Development of Biomimetic PHB and PHBV Scaffolds for a Three Dimensional (3-D) In Vitro Human Leukaemia Model

Leukaemia is defined as a group of haematological diseases (related to blood and blood-forming tissue) characterized by malignant proliferation of myeloblasts or lymphoblasts that replace normal bone marrow elements and infiltrate normal tissues. The study of leukaemia has been hindered by the lack of appropriate in vitro models, which can mimic this microenvironment. It is hypothesized that the fabrication of porous 3-D scaffolds for the biomimetic growth of leukaemic cells in vitro could facilitate the study of the disease in its simulated native 3-D niche. In this study, polyhydroxyalkanoate (PHA), in particular poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate-co-valerate) (PHBV) porous 3-D scaffolds with an improved thickness (in relative to the conventionally made PHA matrices) are utilized and investigated to model the abnormal 3-D leukaemic cellular growth system in the absence of exogenous cytokines. The polymeric porous 3-D scaffolds were fabricated using an ideal polymer concentration of 4% (w/v). The salt-leaching efficacy and the effect of salt residual on the cell growth media were carried out to validate the significant amount of salt remnant inside the porous materials. The physico-chemical characteristics of the porous 3-D scaffolds such as surface wetting, porosity, BET surface area and pore size distribution were studied by means of drop sessile analyzer (DSA), helium gas pycnometry, mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM). To increase probability of cellular attachment and proliferation, the polymeric scaffold surfaces were treated with O2-rf-plasma (100 W at 10 min) and NaOH (0.6M). Next, in order to improve the in vitro 3-D leukaemic cell culture, two main bone marrow extracellular matrix (ECM) proteins which are collagen type I or fibronectin were immobilized via physical adsorption on the treated surfaces of the polymeric porous 3-D scaffolds. Meanwhile, the in vitro degradation studies were conducted on both polymeric scaffolds with the hydrolytic degradation media of phosphate buffered saline (PBS) and cell growth media. The scaffolds were analyzed and compared for mass loss, morphology and pH changed of the PBS and cell growth media throughout 45 weeks and 9 weeks of the study respectively. Overall, PHB and PHBV displayed a good seeding efficiency (24 h) and excellent leukaemic cellular growth for up to 6 weeks (protein-coated scaffolds), assessed by MTS assay and SEM. Once the abnormal hematopoietic 3-D model (cell lines) was established, a new model to culture human primary acute myeloid leukaemia mononuclear cells (AML MNCs) was studied, compared and validated. All leukaemic cells grew better in PHBV scaffolds coated with 62.5 μg/ml collagen type I and sustained cell growth in the absence of exogenous cytokines. As a result, it was concluded that PHBV-collagen scaffolds may provide and could be used, as a practical model with which to study the biology and treatment of primary AML in an in vitro mimicry without the use of 2-D culture system and animal models

Standardization of the bio-active compounds (rotenoids) from the extract of local plant species (derris elliptica) using the internal standard method of high performance liquid chromatography (HPLC)

It is well known now that some plant species represent an efficient factory of chemicals, which are manufactured and used as bio-weapons against pest attacks. Extensive work has been done during the last few decades on these potentially useful compounds. During the last few decades a growing interest has been paid for safe agricultural production i.e free residual toxicity hazards to human beings and to the environment. Plant extracts-based biocides possess a great advantage compared with the chemical ones. Their efficacies are also acceptable. Research carried out was to standardize and determine the bio-active compounds from the extract of local plant species (Derris elliptica) using the internal standard method of the isocratic High Performance Liquid Chromatography (HPLC) analysis system. The raw plants were collected from Kota Johor Lama, Johor and sorted to collect the root and stem. Only the root and stem were utilized as a raw material of the extraction process. The root and stem were extracted by using the Normal Soaking Extraction (NSE) method at 28 0C to 30 0C with 95.0 % (v/v) of acetone as a solvent and the solvent-to-solid ratio of the extraction is (10.0 ml/g). The extraction was carried out for 24 hours and further cleaned up to remove fine debris of root and stem prior to determination of the rotenone and its derivatives content. The rotenone cube resin of SAPHYR S.A.R.L (France) was used to verify the appearances of the compounds in the extract. The employed method of analysis shows significant appearances of the bio-active compounds in the extract compared with the commercial grade of rotenone cube resin

Imidazolium-Based Ionic Liquid Binary Solvent System as an Extraction Medium in Enhancing the Rotenone Yield Extracted from Derris elliptica Roots

Rotenone, is a biopesticide which can be isolated from Derris species roots. However, procuring significant amount of rotenone using green alternative solvent rather than harmful organic solvents for commercialization is a challenge to be faced. Therefore, an approach using imidazolium-based ionic liquids (ILs) as an extraction medium was employed in this study. Five different types of binary solvent systems comprising a combination of acetone and five respective ionic liquids (ILs) of (1) [BMIM] Cl; (2) [BMIM] OAc; (3) [BMIM] NTf2; (4) [BMIM] OTf; and (5) [BMPy] Cl were used in the normal soaking extraction (NSE) of rotenone for a 24-hour extraction. The yield of the rotenone, % (w/w), and its concentration (mg/mL) in the dried roots was quantitatively determined by means of the reversed-phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). The results showed that a binary solvent system of [BMIM] OTf:acetone was the best solvent system combination compared to other solvent systems (p < 0.05). It contributed to the highest rotenone content of 2.69 ± 0.21% (w/w) (4.04 ± 0.34 mg/ml) at the 14th hour of the exhaustive extraction time. In conclusion, a combination of certain ILs with a selective organic solvent has been proven to be able to increase a significant amount of bioactive constituents in the phytochemical extraction process

A preliminary study on mosquito larvicidal efficacy of rotenone extracted from Malaysia Derris sp

Rotenone is a bio-active compound extracted from Derris elliptica (locally known as ‘Tuba’ plant). It has long been used as bio-pesticide, which is more environmental friendly than the commercially available pesticides and has the potential to be used in eliminating mosquito larvae. Therefore, the objective of this study is to determine the mosquito larvicidal activity (LC50) through the usage of liquid crude extract of Derris plant root. The rotenone liquid crude extract was extracted using normal soaking extraction (NSE) method. Two different solvent ratios were used to extract rotenone namely: (A) methyl chloride: methanol (1:1) and (B) methyl chloride: methanol (1:9). The extracts were concentrated using rotary evaporator at 40 °C with vacuum pressure of 800 mbar prior to the reverse-phase high performance liquid chromatography analysis (RP-HPLC) and biological activity (LC50) study. Next, the diluted extracts were subjected to the biological activity treatment for 6 hrs. The results showed that the concentrated liquid crude extracts of methyl chloride: methanol (1:1) which contained the highest rotenone content produced the lowest treatment concentration of 0.024 mg/ml to achieve 50% mortality within 3 hrs of treatment (p<0.05). The rapid mortality (as indicated by the LC50 value) of the mosquitoes’ larvae against rotenone extracted from Derris plant roots has proven that it has the potential to be used as larvicide to control vector-borne diseases especially from mosquitoes

Efficacy Study of Carrageenan as an Alternative Infused Material (Filler) in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Porous 3D Scaffold

Polymeric porous 3D scaffold plays an important role in culturing mammalian cells as ex vivo model. However, the scaffold used is ineffective due to its structural and cell acceptability weaknesses. Therefore, this research attempts to overcome the weaknesses by using carrageenan from red seaweed Kappaphycus alvarezii as an alternative infused material (filler) of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) porous 3D scaffold. The 3D scaffold was conventionally fabricated using the solvent-casting particulate-leaching (SCPL) method. Carrageenan was later infused into 3D porous scaffolds under vacuum pressure and freeze-drying process. Five carrageenan concentrations were prepared and its physicochemical properties such as pH and viscosity were carried out on each concentration to determine the best solutions to produce a new composite 3D structure. The preliminary result shows that carrageenan concentrations of 2, 4, and 6% (w/v) were considered the best solutions for the infusion process due to its stable rheology properties. The pH and viscosity profiles of three selected carrageenan solutions were exhibited in the range of 9.00–9.20 and 0.047–1.144 Pa·s, respectively. Moreover, the incorporated carrageenan gel fraction was in the range of 4.30% to 14.95% (w/w) which was determined by gravimetric analysis and dye staining method (visual assessment). The well-infused carrageenan 3D scaffold was further characterized based on its internal morphology and degradability study. The vertical cross-sections of the scaffolds revealed homogeneous accumulation of dried gelatinous carrageenan which was covered throughout its pores wall. The degradation rate (K) of the carrageenan infused 3D scaffold was between 0.01±1.66 (mg/day) and 0.03±3.23 (mg/day). The higher the carrageenan concentration used, the faster the degradation rate occurring (p2 weeks). In conclusion, the usage of carrageenan as a composite material exhibits its great potential to be used in tissue engineering application and 3D cell culture model

Normal soaking extraction (NSE) of rotenone from Derris elliptica

Derris elliptica or the tuba plant contains rotenone, bio-active compound known that has the potential to be used as bio-pesticide. Bio-pesticide is the best-known alternative bio-pesticide which has the potential to replace the use of conventional pesticides, as it is more environmentally friendly. The main objective of the paper is to obtain the best extraction solvent for optimizing the yield in rotenone extraction. Rotenone was extracted from Derris root using three different parts of roots and three types of solvent in Normal Soaking Extraction (NSE). The types of Derris roots are: (1) Fine root (2) Coarse root (3) Stem. The three types of solvent system are: (1) Acetone 95 % (v/v), (2) Chloroform 99.9 % (v/v) and (3) a mixture of ethanol: H2O (9:1) added with oxalic acid (1mg/ml). The liquid crude extracts were further cleaned up to remove the fine debris of roots. The presence of rotenone was confirmed using qualitative analysis Thin Layer Chromatography (TLC) and thereupon the determination of rotenone content was carried out using High Performance Liquid Chromatography (HPLC). From the results obtained, it was found that the Normal Soaking Extraction (NSE) using acetone 95 % (v/v) was the best method to extract the highest yield of rotenone; 1.14 % (w/w)

Thermal treatments on the oil palm fruits: response surface optimization and microstructure study

Sterilization is the most important steps in the palm oil milling process prior to oil extraction. Experiments involving dry heating sterilization (SD) couple with solvent extraction of palm fruits were done to determine the relationship of palm oil yield and deterioration of bleachability index (DOBI). As a comparison, the conventional method of wet heating was used (SW). The optimum sterilization treatment parameters were determined by using response surface methodology (RSM). Central composite rotatable design (CCRD) was used to study the effects of sterilization temperature, X1 (°C) and treatment time, X2 (min) to oil yield (%), and DOBI. The sterilization temperature and time were conducted between 70 and 90 °C and 20 to 90 min, respectively. Preliminary results proved increasing temperature and time of sterilization process increased oil yield for both SD and SW. Furthermore, the DOBI showed a similar trend as the oil yield. Optimization study using SD gave the optimal response through a combination of parameters, SD: X1 = 90 °C and X2 = 68 min, where the oil yield obtained was 43.21% and DOBI 4.05. However, sterilization treatment using SW showed insignificant results (p>0.05) between temperature and time since R2 value was 0.4368 and the low degree of agreement between adjusted R2 (0.03) and predicted R2 (-2.43). It was also found SD treatment produced high DOBI value though the oil yield was lower than SW

Kinetics Extraction Modelling and Antiproliferative Activity of Clinacanthus nutans

Clinacanthus nutans is widely grown in tropical Asia and locally known “belalai gajah” or Sabah snake grass. It has been used as a natural product to treat skin rashes, snake bites, lesion caused by herpes, diabetes, fever, and cancer. Therefore, the objectives of this research are to determine the maximum yield and time of exhaustive flavonoids extraction using Peleg’s model and to evaluate potential of antiproliferative activity on human lung cancer cell (A549). The extraction process was carried out on fresh and dried leaves at 28 to 30°C with liquid-to-solid ratio of 10 mL/g for 72 hrs. The extracts were collected intermittently analysed using mathematical Peleg’s model and RP-HPLC. The highest amount of flavonoids was used to evaluate the inhibitory concentration (IC50) via 2D cell culture of A549. Based on the results obtained, the predicted maximum extract density was observed at 29.20 ± 14.54 hrs of extraction (texhaustive). However, the exhaustive time of extraction to acquire maximum flavonoids content exhibited approximately 10 hrs earlier. Therefore, 18 hrs of extraction time was chosen to acquire high content of flavonoids. The best antiproliferative effect (IC50) on A549 cell line was observed at 138.82 ± 0.60 µg/mL. In conclusion, the flavonoids content in Clinacanthus nutans water extract possesses potential antiproliferative properties against A549, suggesting an alternative approach for cancer treatment

Interaction study of binary solvent systems ionic liquid and deep eutectic solvent with rotenone

[BMIM]OTf and alcohol-based DES combination with a selected organic solvent (acetone and acetonitrile) have been proven to efficiently extracting rotenone (isoflavonoid biopesticide) compound compared to individual organic solvents. Their efficiency builds up interest to study the solvent-solute interaction that occurs between both selected solvent systems with rotenone. The interaction study was analyzed using FTIR, 1D-NMR and 2D- NMR (NOESY, HMBC). Correlation portrayed by NOESY and HMBC of [BMIM]OTf - standard rotenone mixture predicted probable hydrogen bonding between the oxygen of rotenone with acidic proton C2-H of [BMIM]OTf. While for the alcohol-based DES-rotenone mixture, the correlation shows probable interaction to occur between methyl and methoxy group rotenone with the hydroxyl group of 1,4-butanediol. In conclusion, potential hydrogen bonding that occurs between solvent and solute aid towards the solvent efficiency in extracting rotenone compound while emphasizing on the low cost and green mediated solvent systems