SYNTHESIS AND CHARACTERIZATION OF MOLECULARLY IMPRINTEDPOLYMER MICROSPHERES AND ITS APPLICATION FOR PURIFICATIONOF ANDROGRAPHOLIDE IN METHANOLIC EXTRACT FROMANDROGRAPHIS PANICULATA (BURM F.) NEES HERB

• Main Author: Winingsih, Wiwin
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/63987
• Institution: Institut Teknologi Bandung

Andrographolide is a bioactive compound widely found in bitter herb (Andrographis paniculata Burm.F) Nees. Based on various studies, andrographolide has several diverse pharmacological activities. Therefore, various attemps have been made toisolate and purify it from bitter leaf. The andrographolide isolation method is usuallycarried out by extraction and then purification by chromatography or re- crystallization. The purification process by re-crystallization usually takes several stages and several days for crystal formation. Purification by columnchromatography requires a lot of organic solvents which can have a negative impact on the environment. So that, the simpler and more time-saving purification methods are needed to improve. Currently, molecular imprinted polymers (MIPs) are widelyused for the isolation and extraction of synthetic compounds and natural compounds from various matrices because of their high selectivity. MIPs are polymer that have a selective cavity for theri template molecules. Molecular imprinted microspheres polymers (MIPs) have been used successfully for the isolationof andrographolide from plasma. Based on this, a hypothesis was made that molecularly imprinted microspheres polymers could be used for the isolationandpurification of andrographolide from the methanol extract of the bitter plant. This study aimed to prepared MIPs that can be applicated for andrographolidepurification from Andrographis paniculata (Burm.F) Ness methanolic extract. The study began with a preliminary test using an in-silico approach. The next stepwas the synthesis of silica microspheres. Then, MPMC was synthesized by surfaceimprinting method with silica microspheres as solid supports (MPMCs) andprecipitation polymerization (MPMCp), as a contrast, N-MPMCs and N-MPMCpwere synthesized using the same method without the addition of template molecules. The synthesized MPMC was characterized using SEM, FTIR, High PerformanceLiquid Chromatography. The adsorption capacity of MPMC was evaluated usingtheadsorption isotherm principle. MPMC performance was determined by the imprintingfactor and selectivity factor. The purified isolates were characterized by FTIR, HPLC, UV-Vis spectrophotometry, and melting point. Preliminary in silico study was used to determine the best functional monomer, activesite of reaction of andrographolide and selected monomer, ratio of functional monomer: template molecule, and determine the best porogen solvent so that MPMCwith high selectivity was obtained. Gaussian 09W Version 8.0, and Gaussian View5.0.8 (Gaussian Inc., Wallingford, CT) software were used to calculate all in silicostudy. Determination of the best functional monomer interaction withandrographolide, Density Functional Theory (DFT), B3LYP method, with a basis set of 6-31G+. (d, p) was used. Base of their (?G) and (?E), from the 16 functional monomers, there were 4 functional monomers that have good interactions withandrographolide, namely 3-aminopropyl triethoxylan (APTES) (?G= -44,6 kcal/mol; ?E= -44,5 kcal/mol), 1-viyilimidazole (?G= -17,6 kcal/mol; ?E= -23,1 kcal/mol), methacrylamide (?G= -3,2 kcal/mol; ?E= -16,7 kcal/mol, and methacrylic acid(MAA)(?G= -24,6 kcal/mol;?E= -24,7 kcal/mol). Among the four monomers, APTESand methacrylic acid interacted better than the others, so they were chosenas functional monomers used in this study. Based on the results of the preliminary test, it was shown that the functional monomer containing silica with the best interactionwith andrographolide was APTES. Determination of the active site interaction of the andrographolide aims to predict the ratio of functional monomers to template molecules and to explain the mechanismof interaction between template molecules and functional monomers. Determinationof the active site of the interaction was carried out using full NBO (Natural BondOrbital) command. Based on the results of the determination of the active site, andrographolide has 2 hydrogen atoms which have a more positive partial chargethan the other atoms (H 55 and H 40 atoms) and 3 oxygen atoms which have a morenegative partial charge than other molecules (O 20, O 30, and O 50 atoms thus andrografolid has five reaction sites. The mole comparison of functional monomers with template molecules was carriedout by calculating the bond energy between andrographolide and APTES at a moleratio of 1:1 to 1:5 using Gaussian 09W Version 8.0 software and Gaussian View5.0.8(Gaussian Inc., Wallingford, CT), semiempirical theory, PM3 base set and job plot. The results of the in silico test showed that the most stable compositionof andrographolide: APTES was at a ratio of 1:5, while based on the results of ajobplot of 1:1. The mole ratio between andrographolide and methacrylic acid used is 1:3. The results of optimization by in silico studies were used for the synthesis of MPMCs. The surface imprinting technique was carried out using the sol gel method usingAPTES as a functional monomer, tetraethyl orthosilicate (TEOS) as a crosslinker, acetic acid as a catalyst, silica microspheres as a solid support and methanol as aporogen solvent. Silica microspheres were synthesized by the modified Stober method. The synthesized silica microspheres were then characterized using a ScanningElectron Microscope (SEM), Fourier Transform Infra Red (FTIR), and a particle sizeanalyzer (PSA). The results of the characterization of silica microspheres with SEMshowed a smooth and spherical surface. The FTIR spectrum of silica mikrospheres shows O-H stretch absorption at wave numbers 3500-3000 cm-1 , Si-O-Si stretchat 1100-1000 cm-1, Si-O bending at 805 cm-1, and Si-C stretch at 2000 – 2400 cm-1 . PSA measurement results showed silica microspheres had average diameter sizebetween 400 – 758.8 nm. Silica microspheres with a size of 758.8 nm were used as solid supports for thesynthesis of MPMCs. The synthesized MPMCs were further characterized by usingSEM and FTIR. In addition, MPMCs adsorption isotherm was studied to obtainadsorption capacity, imprinting factor and selectivity factor. The results of the SEMMPMCs characterization showed a rougher and stacked surface. The results of FTIRcharacterization of MPMCs showed loss of absorption at wave numbers causedbythe presence of alkyl groups from APTES and TEOS, namely 2900 – 2800 cm-1 (C-Hstretching of CH3 and CH2), 1446 cm -1 (CH bending asymmetry of CH3,), 1381cm-1 (C-H bending symmetrically CH3), 1416 cm-1, 1324 cm-1 CH wagging fromCH2, 1275 cm-1 C-H twist/wagging from C-H, 1168 cm-1 (CH rocking fromCH3) and960cm- 1(CH rocking from CH3). The presence of a peak at wave number 1600 – 1500cm- 1 (N-H stretch of APTES) in MPMC indicates that APTES has been bound totheMPMC polymer matrix. Based on the FTIR spectrum, it shows that the synthesis process has been successful. MPMCs adsorption isotherm was evaluated with various models, namely theFreundlich, Langmuir, Temkin, and Henry models. Based on the experimental results, the MPMCs adsorption process is best described by the Temkin model (R2 0.9848) where the adsorption process occurs through pore filling. The adsorption capacity of MPMCs was 0.5679 mg/g. The imprinting factor and selectivity factor for MPMCs were 3.9305 and 3.4305, respectively. MPMCp was synthesized using andrographolide as a template molecule, MAAas amonomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinker, benzoyl peroxide as an initiator, and acetonitrile: toluene (3:1) as a porogen solvent. Thecomparison polymer N-MPMCp was prepared by the same method without theaddition of andrografolid. Like MPMCs and N-MPMCs, MPMCp and N-MPMCpwere characterized by the same method. SEM characterization results showedarough surface, spherical shape with a size ranging from 200 - 400 nm. The results of MPMCp FTIR characterization showed loss of absorption at a wave number of 1600cm-1 (streching C=C)indicating the success of the polymerization process. TheMPMCp adsorption isotherm was 1.2486 mg/g, the most suitable adsorption isothermmodel to describe the MPMCp adsorption process was the Freundlich isothermmodel (R 2 0.9981) thus the adsorption was chemical (chemisorption). Imprinting factor andselectivity factor of PMTPp are 1.148 and 12.37. MPMCs and MPMCp were applied for the purification of bulk andrographolide bydirect mixing. Purity of bulk andrographolide increased from 55.37%± 0.69to94.79% ± 0.34 after purified by MPMCp and to 92.92% ± 0.35 after purificationbyMPMCs. The purification results were then characterized by FTIR, UV-Vis spectrophotometry, HPLC and the melting point showed similarities tothecharacteristics of standard andrographolide. From this study we can conclude that MPMC have been successfully synthesizedandcan be applied for purification of andrographolide from methanolic extract of sambiloto.