Palm olein emulsions were produced using the combinations of Span® and Tween® surfactants by mechanical homogenisation. Effects of the types of surfactants, concentrations, effective HLB and the types of viscosity modifiers and concentrations on the characteristics of the emulsions were investigated. With palm olein content of 20% (w/w), stable oil droplets were produced at HLB values ranging from 8.5 to 11.0. Optimal concentrations of surfactants ranged from 25 to 30% (w/w to oil) depending on the types of the Span®/Tween® mixtures. Among the viscosity modifiers used, Carbopol®940 was the most effective. Suitable concentrations of Carbopol®940 for the emulsions prepared with Span®20/Tween®20 ranged from 0.1 to 0.3% (w/w). Beyond this concentration, destabilisation of emulsion due to at least depletion of water molecules could have occurred as a result of competitive hydration between Carbopol®940 and the surfactants. The emulsions produced exhibited viscoelastic and pseudoplastic behaviour, with yield value ranging from 0.1 to 35.2 Pa. Depending on the concentration of Carbopol®940 and within the linear viscoelastic region, the emulsions were elastic in nature as shown by domination of storage modulus (G’) over the loss modulus (G”) and with tan δ < 1 in the frequency range of 0.01 to 10 Hz. These favourable rheological properties were induced by the formation of threedimensional network of Carbopol®940 molecules in the continuous aqueous phase, which also entrapped the oil droplets and thus increased the stability of the emulsion. To the optimised o/w emulsion formulation, active pharmaceutical ingredients and extracts of Cassia alata were incorporated. Betamethasone dipropionate and tolnaftate at concentrations of 0.5 mg/g and 1 mg/g respectively, did not affect the size of droplets and stability of the palm olein emulsions. Nonetheless, betamethasone dipropionate increased the viscosity and elasticity of the emulsion; while tolnaftate slightly reduced the viscosity, thixotropy and elasticity of the emulsion. Cassia alata extracts affected the emulsion stability. With up to 0.5 mg/g of ethanol extract and 0.25 mg/g of ethanol/water extract of Cassia alata, emulsions were stable. Further, Cassia alata extracts did not alter the pseudoplasticity of the emulsion despite decreasing viscosity with increasing concentration of the extracts. Desired rheological properties for the development of topical cream and lotion can be attained by changing the concentration of Carbopol®940 in palm olein-in-water emulsion, which is a potential vehicle for drug delivery.

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Amphiphilic molecules play a key role in the stabilization of many of the colloids. It is, therefore, very important to understand the interfacial behaviour of these molecules and to select the proper ones for the proper activity. Synthetic surfactants and emulsifiers are widely used in many of our foods and pharmaceutical formulation, but, it becomes very important to replace them by natural molecules with good health records. Five medicinal plants which are Syzygium aromaticum, Entada spiralis, Trigonella foenum-graecum, Elephantopus scaber and Andrographis paniculata were selected for this study. The crude extract of the plants were prepared by maceration method. Solvents with different polarity were used for the extraction. The physical properties, in particular the surface activity of the extracts were evaluated and compared. Properties of emulsions prepared from the crude extracts were then investigated. Homogenization was carried out from 20% palm oil with 10% crude extract. The antimicrobial activities of the extracts against two Gram-positive Bacillus subtilis and Staphylococcus aureus and two Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli were investigated. Both the disc diffusion (qualitative) and tube macrodilution (quantitative) assays were employed for the determination of antimicrobial activity. The extracts of E. spiralis and S. aromaticum from ethanol-water 1:1 gave stable emulsions at least up to six months when kept at room temperature. The surface active compounds, if present among the components extracted will be adsorbed differently at the interface producing different extent of emulsion stability. All extracts were able to inhibit the growth of one or more of the bacteria. The patterns of inhibition varied with the type of plant extract, the solvent used for extraction and the organism tested. S. aureus, was the most susceptible to all plant extracts while E. coli was the most resistant microorganism. The highest antibacterial activity was observed from S. aromaticum extract with lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 0.39 mg/mL and 0.78 mg/mL, respectively against B. subtilis. It can be concluded that the extracts from S. aromaticum and E. spiralis have the potential to be used for the preparation of stable pharmaceutical emulsions by providing both emulsifying and antimicrobial actions.

Nicotine dependence is a life threatening disorder and efforts have been focused to identify new mechanisms to assist smokers to quit. The need to find new interventions is vital. The use of herbals as alternative to current treatments is limited and most studies were without randomized control trial (RCT). To our knowledge, this is the first study in Malaysia using randomized, double-blind, placebo-controlled trial to study herbal medication for smoking cessation. Viva® was first introduced in Malaysia to help drug addicts managed withdrawal symptoms. Distaste for cigarettes were noticed in patients undergoing treatment - which led to development of Viva QS® for smoking cessation. Viva QS® is a preparation with twelve herbs from Korea and China.The objectives of the study were to determine the efficacy and safety of Viva QS® for quit smoking. A mobile smoking cessation programme (MSCP) was employed to reach smokers at workplaces. Workers who attended smoking cessation talk and fulfilled study criteria were enrolled upon written consent. Demographic and smoking history information, modified Fagerstrom test for nicotine dependence (FTND) score and carbon monoxide (CO) level were obtained at baseline. Follow-up were conducted by phone calls and/or face-to-face meetings at week 4, 12 and 24. During follow-up, subjects also received brief counselling to continuously promote cessation. Viva QS® and placebo were supplied for 24 weeks. Self-reported abstinence at week 24 was verified using saliva or urine cotinine test in addition to CO test.Biochemically verified 7-days point prevalence demonstrated Viva QS® significantly increased quit rate vs. placebo; 42.7% vs. 26.2% (p = 0.038) at week 4, 32.0% vs. 16.7% {p = 0.031) at week 12 and 30.7% vs. 13.9% (p = 0.015) at week 24. Adverse events reported were similar between groups (p > 0.05). The most frequently reported adverse events were sore throat and dry mouth (36.6% and 17.8%, respectively for Viva QS® vs. 28.8% and 16.9% for placebo). For non-quitters, results showed reduction in the number of daily cigarettes smoked in both groups but there was no significant difference between both arms (p > 0.05). We suggested Viva QS® is safe and effective for smoking cessation.

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The work embodied in the present thesis relates to the studies on the biological activity and chemical constituents of the leaves of Tetracera indica Merr. (Dilleniaceae) (mempelas paya) and is presented in two parts. Part A involves studies on the antidiabetic activity of aqueous (AQ) and methanol (MeOH) extracts of the leaves of T. indica Merr. in vivo and isolation of the active principle (wogonin). Part B describes the isolation and characterization of chemical constituents from MeOH extracts of the leaves of T. indica Merr. Part A comprises of investigation of antidiabetic activity of the aqueous (AQ) and methanol (MeOH) extracts of the leaves of T. indica along with the active principle isolated from MeOH extract in vivo in alloxan induced diabetic and normal male albino rats (Sprague Dawley strain (SD)). Two doses of each extract 250 and 500 mg/kg b.w. and 0.5 and 1mg/kg b.w in the case of active principle (wogonin) were evaluated. Both AQ and MeOH extracts exhibited significant anti-hyperglycemic activity in alloxan induced diabetic rats, however in normal rats, no hypoglycemic activity was observed, when compared with both +ve and –ve controlled groups except that of AQ extract which exhibited hypoglycemic activity in normal rats at 250 mg/kg b.w. The antidiabetic activity was also found to be comparable to that of the effect produced by glibenclamide (GLBC) (0.25 mg/kg b.w.). The LD50 of both AQ and MeOH extracts was found to be more than 5000 mg/kg body weight and no lethal toxicity was observed within this range. Wogonin was isolated as an active principle from MeOH extract which revealed antihyperglycemic activity in vivo comparable to GLBC. Part B deals with the isolation and structure elucidation of the active principle (wogonin) along with other chemical constituents from MeOH extract of the leaves of T. indica Merr. MeOH extract upon repeated silica gel and sephadex LH-20 column chromatography afforded two terpenoids and six flavonoids viz., mixture of glycosides of ?-sitosterol and stigmasterol, betulinic acid; 5,7-dihydroxy-8-methoxyflavone (wogonin); 5,7,8- trihydroxyflavone (norwogonin); 4’,5,7-trihydroxy-8-methoxyflavone (isoscutellarein methyl ether); 4’3,5,7,-tetrahydroxyflavone (kaempferol); 3’,4’3,5,7- pentahydroxyflavone (quercetin) and 5-hydroxy-7-methoxyflavone (techtochrysin). Occurrence of norwogonin, isoscutellarein methyl ether, kaempferol, quercetin, techtochrysin and isomeric mixture of ?-sitosterol and stigmasterol is being reported for the first time from the leaves of T. indica Merr. Keywords: Tetracera indica Merr., Dillineceae, Antidiabetic activity in vivo, active principle, other phytoconstituents.

As a cellular gatekeeper, p53 plays a major role in maintaining the cellular homeostasis. The existence of mutations may change the gene conformation and may affect its major function as the regulator of the cell proliferation. The p53 therapy based approach is a good candidate for treatment of TP53-defect cancers. However, this treatment is unsuitable for some cancer cases, especially those caused by dominant-negative activity of the mutant p53 protein. Dominant-negative occur in the presence of a mutated allele which results in the formation of heterotetramer of endogenous wild-type/mutant p53 that are unable to transactivate certain p53 downstream target genes, which are important for the cells regulation. Thus, more effective treatment is needed to overcome this problem and DNA vaccination may be the suitable candidate. A recombinant mutant p53 designated as R248Q has been put forward as a potential antigen for the DNA vaccination strategy. Therefore, the current research aims to produce mutant TP53R248Q through PCR site–directed mutagenesis and to confirm the tumor suppression ability of TP53R248Q. The mutation of R248Q was generated via Polymerase Chain Reaction (PCR) site-directed mutagenesis to pCMVp53 plasmid by using a set of specifically constructed primers. Subsequently, these constructed TP53R248Q and TP53 gene was transfected into the TP53-null H1299 cell lines. Following that, phenotype and genotype expression studies on the cell lines were performed by colony formation assay and quantification of a TP53 downstream target gene p21waf1, respectively, in order to investigate the tumor suppression ability of the mutated TP53. In phenotype study, the transfection with exogenous TP53 suppressed the colony growth while the treatment with TP53R248Q confirmed the loss of p53 original function by its inability to restrain cell proliferation. The result of phenotype study was confirmed by the expression analysis of downstream TP53 gene, p21waf1. Based on the analysis, it was found that the expression of p21waf1 was upregulated in TP53 and downregulated in TP53R248Q treatments. These data therefore confirmed that the PCR site-directed mutagenesis technique has been successfully carried out to induce the desired mutation in the TP53 gene. Thus, this technique may become an interesting option to generate novel recombinant proteins, which may be useful for the development of specifically designed DNA vaccine as a gene therapy strategy for cancer prevention in the future.

Different parts (leaves, stems and roots) ofT. scandens L. are used in folk remedies by various indigenous peoples in different countries for the treatment of rheumatism, lowering hypertension, lowering blood pressure, inflammatory diseases, hepatitis, internal pains, urinary disorders, dysentery, child birth, sore throat, gout and diabetes related infirmities. The present work investigates the anti-diabetic activity of the polar and non-polar extracts of T. scandens L. leaves in vivo in alloxan induced diabetic and normal rats. This study was conducted in vivo and comparison was made with standard antidiabeteic drug, glibenclamide (GLBC). Glucose levels in male albino rats (Wister strain) with hyperglycemia induced by alloxan (160 mg/kg b.w.) were determined after the oral administration of the aqueous (AQ) methanolic (MEOH), butanol (BuOH), ethyl acetate (EtOAc) and dichloromethane (DCM) extracts. Four doses of AQ and MEOH extract (250, 500, 1000 and 2000 mg/kg b.w.) and a fixed dose of 500mg/kg b.w of BuOH, EtOAc and DCM extracts were evaluated. All extracts exhibited significant anti-hyperglycemic activity in alloxan induced diabetic rats, except DCM, however in normal rats no hypoglycemic activity was observed among all the extracts, when compared with both +ve & -ve controlled . The antidiabetic activity was found to be comparable to that of the effect produced by GLBC (0.25 mg/kg b.w.). The LD50 of both AQ and MEOH extracts was found to be more than 5000 mg/kg body weight and no lethal toxicity was observed within this range. This study provides scientific evidence about the leaves of T. scandens L. which possess antidiabetic agents and justifies its utility by the local herbalists to treat diabetes in Malaysia. Phytochemical investigation and chromatographic fractionation of MEOH extract of the leaves of T. scandens L. led to the isolation and structure elucidation of two new and three known flavonoids, and two known terpenoids. The occurrence of kaempferol, quercetin, isoscutellarein, 5, 7,8,3 `,5,-pentahydroxyflavone, 2, 3, 5, 6, 4` -pentahydroxy stilbene-( 4-----0-----4` ")-kaempferol-3 "-0-,8-Dglucopyranoside, stigmasterol and betullinic acid in the leaves of T. scandens L. is being reported for the first time. Keywords: Tetracera scandens Linn .. Dilleniaceae, Antidiabetic activity, Phytoconstituents

The study was done for the purpose of developing biodegradable gentamicin-loaded microspheres fabricated using poly(D.L-lactic-co-glycolic acid) (PLGA). The microspheres were fabricated by manipulating several variables i.e. molecular weight of PLGA, types of surfactant/emulsifier, different concentrations of polyvinyl alcohol (PVA) as well as the oil phase and different HLB values of surfactants in order to achieve the best formulation for W /0/W emulsion during the fabrication process. Antibiotic treatment of orthopaedic infection is complicated by systemic toxicity and the need of effective therapeutic concentration necessary to ensure optimum killing of bacteria. To overcome the problem of systemic toxicity and to achieve a high initial release followed by sustained release of antibiotics, a new method of delivering gentamicin is attempted by encapsulating gentamicin into PLGA using multiple emulsion, solvent-evaporation method. Gentamicin was first extracted from the microspheres and quantified using Ninhydrin assay before the concentration was measured using UV spectrophotometer. Gentamicin loading after encapsulation was preserved when CTAB (83.51 ± 1.42%) and low molecular weight (LMW) PLGA (82.38 ± 9.08%) were used as indicated by drug loading of more than 80% in the discdiffusion assay. LMW PLGA enabled high burst release (-90%) of gentamicin within the first 10 hours corresponding to zone inhibition of 13.78 ± 0.86 mm, only 30% smaller than the positive control (10 mg/ml gentamicin). The effects of Tg and molecular weight rather than surfactant types influence the initial burst release. The in vitro release profile suggests that by having a mixture of various PLGA microspheres in one dosage implant system, the high burst release can be sustained within therapeutic concentration for a prolonged period (> 1 months). This biodegradable delivery system does not entail another surgery to remove the implant hence reducing the high treatment cost usually associated with the non-bidegradable proprietary gentamicin-polymethyl-methacrylate (PMMA) beads currently in use.

Insulin resistance and pancreatic β-cells defect are central features of diabetes disorder that may progress to several serious complications. Some of medicinal plants are potential sources for antidiabetic agents. Pluchea indica (beluntas) is widely distributed in Malaysia and it is believed to have antidiabetic properties. A hypoglycemic effect of P. indica in normal rats was reported in the previous study. This research was aimed to study the effects of P. indica in glucose and insulin regulation through cell-based in vitro model by using 3T3-L1 adipocytes and RIN-5F pancreatic β-cells. P indica was extracted using soxhlet apparatus with n-hexane, dichloromethane, ethyl acetate and methanol consequtively. The plant also was macerated using water to yield water extract. In cell viability test, the concentration of 0.2 mg/mL was found to be the maximum concentration of P. indica extracts in the absence of cytotoxicity. The preadipocytes were induced to differentiate into mature adipocytes prior to assay. The methanol extract at concentration of 0.05 mg/mL increased glucose uptake in adipocytes (p<0.05), as indicated by up regulation of adipogenesis-regulator Pparγ and insulin-sensitive glucose transporter 4 (Glut4) mRNAs. The n-hexane and water extracts at concentration of 0.05 mg/mL and 0.1 mg/mL respectively stimulated insulin release in β-cells (p<0.05). Moreover, these extracts elevated the transcription level of insulin receptor substrate 2 (Irs2) and glucose-transporter Glut2 in β-cells. Taken together, this in vitro study was useful for a screening model of P. indica extracts to demonstrate the glucose uptake in adipocytes and insulin secretion activity in β-cells. These findings also suggest that P. indica extract deserves further investigation as a potential agent for diabetes management.

Leech saliva contains biologically active compounds that are mainly proteins and peptides. In Malaysia leeches have been used for traditional medicine for a long time. However, there are scanty studies about the isolation and characterisation of Malaysian leeches` saliva. This study aimed to isolate and characterize leeches saliva extract. A modified and smooth extraction method of leeches` saliva without leeches` scarification is used. UV and Bradford assay protein methods showed that the saliva extract contains high concentrations of protein. RP-HPLC chromatogram revealed that more than 30 different peaks were there in leech saliva extract.. Gel electrophoresis revealed the existence of protein and peptides with different molecular weights. The gel showed up to 25 different bands. Comparison of gel electrophoresis data with protein database revealed the closeness of many molecular weights to known proteins isolated from the Hirudinaria leech family. Other proteins detected by gel electrophoresis may be related to completely new biologically active proteins and peptides or to a modification (isoforms) of the existing ones. It was observed that the period of starvation has a vital role in the concentration of saliva proteins and 12 weeks of starvation gave the highest concentration of proteins in the saliva. It was found also that 4 weeks of starvation after first feeding is enough for leeches to recover 42% of their protein concentration. Two anticoagulant proteins (protein 1 and protein 2) were isolated from leeches saliva extract by using RP-HPLC, and their molecular weights were identified (6.289kDa and 14.255kDa) respectively using tricine SDS-P AGE. These two proteins increased the thrombin time by 29 .11 % and 44.13% respectively. In addition, they inhibited the amidolytic activity of thrombin, evaluated by measuring the conversion of the chromogenic substrate (S-2238). The result showed a decrease in the conversion of the substrate (S-2238) by 30.61% and 41.22 % respectively. Traces of heavy metals concentrations were investigated in the water (natural habitat of the leeches), leeches` tissues and leeches` saliva extracts. The concentrations of heavy metals in the leeches` habitat water were found high. Hence the water specification is a class IV (INWQS). Furthermore, traces concentrations of heavy metals were found in leeches` tissues as well as in their saliva extracts. Such concentrations of heavy metals may cause health hazard, especially when leeches from such contaminated environment are applied in treatment and therapy directly without any precautions. Clearing leeches and their saliva extracts from these traces is investigated in this study. It was found that these high concentrations can be mitigated by successive replacement of the lake water by clean and non-chlorinated or distilled water for three weeks. A significant decrease for certain heavy metals concentrations was achieved depending on the type of metal. For instance in the saliva extract, undetected level of cadmium (Cd) was observed while a marginal 7.3% decrease in the case of arsenic (As) was reported after washing. In the case of leeches` tissues, the concentration of cadmium (Cd) increased unexpectedly, while a decrease of 92.38% and 20.01% in the concentration of lead (Pb) and arsenic (As) were recorded respectively.

Glycosmis pentaphylla (Retz) DC contains anti-microbial alkaloids, known as arborine and arborinine. Both of the alkaloids were obtained from bioassay-guided isolation from the crude extract of the leaves. Arborinine, which is non-polar alkaloids can be isolated by using dichloromethane: hexane 80:20 – 95:5, 100% dichloromethane, dichloromethane: ethyl acetate 95:5 – 55:45, giving bright yellow and fine needle- like structure. Combinations of medium and polar solvents dichloromethane: acetone (82: 18 to 70:30 and 45: 55 to 40:60) yielded arborine in the form of white crystal structure. Octanol-water partition coefficients (POW) of arborine and arborinine were determined by the shake-flask method. Each compound was dissolved in octanol to a known concentration and placed in octanol-water system and allowed to equilibrate for at least 72 hours. The octanol-water partition coefficients were determined at varied temperatures (298.15, 303.15, 308.15 and 313.15 K ), in different solvents, at various concentrations and in the presence of non-ionic surfactants. The concentrations of arborine and arborinine in the octanol phase were then determined using standard calibration curves based on the absorbances measured using UV spectrophotometer. The POW was calculated as the ratio of the concentration in octanol phase to the concentration in aqueous phase. With the addition of cosolvents (methanol and ethanol) and non-ionic surfactants (Tween®20 and Span®20), partition coefficients decreased with increasing temperature (except at 40°C) in the presence of non-ionic surfactants. A concentration of 0.1% of either arborine and arborinine was used in palm olein-in-water emulsion and modified to a cream consistency. The cream from F3 containing 25% palm olein, 1.5% Cremophor A25 and 1.0% sodium CMC showed the most stable cream up to at least nine months (up to date) when kept at room temperature 28°C. The particle size distribution of both cream formulations did not show any change throughout storage suggesting good chemical physical stability.

The work presented in this thesis describes selected biological activities of the salivary gland secretion of the medicinal Malaysian leech Hirudinaria manillensis (Lesson, 1842). A new device for leech feeding was developed using common laboratory tools consisting of a glass funnel wrapped with a parafilm membrane and filled with the phagostimulatory solution. Many phagostimulatory solutions were examined and only those containing sodium chloride and arginine were accepted by leeches. It was found that 71% of the tested leeches continued sucking for 15-45 min and 86% of them experienced 1-5 folds increments in body weight. For collection of a high quality and quantity LSE, we starved leeches for three weeks. A new method for LSE collection was described. The fed leeches were forced to regurgitate whatever they sucked by immersing them in ice containers. By using this method, leeches stayed alive and regain their activity after 15-30 min. The maximum concentration (A280 = 0.342) was provided within the first five minutes of sucking action. Gel electrophoresis (SDSPAGE and Tricine-SDS-PAGE) methods revealed the existence of various peptides and proteins with molecular weights ranging from 2.3-250 kDa. Amidolytic activity assay showed that LSE inhibited thrombin-induced release of p-nitroanilide from the synthetic substrate S-2238 with IC50 of 49.391±2.219 ?g/ml. LSE was shown to prolong thrombin time in vitro. The maximum antithrombin activity was obtained during the dry season (IC100 =16.081±0.079 ?g/ml). A longer starvation period yielded a lower antithrombin activity. An optimization of lyophilization conditions revealed that pre-freezing at -20

Highly Efficient Activated Carbon (HEAC) as an adsorbent of toxins has been successfully produced from palm shell through chemical activation process using phosphoric acid as an activating agent. Palm Kernel shell used as the main raw material for activated carbon production, was purchased from a local oil palm mill in Pahang, Malaysia. Temperature range 550 ºC – 650 ºC was used during the activation process. The effect of temperature variation on the pore size and surface morphology of the activated carbon were studied. Well developed pore size and low number of functional groups observed on activated carbon at 600 ºC were determined by Scanning Electron Microscope (SEM) and Fourier- Transform Infrared (FTIR) spectroscopy, respectively. The surface area and pore volume were determined by Brunauer, Emmett and Teller (BET) method using N2 gas adsorption. The highest surface area (1287 m2g-1) and pore volume (0.74 cm3 g-1) was observed with sample HEAC-2. The adsorption efficiency of HEAC-2 was studied in vitro for paraquat as toxin using distilled water and NaCl (0.9%) solution. These study shows paraquat was adsorbed more on HEAC-2 in the presence of sodium chloride solution (4.68 mgL-1) than in distilled water (3.62 mgL-1). Furthermore, a comparision was done between HEAC-2 (4.68 mgL-1) and commercially available activated carbon (4.18mg L-1) which proved HEAC-2 is more effective than commercially available activated carbon

Traditional medicine (TM) is the oldest form of health care style known to humanity; it has been used in different cultures throughout the history. According to the World Health Organization (WHO) reports, more than 70% of the world population use TM. The broad use of TM is often attributed to the accessibility, affordability and availability of such products to the majority of the world`s population. Asians are well-known for their reliance on TM. Malaysia has a long tradition and rich legacy from three main cultural groups (Malay, Chinese and Indian) of using TM, a large section of the population in this country is depending on TM for their healthcare needs. The huge demand for TM in Malaysia has significantly increased the Malaysian TM market from US$ 385million in 2000 to US$ 1.29 billion in 2005. Due to the global wide diffusion of TM the safety, efficacy and quality control of such products became significant concern from various health institutes. Presence of toxic substances such as heavy metals is often reported in TM products. This study has been initiated with a prime focus of detecting the amount of heavy metals namely arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn) and iron (Fe) in locally available traditional medicines both registered and unregistered medicinal products in various dosage forms from the East Coast region of Malaysia. The determination of Zn and Fe were conducted using Flame Atomic Absorption Spectrometer (FAAS), while Pb, Cd and Ni analysis were conducted using Graphite Furnace Atomic Absorption Spectrometer (GFAAS) and As detection was performed with Hydride Generation Atomic Absorption Spectrometer (HGAAS).TM samples were collected from three states of Malaysia namely Pahang, Terengganu and Kelantan. Total of sixty TM samples from various dosage forms such as capsule (50%), pill (25%), powder (21.6%) and tablet (3.4%) were analysed to determine the content of heavy metal using AAS. Three different acid digestion methods were compared to optimize the best sample preparation technique for analyzing TM samples. They were nitric –perchloric acid digestion (Method-A) nitric acid digestion (Method-B) and hydrochloric –nitric acid (Method-C) digestion respectively. It was found that Method-C showed the highest recovery compared to the other two methods (Method-A and Method-B) and the difference was found statistically significant (p< 0.05). Method validation was performed using QC standard sample, spiked TM samples and standard reference material (SRM). It was found that the limit of detection (LOD) for As, Cd, Pb, Ni, Zn and Fe were 0.11ppb, 0.1ppb, 1.17 ppb, 2.01 ppb, 0.01 ppm and 0.09 ppm respectively. Limit of quantification (LOQ) were 1.1 ppb for As, 1ppb for Cd, 11.7 ppb for Pb, 20.1 ppb for Ni, 0.1 ppm for Zn and 0.9 ppm for Fe. The recovery percentages for QC samples were ranged from 95.12- 102.4 which reflects the accuracy of the method. While the relative standard deviation (RSD) that represents the precision of the method for QC samples were in the range of 3.23-0.2. For spiked TM samples the recovery range and the RSD range were 95- 105 and 0.11-5.0 respectively. All the validation results were within the specification limit of American Organization of Analytical Chemistry (AOAC) guideline. The accuracy of the method was further checked by the analysis of SRM. The recovery percentages of all metals were in the range of 94.5-108. Among the sixty TM samples it was found that As, Cd, Pb, Ni, Zn and Fe were present in 43%, 81%, 90%, 100%, 100% and 93% of the total samples with a concentrations range of 0.214-1.325, 0.1-1.23, 1.2-19.3, 2.01-36.3, 13.2- 391 and 103.3- 1484.7 ?g/g respectively. The results further revealed the fact that 36 % of samples contain Cd higher than the permissible limit and 10% of the samples were found having Pb above the permissible limit set by NPCB.

Significant finding of Rutaceae alkaloids in search of new drugs have led to an important strategy to overcome the problems of resistance and side effects associated with conventional antibiotics. In this study, leaves of the plant Ruta angustifolia (L.) Pers. was extracted and fractionated by using column chromatography. Through bioassay-guided isolation, combined fraction of R33 and R48, fraction RC-8 and Rd-10 yielded a total of three antimicrobial active alkaloids. These isolated alkaloids were then identified by means of Thin Layer Chromatography profile, melting point and maximum wavelength for UV absorption in methanol (UV? max-MeOH) in comparison with authentic alkaloids. The identification was further confirmed by 1H NMR and 13C NMR spectroscopic data and was characterized as acridone, furoquinoline and 4-quinolone so named arborinine, skimmianine and graveoline respectively. The antimicrobial activites of arborinine and graveoline were tested against Staphylococcus aureus, Enterococcus fecalis, Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa and Candida albicans of ATCC strains. Broth microdilution assay of both alkaloids gave Minimum Inhibitory Concentration (MIC) values ranging from 250 µg/ml to 1000 µg/ml. Minimum Bactericidal Concentration (MBC) values were recorded at 1000 µg/ml and more than 1000 µg/ml. The MIC and MBC of the compounds was compared with that of the standard antibiotics namely norfloxacin, ciprofloxacin, vancomycin, erythromycin and ketoconazole. Antibacterial combination effects of graveoline with erythromycin or vancomycin were studied against S. aureus, E. fecalis and E. coli by means of Fractional Inhibitory Concentration (FIC) index. All the tested combination resulted in additive effects with FIC index ranged from 0.75 to 1.02. Antifungal combination effects of arborinine and ketoconazole was experimented against C. albicans and showed synergistic interaction with FIC index of 0.5. Bacterial DNA-binding properties of the three alkaloids were investigated against double-stranded DNA with various restriction enzymes. The investigation revealed that these three alkaloids mostly affect the cleavage activity of the restriction enzymes which contains 5’-TpA sequence rather than 5’-ApT sequence in their recognition pattern and potential crosslink sites under UV exposure. These isolated alkaloids were screened to possess antimicrobial activity through DNA synthesis inbition mechanism and might need to combine with other agent to enhance its inhibitory effects.

Senna alata or Cassia alata (C. alata) is a plant belonging to the family Leguminosae and has been documented to have antifungal and antimicrobial activities. This research aims to extract C. alata using known extraction procedure, and to find optimum condition for microencapsulation by employing double emulsion solvent evaporation method. Biodegradable poly (D, L-lactide-co-glycolide) (PLGA) was the polymer of interest to encapsulate the C. alata extracts owing to its ability to give a controlled-release profile. Resultant microspheres were characterized for size distribution and external morphology by laser diffraction technique and scanning electron microscopy, respectively. Encapsulation efficiency (EE) was also calculated and the C. alata was quantified by UV absorbance. Several parameters have been investigated to optimize the characteristic of fabricated microspheres during the preparation process including different homogenization times for the primary emulsion, different volume ratios of aqueous/oil phases, several types and concentrations of surfactants, co-solvent in aqueous or oil phase and buffer systems (at varying pH and different concentrations of PBS). It was found that, most of the parameters employed resulted in low EE (<11%), however the encapsulation efficiency (64%) was significantly improved when the hardening tank is buffered to pH 7, with minimal effect on particle size. The particle size range obtained was between 6-30 ?m. Subsequent in vitro analysis displayed usual release pattern attributed to PLGA that is initial burst release followed by a gradual release phase up to the period of study. Although our C. alata extract did not show antimicrobial and antifungal activities against various strains tested but it appeared active against Escherichia coli. It is hereby suggested that our C. alata microspheres may be useful as preservative and antidote related to food-poisoning commonly caused by Escherichia coli.

The increase incidence in diabetes-and obesity-related diseases in developed and developing countries has driven serious efforts towards the discovery of adipogenic differentiation-inhibitory compounds in natural products. Mangostins and triterpenoids from Garcinia have been reported to contain a wide range of bioactivities. However, its antidiabetic and antiobesity activity has not been previously addressed. With regard to the fact, this research is designed to study the antihperglycaemic and antiobesity effects of selected phytochemicals isolated from Garcinia malaccensis and G. prainiana namely alpha-mangostin, beta-mangostin, cycloartane, friedelin and lanosterol. The compounds were tested for their antihyperglycaemic and antiobesity effects in 3T3-L1 adipocytes. In this study, we first elucidated the effects the compounds on the lipid accumulation of 3T3-L1 preadipocytes by using Oil Red O staining. The ability of the cell to uptake glucose was measured by liquid scintillation counter. Peroxisome proliferator-activated receptors gamma (PPARy), glucose transporter four (GLUT4) and leptin genes expression was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The results showed that α- and β-mangostin reduced the triglyceride accumulation in 3T3-L1 adipocytes with highest lipid inhibition activity was observed at 50 μM concentration (p<0.05) when compared to MDI (0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 0.25 mM dexamethasone, 1 μg/mL insulin) treated cells. On the other hand, treatment with other compounds significantly induced lipid accumulation. Analyses of 2-deoxy-D-[3H] glucose uptake activities showed that all the compounds including the MDI, metformin and sodium orthovanadate (positive control) significantly (p<0.05) improved the glucose uptake when compared to the basal cell. In addition, evaluation by using the adipolysis kit (Cayman Chemicals) showed that α-mangostin and β-mangostin increases the amount of free fatty acid (FFA) release. Further evaluation by the gene expression analysis showed that α-mangostin and β-mangostin may reduce lipid accumulation by inhibition of the expression of PPARγ genes. In addition, induction of glucose uptake and free fatty acid release by α- and β-mangostin was accompanied by increasing of mRNA expression of GLUT4 and leptin. Interestingly, mature 3T3-L1 cells treated with cycloartane triterpenoid was shown to enhance PPARγ and GLUT4 gene expression and decreased leptin expression. Besides, our results for cells treated with friedelin and lanosterol shows that both compounds could stimulate adipocyte differentiation and stimulate glucose uptake in adipocytes. Since, enhance PPARγ and GLUT4 genes was found to be involved in that process, we suggest that friedelin and lanosterol also will stimulate PPARγ and GLUT4 genes expression in adipocyte differentiation and glucose uptake process, respectively. As a conclusion, these line of evidences indicated that α-mangostin, β-mangostin and cycloartane triterpenoid together with friedelin and lanosterol derived from Garcinia may become an interesting candidate for the prevention of metabolic disorders such as diabetes and obesity.

Apolipoprotein E gene (APOE) has been known for more than 30 years and has been widely studied around the world for its role in the pathogenesis of diseases that are closely related to lipid and lipoprotein metabolism. Studies regarding the association between the APOE gene and type 2 diabetes mellitus (T2DM) are scarce. Therefore, this case-control study was aimed to investigate APOE allelic frequencies among the diabetic and non-diabetic subjects and associations between the alleles and selected bio-chemical markers between them. A total of 102 subjects were recruited, 51 were diabetic, and 51 were non-diabetic. Their fasting blood samples were analyzed for fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL). Restriction Fragment Length Polymorphism technique was used to identify the APOE alleles. Statistical analyses were performed using the Predictive Analytics SoftWare (PASW) Statistics, Version 18.0. Ɛ2 and Ɛ4 alleles were slightly higher, and the Ɛ3 allele was slightly lower in the diabetic group compared to the non-diabetic group (12.7% vs 8.8%, 24.5% vs 22.6%, and 62.8% vs 68.6% respectively). Diabetics with Ɛ2 allele had the highest mean values of all selected bio-chemical markers, and the trend was followed by Ɛ4 and Ɛ3 alleles. Both the Ɛ2 and Ɛ4 allelic diabetic subjects had significantly higher FBG compared to the Ɛ3 allelic diabetic subjects (10.11 vs 8.18 and 9.89 vs 8.18 mmol/L respectively, p<0.05). In contrast, diabetic subjects with Ɛ2 and Ɛ4 alleles had significantly higher TC (6.57 vs 4.58 and 5.41 vs 4.07 mmol/L respectively, p<0.05) while only Ɛ4 allelic diabetic subjects had significantly higher TG (1.57 vs 0.97 mmol/L, p<0.05) compared to the non-diabetic subjects. In conclusion, the APOE gene polymorphism influences blood levels of the selected bio-chemical markers in subjects with T2DM.

Alpha-mangostin (AM) is a lipophilic material and has been reported to have multitherapeutic activities. However, many obstacles have been documented that limit its medical applications. Therefore, this study aimed to encapsulate this compound in PLGA-based mico/nanoparticles using two different protocol namely single and double emulsion solvent evaporation techniques. Different experimental variables had been manipulated during the fabrication processes in order to investigate their effects on the particles’ characteristics and to obtain optimized formulations for both employed protocol. Reverse-phase high performance liquid chromatographic method had also been developed and validated for their linearity, precision, accuracy, limit of detection and limit of quantitation for reliable quantification of AM content in the particles. In the double emulsion technique, levels of evaluated factors included polyvinyl alcohol (PVA) concentration, ratio of oil to aqueous phases and emulsification time for both primary and secondary emulsion were optimized by using response surface methodology (RSM). The optimized formula that was prepared by 1% PVA, 1:10 ratio of oil to aqueous phases, and sonicated at 2 and 5 min time for primary and secondary emulsions, respectively, showed an encapsulation efficiency of 39.1%, a particle size of 2.06 μm and with desirable polydispersity index value (0.95). Interestingly, this optimized formula had an aerodynamic diameter of about 784.3 nm that is desirable for pulmonary delivery. Thermal analysis of these microspheres showed a physical conversion of AM from crystalline to amorphous state due to strong hydrogen bonds formed between carboxylic groups of polymeric material and hydrogen groups of AM as shown by FTIR. Thereafter, influences of other factors including AM concentration and type of stabilizer were also studied on the particles features. The resultant microspheres were characterized for their encapsulation efficiency (EE), loading efficiency (LE), particle size (PS), polydispersity index (SPAN), external morphology, in vitro release profile and in vitro cytotoxicity against non-small lung cancer cells line (A549). Our data revealed that replacement of PVA with Tween 20 or Span 20 showed significant decrease in the EE at low AM concentration (1% w/v), while increasing the AM concentration up to 2.5% showed significant increment in LE. Addition of Tween 20 also improved both EE and LE percents. Most of AM-loaded microspheres exhibited cell inhibitory activities close to that of free AM despite their release profile showed sustained release of AM over four weeks with total cumulative release of about 36.9-44.3%. In single emulsion technique, the evaluated experimental variables were type of emulsification process, type of surfactant and PVA concentration. Our data revealed that Tween 20 and Span 20 were unable to stabilize water-in-oil (o/w) emulsion system at the ratio were 1:1 and ultrasonic processor had no disruptive effect on the AM stability. Optimized formula prepared with 1% PVA, 0.3% chitosan hydrochloride and emulsified by ultrasonic processor showed EE of 79.1%, LE of 26.4% and with a diameter of 156.6 nm (based on SEM. Cytotoxicity of these particles (IC50; 24.6 μM) was lower than free AM (IC50; 12.8 μM) that consistent with their cumulative release after same incubation time. Incorporation of chitosan modified zeta potential of the nanoparticles to positive charge. The fabricated particles may be used as promising nano/microcarriers system to deliver AM to the lung cancer tissue.