The result from this study can be applied for the development of azelaic acid for topical use.     

Enhancement of Transdermal Absorption,Gene Expression and Stability of Tyrosinase Plasmid (pMEL34)-Loaded Elastic Cationic Niosomes: Potential Application in Vitiligo Treatment

The pMEL34 was loaded in elastic cationic niosomes (Tween61/Cholesterol/DDAB at 1:1:0.5 molar ratio) by chloroform film method with sonication and rehydrated with 25% ethanol. The amount of pMEL34 was determined by gel electrophoresis and gel documentation. The maximum loading of pMEL34 in elastic cationic niosomes was 150 mg/16 mg of the niosomal compositions. At 8 weeks, the remaining plasmid in the elastic niosomes kept at 4 ± 2 °C, 27 ± 2 °C were 49.75% and 38.57%, respectively, whereas at 45 ± 2 °C, all plasmids were degraded. For transdermal absorption through rat skin investigated by Franz diffusion cells at 6 h, the fluxes of pMEL34 loaded in elastic and nonelastic niosomes in viable epidermis and dermis (VED) were 0.022 ± 0.00 and 0.017 ± 0.01 μg/cm²/h, respectively, whereas only pMEL34 loaded in elastic cationic noisome was observed in the receiver solution. The pMEL34 loaded in elastic cationic niosomes showed the highest tyrosinase gene expression demonstrating higher tyrosinase activity than the free and the loaded plasmid in nonelastic niosomes of about four times. This study has suggested the potential application of elastic cationic niosomes as an efficient topical delivery for tyrosinase gene in vitiligo therapy. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3533-3541, 2010     

Influenza virus (H5N1) in live bird markets and food markets, Thailand

A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006-August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004-2005.     

Potent anti-cervical cancer activity: Synergistic effects of Thai medicinal plants in recipe N040 selected from the MANOSROI III database

Ethnopharmacological relevances

One of the prestigious Thai/Lanna folklore wisdoms is the medicinal plant recipes. Thai/Lanna medicinal plant recipe database MANOSROI III has been developed by Prof. Dr. Jiradej Manosroi. It consists of over 200,000 recipes covering all diseases including cancer.

Aim of this study

To investigate the in vitro and in vivo anti-cervical cancer activity and the active constituents of the Thai medicinal plant recipe N040 selected from the MANOSROI III database.

Materials and methods

The extracts of recipe N040 and single medicinal plants in the recipe were prepared by hot water and methanol extraction, respectively. The n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH) and water fractions of Caesalpinia sappan, the plant which showed the highest anti-proliferative activity were prepared by liquid–liquid partition extraction. The fraction which showed the highest anti-proliferative activity was further isolated for active constituents. Anti-proliferative activity of recipe N040, methanolic extracts, fractions of Caesalpinia sappan and brazilin, an active constituent on HeLa cell were investigated using sulforhodamine B (SRB) assay. Anti-oxidative activities including free radical scavenging and metal ion-chelating activities, as well as the phenolic and flavonoid contents of these fractions were also determined. The in vivo anti-cancer activity of recipe N040 on HeLa cell xenograft and the subchronic toxicity were performed in nude mice and rats, respectively.

Results and conclusions

N040 showed the potent in vitro anti-proliferative activity on HeLa cell with the IC₅₀ value of 0.11 µg/ml. Phytochemicals detected in the plants were steroids/triterpenoids, tannins, flavonoids, saponins and alkaloids. For the single plant, methanolic extract of Caesalpinia sappan gave the highest anti-proliferative activity with the IC₅₀ of 33.46 µg/ml. EtOAc fraction of Caesalpinia sappan showed the highest anti-proliferative and free radical scavenging activities with the IC₅₀ and SC₅₀ of 17.81 and 21.95 µg/ml which were 1.88 and 0.83 folds of its methanolic extract and ascorbic acid, respectively. Poor metal chelating activity (MC₅₀>500 µg/ml) was observed in methanolic extract and all fractions. The highest phenolic and flavonoid contents were observed in the methanolic extract. Brazilin, the known compound isolated from the EtOAc fraction exhibited potent anti-proliferative activity with the IC₅₀ of 0.28 µg/ml which was higher than its methanolic extract and EtOAc fraction of 119.50 and 63.61 folds, respectively, but only 0.39 fold of the recipe extract N040. The tumor size of the HeLa cell xenograft nude mice treated with the recipe N040 at the dose of 44.50 mg/kg body weight per day was significantly smaller (p<0.05) than that of the control with the relative tumor weight inhibition of 57.23% which was 0.65 fold of cisplatin. In the subchronic toxicity study, N040 given orally at the dose of 1000 mg/kg body weight per day for 90 days showed no alteration in body weight gain, hematology [except the increase mean corpuscular hemoglobin (MCH) in the treated male rats] and clinical blood chemistry (except the increase blood glucose in the treated male rats) both in female and male rats. Only minor lesions of the organs including lung, liver, kidney and small intestine were observed in both sexes. This study has demonstrated the synergistic effect of the plants composed in the recipe which resulted in the potent anti-cancer activity and confirmed the traditionally use of the recipe N040. In addition, this study has also suggested the compound brazilin isolated from Caesalpinia sappan for its high potential to be further investigated as a novel anti-cervical cancer drug.     

Anti-proliferative and apoptosis induction activities of extracts from Thai medicinal plant recipes selected from MANOSROI II database

Ethonopharmacological relevances

Traditional medicines have long been used by the Thai people. Several medicinal recipes prepared from a mixture of plants are often used by traditional medicinal practitioners for the treatment of many diseases including cancer. The recipes collected from the Thai medicinal text books were recorded in MANOSROI II database. Anticancer recipes were searched and selected by a computer program using the recipe indication keywords including Ma-reng and San which means cancer in Thai, from the database for anticancer activity investigation.

Aim of study

To investigate anti-cancer activities of the Thai medicinal plant recipes selected from the “MANOSROI II” database.

Materials and methods

Anti-proliferative and apoptotic activities of extracts from 121 recipes selected from 56,137 recipes in the Thai medicinal plant recipe “MANOSROI II” database were investigated in two cancer cell lines including human mouth epidermal carcinoma (KB) and human colon adenocarcinoma (HT-29) cell lines using sulforhodamine B (SRB) assay and acridine orange (AO) and ethidium bromide (EB) staining technique, respectively.

Results and conclusions

In the SRB assay, recipes NE028 and, S003 gave the highest anti-proliferation activity on KB and HT29 with the IC₅₀ values of 2.48 ± 0.24 and 6.92 ± 0.49 μg/ml, respectively. In the AO/EB staining assay, recipes S016 and NE028 exhibited the highest apoptotic induction in KB and HT-29 cell lines, respectively. This study has demonstrated that the three Thai medicinal plant recipes selected from “MANOSROI II” database (NE028, S003 and S016) gave active anti-cancer activities according to the NCI classification which can be further developed for anti-cancer treatment.     

Determination and geographic distribution of Orientia tsutsugamushi serotypes in Thailand by nested polymerase chain reaction

Clotted blood samples of 240 scrub typhus patients were collected from 8 Regional Medical Sciences Centers in Thailand during 1999 to 2002. The serotypes of Orientia tsutsugamushi and their geographic distribution were determined. A nested polymerase chain reaction (PCR) was used to identify the serotypes of O. tsutsugamushi. The number of patients with positive results for O. tsutsugamushi was 25.0%. Two serotypes, Karp and Kato, were detected in these samples. No Gilliam serotype was detected from any of the study locations. The PCR products were sequenced using an automated DNA sequencer. The nucleotide sequence of gene encoding 56-kDa protein from these samples showed a high sequence homology with the reference sequence of O. tsutsugamushi Karp and Kato serotypes. O. tsutsugamushi Karp serotype was predominant throughout Thailand with the percentage of 96.8% of the total serotype-positive patients, whereas 3.2% for Kato serotype was observed only in the south. The highest number among the region of Karp serotype-positive patients of 31.6% was found in the northeast.     

Characteristics and anti-proliferative activity of azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines

The hydrophilicity and lipophilicity of azelaic acid (AA) were modified to diethyl azelate (DA) which was synthesized by Fisher esterification reaction and identified by IR, MS and ¹H NMR and to azelaic acid-β-cyclodextrin complex (AACD) which was prepared by inclusion complexation and identified by IR, DSC and XRD respectively. AA, DA and AACD were entrapped in liposomes and niosomes comprising of l-α-dipalmitoyl phosphatidylcholine (DPPC)/cholesterol at 7:3 molar ratio and Tween61/cholesterol at 1:1 molar ratio, respectively, using a thin-film hydration method with sonication. The size and morphology of these bilayer vesicles were determined by optical and transmission electron microscopy. The particle size was found to be in the range of 90–190 nm. The entrapment efficiency of AA, DA and AACD in all vesicular formulations was more than 80%, as analyzed by HPLC for AA and AACD, and GC for DA. Anti-proliferative activity of AA and its derivatives (DA and AACD) both entrapped and not entrapped in bilayer vesicles, using MTT assay in three cancer cell lines (HeLa, KB and B₁₆F₁₀) comparing with vincristine, were investigated. AACD showed the highest potency comparing to AA in HeLa, KB and B₁₆F₁₀ of 1.48, 1.6 and 1.5 times, respectively. AA entrapped in liposomes was about 90 times more potent than the free AA, and about 1.5 times less potent than vincristine. When entrapped in bilayer vesicles, DA and AACD were more effective than AA in killing cancer cells. AACD entrapped in liposomes gave the highest anti-proliferation activity in HeLa cell lines with the IC₅₀ of 2.3 and 327 times more potent than vincristine and AA, respectively. DA in liposomes demonstrated the IC₅₀ of 0.03 times less potent than vincristine in KB cell lines, while in B₁₆F₁₀ AACD in niosomes showed the IC₅₀ of 0.05 times less potent than vincristine. This study has suggested that the modification of AA by derivatization and complexation as well as the entrapment in bilayer vesicles can enhance its therapeutic efficacy.