Theses (MSc. Analytical & Pharmaceutical Chemistry)

Plants have exceptional abilities to produce secondary metabolites that have medicinal value as well as being cytotoxic agents. With the rise of multidrug-resistant strains of bacteria like Vancomycin-resistant Staphylococcus aureus (VRSA) and Methicillin Resistant Staphylococcus aureus (MRSA) has required the discovery of new antimicrobial drug leads. Currently there is not a single chemical entity plant derived antibacterials being used clinically. Malaysia with its abundant biodiversity has many plants which are used in traditional medicine to treat wound infections and with knowledge gathered from ethnobotany lead to this study of the antimicrobial activity of Alternanthera sessilis Red (locally called Hongtyang wu) leaves ethanolic extract on 11 standard microorganisms namely Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, Enterococcus fecalis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter iwoffii, Shigella dysenteriae, Aeromonas hydrophilia and Yersinia enterocolitica. In the present study, two microorganisms namely, the gram positive S. aureus and B. cereus exhibited susceptibility to the ethanolic leaves extract using the disk diffusion assay method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were performed using the Resazurin assay (broth microdilution techniques) as described by the Clinical and Laboratory Standards Institute (CLSI). The MIC values were 12.5mg/mL and 0.2mg/mL for B. cereus and S. aureus respectively and A. sessilis Red ethanolic leaves extract killed these 2 types of microorganisms at MBC values of 25mg/mL and 0.4 mg/mL, respectively. The results showed that A. sessilis Red ethanolic leaves extract either modified resistance in B. cereus and S. aureus and/or acted as antibacterial agent by possessing bactericidal or bacteriostatic properties. The leaf extract had potent activity against S. aureus which could be exploited to develop new classes of anti-staphylococcal phytochemical agents which should undergo further microbial specificity, cytotoxic and preclinical in vivo evaluation to determine their development for clinically use.

Andrographolide (AGP) is the major ent-labdane diterpenoid isolated from Andrographis paniculata (known locally in Malaysia as Hempedu Bumi). AGP was found to exhibit neuroprotective effects by inhibiting inflammation and oxidative stress. As such, AGP could act as a novel alternative for the treatment of neurological disorder. However, its oral bioavailability is reduced due to inactivation by liver glutathione S-transferase (GST). To tackle this shortcoming, a series of 12,13-dihydroandrographolide (HAGP) analogues were semisynthesised in the previous project where the C12-C13 double bond was reduced to avoid glutathione (GSH) conjugation. Besides that, substituted benzylidene moiety was introduced at C-3 and C-19 hydroxyl groups to increase the lipophilicity of the compounds for better blood-brain-barrier penetration. Among the analogues, 3,19-(3-ethoxy,4-hydroxylbenzylidene)-12,13-dihydroandrographolide (HAGP34) and 3,19-(3-flurobenzylidene)-12,13-dihydroandrographolide (HAGP38) showed better activity in the initial bioassay screening against pheochromocytoma (PC12) cells. In view of this, both compounds were selected to evaluate their in vitro pharmacokinetics and neuroprotective properties in this study. HAGP34 and HAGP38 were shown to avoid GSH conjugation (phase II metabolism). However, in the rat microsomes metabolism experiment to mimic Phase I metabolism in the liver, AGP (intrinsic clearance, CLint = 30 μL/min/mg) was found to be more stable than HAGP34 (CLint = 67 μL/min/mg), but less stable than HAGP38 (CLint = 16 μL/min/mg). These two in vitro pharmacokinetics studies evealed that HAGP38 was more stable than AGP in phase I and phase II metabolism. HAGP38 and AGP showed comparable reactive oxygen species (ROS) inhibition in rat pheochromocytoma cells (PC12), where the percentages of inhibition were 18% and 23% respectively at 10 μM. In mouse microglial cells (BV2), AGP exhibited strong nitric oxide (NO) inhibition where its IC50 value was 5 μM. When compared with HAGP38, the NO inhibition was only 17% at 5 μM. The Trans-AM assay revealed that HAGP38 inhibited NO production via disruption of NF-κB signalling pathway, similar to AGP as reported in the literature. On the other hand, HAGP34 showed weak ROS and NO inhibition in both cell lines. These accumulating findings revealed that the C12-C13 double bond or C-3 and C-19 hydroxyl groups of AGP may play strong roles as the pharmacophore of anti-inflammatory and antioxidant activities. Although the biological activities of HAGP38 were not as potent as than AGP, its improved in vitro pharmacokinetics may give better bioavailability compared to AGP. Therefore, HAGP38 is may be a promising molecule that should be further tested in animal models.

A series of 12 novel 2-(substituted)-5,6-dimethyl/-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]pyrimidine4-(3H)-one derivatives were designed to recognise their hypothetical binding motif required to bind with the enzyme cyclooxygenase-2 (COX-2). The novel compounds were designed based on in silico molecular docking studies by using the crystal structures of COX-1 (PDB ID: 3N8X) and COX-2 (PDB ID: 3LN1) isoenzymes respectively by employing the Glide (Schrodinger Inc.) software program. Docking studies revealed that nonselective non-steroidal anti-inflammatory drugs (NSAIDs) like indomethacin form a salt bridge with the amino acid residue Arg120 inside the binding pocket of COX-1/2 which gives a generalised anchoring point for all the classical NSAIDs (non-steroidal anti-inflammatory drugs) thus limiting their selectivity due to curtailed freedom of movement of the ligand. Interestingly all the designed compounds did not form any such salt bridge and predicted to be comparatively selective towards the COX-2 over the COX-1 isoenzyme. Further, these designed compounds were also found to be aligned with the reference standard drug celecoxib inside the binding pocket of COX-2 by forming significant hydrogen bonding interactions with all the crucial amino acid residues. The compounds which were predicted to have good binding affinity as well as selectivity towards COX-2 were further screened for their in silico pharmacokinetic properties (absorption, distribution, metabolism and excretion: ADME) to test their drug-likeness by using the QikProp (Schrodinger Inc.) software program. The predicted ADME properties of all the designed compounds were found to be in the ranges as predicted by QikProp for 95% of known oral drugs and also satisfy the Lipinski’s rule of five. Two of the 12 designed compounds (compound 7 and 8) were synthesised, purified and characterised by following the appropriate synthetic scheme-1. An attempt was made to evaluate their in vitro COX-1/2 activity; the outcome of the assay is not yet conclusive due to technical error associated with the COX inhibition assay kit. The repetition of COX inhibition assay to confirm the inhibitory binding affinity and selectivity of all the newly synthesised compounds towards COX-2 over COX-1 will be the basis of the future research work. Keywords: Thieno[2,3-d]pyrimidine, docking studies, in silico ADME prediction, COX-1/2 inhibition assay.

A simple, rapid and accurate reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of sildenafil and tadalafil in twenty herbal dietary supplements sold for enhancing sexual performance in Malaysia. Chromatographic separation was achieved with a Hypersil Gold C18 column (150 mm × 4.6 mm i.d., 5 µm particle size) at 30 °C, using a mobile phase consisting of 20 mM of ammonium acetate (adjusted to pH 5.5 with orthophosphoric acid) and acetonitrile at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 245 nm. Tadalafil and sildenafil eluted out from the column at retention times 5.8 min and 6.3 min, respectively. The method was validated in accordance to specificity, accuracy, linearity and range, precision, limit of detection (LOD) and limit of quantification (LOQ). Calibration curves were linear over the concentration range of 10−30 µg/mL and 1−3 µg/mL for sildenafil and tadalafil, respectively. The LOD values of sildenafil and tadalafil were 1.00 µg/mL and 0.07 µg/mL, respectively while LOQ values were 3.02 µg/mL and 0.21 µg/mL, respectively. The recoveries of both compounds were in the range of 99.11−100.87%. The RSD of repeatability and intermediate precision were less than 2%. The proposed method was successfully applied in the screening and quantification of tadalafil and sildenafil in adulterated herbal dietary samples. Keywords: Herbal dietary supplements; Sildenafil; Tadalafil; HPLC; Adulteration.

Hidradenitis suppurativa (HS) has been considered an orphan disease with limited treatments available. The main topical treatment is clindamycin lotion; however, short retention and frequent application is the main setback. The aim of this study was to attain an optimized antibacterial in-situ spray formulation for the skin condition hidradenitis suppurativa, which gels once in contact with the skin surface of around 37⁰C and possesses bioadhesion as well as sustained-release properties. Different concentrations of thermo-reversible gelling polymer Pluronic F-127 were investigated along with the selected bioadhesive polymers, HPMC and SA. The optimized formulation F3 having 18% Pluronic F-127 with 0.2% HPMC and 0.2% SA was characterized based on various physicochemical properties. The gelation temperature of F3 was found to be 29.0 ± 0.50⁰C at a time 1.35 ± 0.40 min. F3 had the viscosity of 178.50 ± 5.50 cP at 25⁰C, and 7,800 ± 200 cP at 37⁰C as the gel set. The detachment force was found at 0.80 ± 0.041 N. More than 80% cell found viable after treating at 10 mg/mL. The pH measurement with 1% clindamycin was obtained at pH 5.8. Cumulative drug release was 73.80% in 8 h; the release pattern of drug followed zero-order kinetics with Higuchi release mechanism. The n-value obtained from Korsmeyer-Peppas model showed Fickian diffusion. Average zone of inhibition was 43.44 ± 1.34 mm. The F3 formulation showed to improve residence time and enhance sustained drug release.

Slimming products based on phytotherapeutic formulations are popular due to their affordability and the misconception that they are safe because they are „natural products‟. However, adulterants have been found to be added into these herbal formulations to improve the weight lost effect of the product. Consumption of these adulterated products may lead to health problems, and even death. We hypothesise that over-the-counter (OTC) slimming phythotherapeutic formulations sold in Malaysia are adulterated with synthetic and natural drugs. Caffeine and phenolphthalein are popular adulterants due to their respective properties as an anorexic (appetite suppressor) and laxative. The objective of this study is to develop and validate a simple, precise and specific high-performance liquid chromatography coupled with ultraviolet detector (HPLC-UV) analytical method for the simultaneous detection and measurement of caffeine and phenolphthalein in slimming phytotherapeutic formulations. The method was then applied to analyse eight OTC slimming phythotherapeutic formulations purchased from the Malaysian market. The method was developed using a HPLC connected to a variable-wavelength detector (VWD). Separation was achieved with a C18 reversed-phase column using gradient elution. Mobile phase used were solvent A (aqueous): 25 mM ammonium acetate buffer (pH 5), and solvent B (organic): methanol. Gradient elution was achieved by a ratio of time (minutes) over methanol (%): 0/10, 1/10, 3/40, 5/55, with a run time of 10 minutes. Flow rate was maintained at 1.1 mL/min and the effluents monitored at 254 nm wavelength. The retention time of caffeine and phenolphthalein were approximately 5.5 minutes and 8.8 minutes, respectively. The developed method was validated for linearity, precision, accuracy and selectivity according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines (Validation of Analytical Procedures: Text and Methodology, Q2(R1)). The calibration curve area versus concentration was found to be linear (coefficient of determination, R2 ≥ 0.999) in the range of 10–100 μg/mL for both caffeine and phenolphthalein. The limit of detection (LOD) which indicates the lowest concentration of caffeine and phenolphthalein that can be detected were 0.77 and 0.47 μg/mL, respectively. The limit of quantification (LOQ) that express the lowest concentration of caffeine and phenolphthalein which can be confidently quantified were 2.35 and 1.44 μg/mL, respectively. The method was found to be accurate for both compounds with percent recovery values of 98–102% for both caffeine and phenolphthalein. The method had good intra-day and inter-day precision with percent relative standard deviation (%RSD) values of less than 2%. The validated method was applied to analysis of eight OTC slimming phytotherapeutic formulations purchased from the Malaysian market. Phenolphthalein was detected and quantified in five of these slimming products, at levels ranging from 0.68–126.83 mg/g. Caffeine was detected in six of these slimming products but only quantified in two products (sample A, 3.39 ± 0.21 mg/g and sample B, 0.99 ± 0.01 mg/g) as most of the products claimed to be of plant origin and hence caffeine detected were likely from natural sources. In conclusion, a simple and fast HPLC-UV method to detect for caffeine and phenolphthalein as adulterants was successfully developed and validated. The method was applied to actual OTC slimming products, and proved the hypothesis that OTC slimming phythotherapeutic formulations sold in Malaysia are adulterated. As future work, the developed method can be used to analyse more variety of slimming products. The analytical technique is also suitable to be used in quality control laboratories for routine detection of phenolphthalein and caffeine in slimming products.

A simple and rapid isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated as per International Conference on Harmonisation (ICH) and United States Pharmacopeia (USP) guidelines for analysis of tiagabine in the presence of its degradation products. The chromatographic separation was achieved on a Vision HT C18 column (150 mm × 4.6 mm, 5 μm) with mobile phase comprising of 11.5 mM sodium dihydrogen phosphate buffer (adjusted to pH 2.0 with orthophosphoric acid)/acetonitrile (50:50, v/v) at a flow rate of 1 mL/min and the UV detection wavelength was set at 254 nm. Stress degradation studies were performed as stated in ICH guidelines on tiagabine bulk drug using acid, base, oxidation, heat and light. The method was validated in accordance to system suitability, linearity and range, specificity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness. Tiagabine was found to degrade under acidic, photolytic, oxidative and thermal condition, but stable under basic hydrolysis condition. The developed method was found to be linear in the concentration range of 50-150 μg/mL (r2 = 0.9983) and the percentage recovery for the accuracy was 98.86-99.35%. The LOD and LOQ obtained were 31.93 μg/mL and 96.76 μg/mL, respectively while the %RSD for precision, robustness and stability studies were less than 2%. The degradation products formed from the stress degradation studies were well separated from tiagabine and hence the method could be regarded as stability indicating. The proposed method can be used in the analysis of tiagabine bulk drug and pharmaceutical formulations in quality control laboratories. Keywords: Tiagabine, RP-HPLC, Stress degradation studies, Validation.

Glimepiride, an antidiabetic drug was classified as Class II drug in Biopharmaceutical Classification System (BCS) due to its low solubility (< 0.004 mg/mL) and high permeability. The aim of this work was to investigate the use of palm-oil polyesteramide (POPEA) and stearic acid-based polyesteramide (SAPEA) as carrier in GMP solid dispersion (SD) to increase dissolution rate and solubility. Glimepiride SDs were prepared by solvent evaporation technique, using POPEA and SAPEA as a polymer carrier. The SDs were characterised for drug content, solubility, dissolution and physico-chemical characteristics by DSC, FTIR and SEM. All SDs demonstrated faster dissolution rate and solubility as compared to the pure glimepiride. The optimized SD B9 showed 4-fold improvement of drug release (77.42 ± 0.79%) against pure glimepiride (18.33 ± 3.10%) at 2 h of dissolution. DSC endothermic peak for glimepiride in optimized SD B9 (90% glimepiride, 10% SAPEA) was broadened and shifted to lower temperature (198°C). FTIR spectroscopy confirmed no chemical changes in glimepiride SD. SEM images showed the irregular, less crystalline and smaller size of SD which improved the solubility and dissolution rate due to the transformation of glimepiride crystalline structure to amorphous form. POPEA and SAPEA as a drug carrier demonstrated good characteristic in improving the aqueous solubility and dissolution rate of glimepiride in SD.

Usage of plant-based compounds has been increasing recently due to their unexpected efficacy in the mitigation of certain diseases, including atherosclerosis. The aims of this study are to investigate the potential anti-inflammatory and anti-oxidative effects of 14-deoxy-11,12-didehydroandrographolide (DDAGP), an Andrographis paniculata derivative, in C57BL/6J mouse model of atherosclerosis by determining relative NF-ĸB p65 and NOX-4 expression levels, in conjunction with biochemical profiling of plasma lipid and liver enzyme levels, in Poloxamer-407 (P-407) challenged C57BL/6J mouse model. Oral administration of DDAGP, with non-toxic concentration of 15 mg/kg/day, 30 mg/kg/day and 45 mg/kg/day, measurably reduced plasma lipid components, specifically LDL-c; but only the 45 mg/kg/day dose was conducive to significant TGL level reduction. Nevertheless, DDAGP did not significantly suppress the Atherogenic Index of Plasma (AIP) and the Atherogenic Coefficient (AC), suggesting that it lacks clinically significant anti-atherogenic ability. However, treatment of P-407 challenged mice with DDAGP significantly inhibited elevation of heart homogenate levels of NK-ĸB and NOX4 in a non-dose-dependent manner. Hence, even though DDAGP may not be an effective anti-atherogenic drug, it instead possesses marked anti-inflammatory and anti-oxidative potential. In conclusion, further research along these lines is warranted, with focus on DDAGP’s efficacy in the prophylaxis and management of inflammatory diseases.

Resveratrol (RSV) is an essential non-flavonoid polyphenolic compound that is frequently present in numerous botanical species. RSV exhibits properties that are associated with anticancer, antioxidant, anti-inflammatory, and cardioprotective effects. Nevertheless, the systemic absorption of the substance is constrained as a result of its lowered water solubility, which consequently reduces its oral bioavailability and its natural chemical instability. Using resveratrol as a model drug, resveratrol-loaded niosomes with two levels of RSV loading concentration (2 mg/mL and 4 mg/mL) were prepared by the thin-film hydration method with different molar ratios of Span 60 (SP 60) / Tween 60 (TW 60) mixed non-ionic surfactants to vitamin D3 (VD3) and to cholesterol (CHL) as vesicular stabiliser, respectively. Their physicochemical properties including shape, vesicle size, polydispersity index (PDI), zeta potential, and stability study, were characterized by zeta sizer and scanning electron microscopy (SEM), while encapsulation efficiency and drug release profile were measured by RP-HPLC and UV-vis spectrophotometer, respectively. It was found that the ratio of stabiliser-to-the mixed non-ionic surfactant and the RSV loading concentration affected particle sizes (125.5 nm to 934.2 nm), PDI (0.492 to 0.794), and zeta potential (-29.9 mV to -42.1 mV) to various extent, with niosomes prepared from resveratrol solution loading concentration of 2 mg/mL having more significant impact. Specifically, VD3-stablised resveratrol-loaded niosomes had a smaller mean particle size and a lower negative zeta potential value than CHL-stabilised ones, suggesting that the type of stabiliser and drug concentration affect the zeta potential but not the stability of the niosomes. The SEM images of selected VD3- stabilised niosomes (Formulation F3; 4 mg/mL RSV) exhibited a quasispherical surface shape, while the CHL-stabilised niosome (Formulation F3; 4 mg/mL RSV) showed a spherical shape. Although the RSV entrapment efficiency (%EE) of the VD3-stabilised niosomes were significantly lower compared with those of CHL-stabilised niosomes prepared at both RSV loading concentrations, the VD3-stabilised niosomes had significantly faster RSV release rate (64.1%) than CHL-stabilised niosomes (56.7%) at 12 hours (F2, RSV = 4 mg/mL loading conc). In the niosomal stability study, the VD3- stabilised niosomes have shown better stability when kept at 4°C than at 25°C over 90 days. Therefore, it can be concluded that VD3 performed better in enhancing favorable characteristics with regard to particle size, stability, and in vitro drug release rate, making it an appropriate replacement for CHL as a stabiliser.

Everolimus is a mammalian target of a rapamycin inhibitor drug approved for treating advanced-stage, metastatic cancer that is non-responsive to endocrine therapy. Like most chemotherapeutic drugs, everolimus comes with numerous side effects, and the severity of these side effects often varies from patient to patient. In hopes of curbing the side effects during the treatment process, it was reported that some patients default to complementary and alternative medicine (CAM) in the form of natural product-based dietary supplements. One of the natural product supplements that was reported to have been consumed by patients undergoing everolimus treatment is curcumin. Following the consumption of curcumin during everolimus treatment is a significant fall in everolimus blood concentration levels (Cmin.). It was speculated that curcumin induces CYP3A4 activity, thus increasing everolimus metabolism. However, whether the effects of curcumin on everolimus metabolism are CYP3A4-related is yet to be proven, as there are other possible ways for curcumin to be involved in decreasing everolimus Cmin in patients. One of them includes the possibility of physicochemical interaction. The primary objective of this project was to investigate the potential chemical interaction(s) that could occur between everolimus and certain selected natural compounds (rutin, quercetin and curcumin) when present in an aqueous matrix under different physiological pH environments via HPLC. The occurrences of said interactions are depicted by the appearance of unknown peaks in sample chromatograms whereby everolimus was paired with other foreign compounds. These unknown peaks were not detected in the control samples, whereby everolimus stands as the lone compound. Not all peaks present in the sample chromatograms are integrated and quantified. As a pre-requisite, the unknown peaks that are isolated in sample chromatograms mustn’t be present in blanks or controls. Only the unknown peaks that share the same spectral profile as the primary molecule, everolimus are captured and quantified. Based on the findings of this investigation, everolimus is most unstable in the presence of the natural compounds when incubated in pH 6.8 medium. The degree of instability of everolimus was depicted by the number of unknown peaks present. This indicates that more chemical interactions take place between everolimus and the natural compounds at a pH 6.8 environment compared to other pHs. Also, in pH 6.8, everolimus is most unstable and degrades rapidly when paired individually with the natural compound quercetin. The extent of interaction that takes place between everolimus and each natural compound differs. When in the presence of all three natural compounds at once, the rate of degradation of everolimus is highest at pH 6.8. The rate of degradation is interpreted as the formation of unknown peaks per unit time. However, in a pH 4.5 environment, everolimus has the most interaction when individually paired with curcumin. Most of the unknown peaks observed in single-paired samples are not found in the control sample. Based on the evidence gathered, the stability of everolimus in different pH environments is highly affected by the presence of natural compounds.

MMP-2 is an important proteolytic enzyme that controls the degradation of extracellular matrix (ECM), inflammation, cancer, cardiovascular diseases, etc. Applying the machine learning technique with quantitative-structure activity relationship (QSAR) modelling could overcome the challenges of classical QSAR modelling, which derives bioactivity through linear regression, elucidating linear relationships from single analogue series. Konstanz Information Miner (KNIME), a free and open-source data analytics platform, was used to develop machine learning models. The bioactivity dataset of MMP-2 inhibitors was downloaded from ChEMBL and PubChem. The ChemBL database was used as a training dataset to develop machine learning models. A total of 15 machine learning models were produced with five algorithms; 1) Random Forest, 2) Extreme Gradient Boosting (XGBoost), 3) Naïve Bayes, 4) Support Vector Machine (SVM), and 5) Probabilistic Neural Network (PNN) using 3 molecular structure fingerprints; 1) Molecular access system (MACCS), 2) Feat Morgan, and 3) Atom Pair. The PubChem dataset was used for external validation of machine learning models. The commercially available compound libraries from Enamine, USA, were used as test datasets for shortlisting the novel MMP-2 inhibitors. The shortlisted MMP-2 inhibitors were further shortlisted using molecular docking studies with Glide - Schrödinger (Schrödinger small molecule drugdiscovery suite version 2022-1). The shortlisted compounds were purchased from Enamine and tested their MMP-2 inhibitory activity by determining RNA and protein expression in human neuroblastoma (SH-SY 5Y) cells. The MMP-2 RNA expression was quantified using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and protein expression was determined using ELISA. The molecular fingerprint, MACCS, showed the best predictive performance in all the ML models, followed by Atom Pair and Feat Morgan. The Random Forest algorithm had shown the best classification (active vs inactive) capability with the highest scores across all performance metrics. The classification performance of Naïve Bayes, XGBoost and Probabilistic Neural Network were equal. The Support Vector Machine showed the least classification capability. The identified hits from Enamine commercial database were shown favourable binding (negative free energy) in molecular docking studies. The shortlisted hits inhibited the MMP-2 gene and protein expression in human neuroblastoma cells. Machine learning methods were successfully deployed to identify the novel potent MMP-2 inhibitors.

Concerns on increasing trends of multidrug resistance (MDR) around the world have triggered the need to investigate and develop new potent antibacterial drugs. Previously Food and Drug Administration (FDA) approved drugs have slowly been outsmarted by bacteria. Hence, 20 Schiff bases and their copper(II) complexes were synthesised and tested in hope to combat MDR. Schiff bases and their copper(II) complexes analogues were characterised by elemental analysis and Fourier-transform infrared spectroscopy (FTIR). The compounds were screened against eight Gram-negative and Gram-positive bacteria: (Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 35923, Acinetobacter baumanni BAA-1797, Escherichia coli BAA-196, Pseudomonas aeruginosa BAA-2108 and Methicillin-resistant Staphylococcus aureus MRSA 43300) to evaluate their antibacterial properties potential based on microbroth dilution. Minimum inhibitory concentration (MIC) results showed the compounds - Cu(SB4CB)2, Cu(SBFH)2, Cu(SBCH)2, Cu(phen)SBFH and Cu(bpy)SBFH have antibacterial potential with MIC values <16 μM indicating good antibacterial potential when tested against susceptible S. aureus ATCC 35923. Among the promising compounds, Cu(SBFH)2 due to its broad spectrum of antibacterial activity was specifically selected for further testing in combination tests alongside membrane permeabilising agent polymyxin B sulfate (POLY) and phenylalanine-arginine-β-naphthylamide (PAβN) against all bacteria. Fractional inhibitory concentration (FIC) index showed improved MIC values with additivity effect for Cu(SBFH)2 in combination with POLY and PAβN. Molecular docking studies had been carried out and demonstrated that Cu(SBFH)2 bound to the active binding site of NorA of S. aureus through hydrogen bonding and pi-pi stacking with Phe140 and hydrophobic interactions with Gly114. Keywords: Schiff bases, metal complexes, antibacterial, molecular docking, multidrug resistance.

Andrographolide (AGP) is the main bioactive component of Andrographis paniculata. It is an important medicinal plant used in the traditional medicine for the treatment of many infectious diseases. AGP has an interesting pharmacophore but its poor water solubility causes lower bioavailability, seriously limited its pharmacological function after oral administration. The objectives of this study were to prepare the self microemulsifying drug delivery system (SMEDDS) formulation to improve the oral bioavailability of AGP and to develop an analytical method to determine its pharmacokinetic parameters. An analytical method using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to quantify AGP in rat plasma. Protein precipitation technique was used to extract AGP from the plasma and then separated on an HSS C18 column with a mobile phase consisting of methanol–water (70:30). Pharmacokinetic investigations were performed in three groups of rats each group consists of 4 rats following oral administrations of unformulated and SMEDDS formulation of AGP (10 mg/kg) and intravenous administration of AGP (2 mg/kg). Cmax of SMEDDS formulation was 5.8-fold greater than Cmax of AGP unformulated. Tmax was decreased about 21 min. AUC0-24 of SMEDDS formulation increased by 5.1-fold compared to the unformulated AGP. SMEDDS formulation improved the absolute oral bioavailability of AGP from 3.3% to 16.7% which was a 5.1-fold increase.

In this study, six chalcones containing methyl and hydroxyl substituents at meta positions in aromatic rings; (E)-3-phenyl-1-(m-tolyl)prop-2-en-1-one (C-01), (E)-1-phenyl-3-(m-tolyl)prop-2-en-1-one (C-02), (E)-1,3-di-m-tolylprop-2-en-1-one (C-03), (E)-1-(3-hydroxyphenyl)-3-phenylprop-2-en-1-one (C-04), (E)-3-(3-hydroxyphenyl)-1-phenylprop-2-en-1-one (C-05) and (E)-1,3- (3-hydroxyphenyl) prop-2-en-1-one (C-06) were synthesised using Claisen-Schmidt reaction of substituted acetophenones and benzaldehydes at room temperature. All the synthesised compounds were characterised by spectroscopic techniques including infrared (IR), ESI-MS, UV-visible and nuclear magnetic resonance (1HNMR). The synthesised chalcones were tested for their anti-inflammatory activities using Greiss assay on mouse macrophages (RAW 264.7 cells). The results showed that presence of methyl (-CH3) and hydroxyl (-OH) substituents improved the anti-inflammatory activity of chalcones. Hydroxyl substituted chalcones showed higher anti-inflammatory activity compared to methyl substituted chalcones. Among the hydroxy-substituted chalcones, the (E)-1,3-bis (3-hydroxyphenyl) prop-2-en-1-one (C-06) was the most potent with an IC50 value lower than 6.25 μM. This compound was further tested for its influence on expression of cytokines using multianalyte ELISA array kit. This chalcone significantly reduced the elevated levels of inflammatory cytokines (IL-6, IFN-γ, TNF-α and G-CSF). Key words: substituted chalcones, anti-inflammatory activity, cytokines, and ELISA array kit.

The discovery of new therapeutic agents for inflammatory disorders has attracted more attention in recent years. Chalcone term is given to the flavonoid compounds bearing the 1,3-diphenyl-2-propen-1-one framework. Generally, chalcones are precursors of flavonoids with two aromatic rings joined together through three carbons, α, β-unsaturated carbonyl system. In plants, chalcones are converted to the respective (2S)-flavanones by enzymatic reaction of chalcone isomerase. Based on the close chemical and biogenetic relationship between flavanones and chalcones, they are considered as natural products. For anti-inflammatory activity of chalcones, activated macrophages play an important role and compounds with that inhibit nitro oxide production by macrophages have been found potential for the prevention and treatment of inflammatory disorders. Some functional groups such as dimethylamine, methoxy and butoxy groups increase the electron density of the B-ring resulting in significant loss of anti-inflammatory activity. Therefore, in this project we synthesised five compounds for chalcones containing halogens (-Cl, -F) at meta-positions on aromatic rings in chalcones and tested for their anti-inflammatory activity. The synthesized compounds were purified by column chromatography and characterised by 1H-NMR, 13C-NMR, FTIR, Mass and UV spectra. Further evaluation of their in vitro anti-inflammatory activity were carried out using RAW 264.7 mouse macrophages. The test dose of chalcones were determined was cytotoxicity (MTT) assay on RAW264.7 mouse macrophages. The results showed that the halogen substitution at meta-positions on aromatic rings improved the anti-inflammatory activity for the compound (E)-1,3-bis(3-chlorophenyl) prop-2-en-1-one (III) shows the best activity. The table below showed the compounds activity with IC50 values. Keywords: Chalcones, m-chlorobenzaldehyde, m-chloroacetophenone, Ethyl alcohol, Anti-inflammatory activity, RAW 264.7 cells

Background and Objective Atherosclerosis and the relative cardiovascular complications remain the primary contributors to the statistics of being the leading cause of death worldwide. The elucidation of molecular and cellular pathways pertaining to oxidative stress and inflammation in atherosclerosis has been elucidated via scientific studies. Phytochemical investigations on Andrographis paniculata have revealed to possesses beneficial effect against various cardiovascular disease. Andrographolide (AGP), as the major bioactive component of A. paniculata, has demonstrated to have various biological properties including anti-oxidant, and hepatoprotective in addition to anti-inflammatory properties. In this study, we aim to demonstrate the anti-atherosclerotic properties of AGP in poloxamer-407 (P-407) induced atherosclerosis in C57BL/6J mice. Methods Atherosclerosis was elicited in C57BL/6J mice using P-407 via intraperitoneal injection. On the other hand, the treatment with AGP (15, 30 and 45 mg/kg/day) was given concomitantly for 6-weeks period. Disease control and normal control were received vehicle treatment throughout the study period. At the end of the study, heart and aorta was harvested and utilised for the subsequent enzyme-linked immunosorbent assay (ELISA) and histological studies. Results The results demonstrated that the treatment of mice with AGP reversed the effects of P-407 induced atherosclerosis. The doses of AGP were in correlation with the reduction of atherosclerosis biomarkers. The high dose (45 mg/kg/day) was the most significant dose observed to reduce the progression of atherosclerosis. The Low-Density Lipoprotein (LDL), Triglycerides (TG), and atherogenic index (AI) were significantly reduced by the AGP treatment in comparison to the disease control. The histological results showed reduction in inflammation, fibrosis and hypertrophy in the heart tissues of the groups treated with AGP compared to the disease control. In addition, AGP treatment significantly reduced marker molecules of oxidative stress (NOX-4) and inflammation (p65 NF-B) in comparison to those in the disease control. Moreover, the aorta of the AGP treated groups showed normal morphological characteristics while the disease control cells were highly damaged. In addition, lipid accumulation was observed to be very clear for the disease control, while the AGP groups showed significant reduction in the lipid accumulation. Conclusion The results of our study demonstrated that AGP is very effective to reduce the symptoms of atherosclerosis through reducing of oxidative stress and inflammation. Thus, AGP is highly recommended to be considered as natural treatment to reduce or prevent the development of atherosclerosis.

Nanofibers for wound dressing application has become the research hotspot due to their excellent properties to make wound dressing. The nanofibers can be loaded with the drugs to kill or inhibit the bacteria on wound area. This study demonstrated the preparation and formulation of polycaprolactone (PCL) nanofibers loaded with curcumin, titanium dioxide and zinc oxide using electrospinning method. Optimisation of the PCL nanofibers for each parameter of electrospinning was done before preparing curcumin-titanium dioxide/zinc oxide nanofiber. Scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, and differential scanning calorimetry (DSC) were then used to characterise the nanofibers. Surface properties measurement, degradation analysis, in-vitro release of drug test, in-vitro cytotoxicity study, and antibacterial activity were evaluated on nanofibers. The optimised parameters to produce smooth nanofibers were found to be 12 wt% polymer concentration, a 6:4 mixture of chloroform and dimethylformamide at room temperature, 15 kV applied voltage, 0.5 mL/h flow rate, and a 20 cm working distance. The addition of curcumin, titanium dioxide nanoparticles, and zinc oxide nanoparticles into the nanofibers can increased the diameter and hydrophobicity of the PCL nanofibers but decreased its crystallisation and melting temperature. The degradation test showed that the PCL nanofibers were able to degrade completely within 14 days with the presence of lipase. In drug release study, the nanofiber exhibited a burst release of curcumin at the beginning of time, then sustained release. In cytotoxicity study, 1 and 3% w/w of curcumin in nanofibers do not show cytotoxic effect to HaCaT cells. The addition of curcumin-titanium dioxide/zinc oxide can suppress the activity of Staphylococcus aureus and Pseudomonas aeruginosa in antimicrobial tests. The curcumin-titanium dioxide and zinc oxide nanofibers with 1 and 3% w/w of curcumin show potential application in wound healing, as they do not harm the cells and able to inhibit the activity of bacteria, which will be useful to protect the wound.

Porphyromonas gingivalis (P. gingivalis) is a Gram-negative, anaerobic rod-shaped bacterium that has been implicated as a keystone pathogen in chronic periodontitis. Such disease is characterized by inflammation and affects the individual’s gums, which eventually leads to tissue destruction and tooth loss. There is an increasing number of evidences on the role of P. gingivalis in Alzheimer’s disease pathogenesis. Inhibition of gingipain K, one of the virulence factors of P. gingivalis, has been considered as an effective strategy for treating Alzheimer’s disease, especially for individuals with concomitant chronic periodontitis. The objectives of this project are to evaluate the binding interactions of a set of FDA-approved drugs in the active site of P. gingivalis gingipain K and to determine their inhibitory activity against the gingipain K. Structure-based virtual screening on gingipain K was performed through molecular docking simulation of 300 FDA-approved drugs obtained from the ZINC 15 database. The top ten compounds (fludarabine, phenylephrine, levetiracetam, midodrine, formoterol, lamivudine, olsalazine, benazepril, ketorolac and diltiazem) with relatively high docking scores were selected for subsequent biological screening. Through gingipain K enzymatic assay, the gingipain K activity was determined by measuring the absorbance at 405 nm over 15 minutes. Results from the molecular docking studies showed that the compounds engaged primarily in hydrogen bonding, π-π stacking, salt bridge and pi-cation interaction with the surrounding residues, namely Trp391, Thr442, Ala443, His444, Gly445, Asn475, Cys477, Ser511, Tyr512, Trp513, Asp516 and His575. The presence of functional groups in the ligands, such as carbonyl groups, amino groups, amido groups, hydroxyl groups as well as aromatic rings was considered important for the binding interaction between ligands and gingipain K. Olsalazine (% gingipain K inhibition = 32.43% at 50 μM and IC50 = 93.44 ± 1.89 μM) was identified as the top hit compound among the ten compounds from the biological evaluation. It is considered as a promising compound for further development as a therapeutic agent for the treatment of AD. Keywords: Porphyromonas gingivalis gingipain K, Alzheimer’s disease, drug repositioning, in silico study, biological evaluation

A simple, selective and rapid stability-indicating RP-HPLC coupled with a photodiode array detector method has been developed and validated for the simultaneous determination of simvastatin and niacin in a combined dosage form. The chromatographic separation of the two cholesterol lowering drugs was achieved by utilizing Inertsil CN, 5µm, 250 mm x 4.6 mm i.d. column (GL Sciences Inc, Tokyo, Japan), maintained at 30 °C throughout the analysis. The drugs were separated in isocratic elution mode with a mobile phase of 0.1% Acetic Acid buffer-methanol (50:50, v/v) at a flow rate of 1.0 mL/min. Both simvastatin and niacin compound were detected by UV-PDA at 237 nm. The method was validated for forced degradation, linearity and range, accuracy, precision and robustness according to ICH guidelines. The linearity and range for niacin and simvastatin were 0.05 to 0.150 mg/mL (R2 > 0.9999) and 0.004 to 0.012 mg/mL (R2 > 0.9992), respectively. The assay mean recoveries observed for niacin and simvastatin were 99.36% and 99.93%, respectively. The precision of the method obtained was 99.66% for niacin and 99.34% for simvastatin with RSD less than 2%. The lower degree of RSD that obtained in robustness and intermediate precision has proven that the method is highly robust. The validated method can be very helpful in the quality control analysis of active pharmaceutical ingredient and pharmaceutical dosage form.