A study on dispersion and characterisation of α-mangostin loaded pH sensitive microgel systems
© Ahmad et al.; licensee Chemistry Central Ltd. 2013
Received: 6 December 2012
Accepted: 13 May 2013
Published: 16 May 2013
α-Mangostin was extracted with methanol from the rind of mangosteen fruit and purified by using silica gel column chromatography technique. The compound is characterised using infrared, 13C and 1H NMR as well as UV–vis spectroscopy. The α-mangostin dispersion in colloidal systems was studied by incorporating it with an ionic microgel, poly (N-Isopropylacrylamide)-co-2VP at different pH.
The DLS result showed the size of microgel-α-mangostin mixture declined from 548 nm to 200 nm upon the increment of the pH. Moreover, it was found the morphology of loaded compound depended largely on the nature of the continuous phase of the microgel system. Interestingly, by manipulating the pH, α-mangostin tends to form crystal at extremely low pH and transforms into spherical shapes at pH 6.
This research shows different structures of the α-mangostin particle that are attributed by adjusting the pH using microgel systems as a template.
Mangosteen, the ‘queen’ of all fruits is a plant native to Southeast Asia that is used as traditional remedy to treating skin infections wound, improve muscle and bone pain, eating disorder, diarrhea and accelerating wound healing [1, 2]. Among the essential phytonutrients found in the rind of the mangosteen, α-mangostin or 1,3,6-trihydroxy-7-methoxy, 2-8-bis (3-methyl-but-2-enyl)-xanthen-9-one stands alone in its impressive benefits. Since it was first discovered by W. Schmid in 1855 , this compound has attracted many researchers due to its biological active properties such as antioxidant , anti bacteria , antifungal , anti inflammatory , anti cancer  and anti tuberculosis , therapeutic drugs  and also being used as mosquitoes larvicide . Furthermore, it has been commercialised as supplement in food products and natural dyes in fabric industries, which are readily available in the worldwide market. However, its poor solubility in aqueous solution and low oral bioavailability are the limiting factors for many applications. Therefore, it is a challenge to the scientists to solve these problems in order to fully utilise this compound deemed suitable for the human body systems. So far, there is only one reported study on the enhancement of the α-mangostin solubility and oral bioavailability by Aisha et al . on the solid dispersion of α-mangostin in water soluble carriers using polyvinylpyrrolidone (PVP). Although this technique showed an improvement on the dissolution of α-mangostin, its commercial use is still very limited, primarily due to manufacturing difficulties and stability problems . As an alternative, a responsive polymer has being used for α-mangostin dispersions.
Much attention has been given to responsive polymers (smart polymers) due to their response ability to external stimuli such as temperature , electrolyte, light and pH . One of them is microgel, a polymer colloid particle with three-dimensional network structure that offers many applications from the viewpoint of drug delivery. It can be manipulated as nanoreactor which controls the size property, from macrometers to nanometers [13–15]. Moreover, this polymer is responsive and having large surface network area enables to incorporate with bio-related molecules. This ‘smart’ system is used for bioactive molecules entrapments including drugs, proteins, carbohydrates and DNA [14–16]. Its applications are not only limited for biomedical purposes but are widely applied for the incorporation of inorganic nanocrystals, quantum dots [16, 17], magnetic nanoparticles [18, 19], optical imaging for living cells and photodynamic therapy .
Ionic microgels are formed when at least one of the co-monomer becomes charged specifically when the pH reaches the pKa of that species. Generally monomer such as 2-vinylpyridine , acrylic acid  and methacrylic acid  are used to produce a pH responsive microgel, which can be synthesized via emulsion polymerisation or surfactant free emulsion polymerisation . Xu et al  reported on the utilization of poly (ϵ-caprolactone)-pluronic–poly-(ϵ-caprolactone)-dimethylacrylic (PCFC-DMA) as a vitamin B12 carrier. These modified microgels were discovered to be very sensitive towards pH. They found the vitamin B12 could be released from the microgel faster at pH 7.4 than at pH 12. Wang et al . studied the utilization of poly (N-isopropylacrylamide-co-acrylamide) as bleomycin drug carrier. The result showed the releasing rates of bleomycin from the microgel exhibited diffusion control at human body temperature.
Both experiments discussed on the successful of using microgel as a carrier for commercially bioactive molecules yet, there was no attempt of incorporating microgel with any organic bioactive compounds. Therefore, this paper discussed on the characterisations of extracted α-mangostin and the attempt of its dispersion in an ionic microgel system, poly (N-isopropylacrilamide) PNIPAM with a co-monomer 2-vinylpyridine (PNIPAM-co-2VP). The stability over a range of pH in microgel system is also reported
Results and discussion
Incorporating α-mangostin into PNIPAM-co-2VP dispersions
In both cases, the particle size reached its optimal de-swollen state at pH 5 (Dh ~200 nm). Any further increase in pH had no significant effect on the particle size. The exact mechanism by which the α-mangostin associates with the microgel is still unclear. However, Bradley et.al  proposed that the step involved would be the electrostatic interaction between the negatively charged of the additional compound with the cationic groups on the microgel network. This would act to reduce the electrostatic repulsion screening of the charges that would otherwise exists in the network .
Yellow compound was successfully extracted from the rind of Garcinia Mangostana Linn., which has similarity with α-mangostin characteristics and supported by data that is highly significant with previous literature. The α-mangostin dispersions in microgel systems as a function of pH were investigated. The DLS results showed that there was an interaction in the presence of α-mangostin in PNIPAM-co-2VP microgel systems in swollen state, where the neutralization occurred which then affected the particle sizes. On the contrary, at the collapsed state (pH 6 and higher) there was no contribution to any significant changes to the structure of PNIPAM-co-2VP microgels since it was only a surface interaction. Interestingly, the extreme of pH might not only affect the colloidal dispersion but also the α-mangostin morphology. At pH 2, it resulted as crystal-like shape. However, by increasing the pH value it turned to be agglomerated as big spherical forms. This result showed PNIPAM-co-2VP could be potentially used as a controlled-reactor triggered by pH for α-mangostin particles. Over a variation of pH, interaction of the microgel polymers with distinct crystallographic planes or area of the growing nuclei permits control of the size, shape and structure of the organic compounds. Moreover, it can also be used to overcome its weakness in the aqueous solubility. The future research for investigating the retention, release, the kinetics and its mechanism might be useful for the application of PNIPAM-co-2VP as drug delivery agent for α-mangostin particles to the body system.
Material and instrumentation
All experiments utilized purified water which was milli-Q water standard (PureLab, Elga), with resistivity of 18.2 MΩ cm. Dialysis tubing (Fisher) with a Mw cut-off of 12,000-14,000 Daltons was used for microgel purification. For all samples, pH was measured by using a waterproof pH meter (HI98127, pHep Hanna). For poly (N-isopropylacrylamide) co-2-vinylpyridine (PNIPAM-co 2VP) microgel synthesis, the cross-linking monomers divinylbenzene (DVB, Aldrich, 80%) and N, N-methylenebisacrylamide (BA, Aldrich, 99%), were used without further purification. The initiator used for the cationic microgels was 2, 2’-azobis (2-methylpropionamidine) dihydrochloride (V50, Waco, 95%). Aqueous solutions of HCl and NaOH were used to adjust pH. All chemicals and solvents used were reagent grade and used without further purification. Infrared spectra were recorded on a Perkin Elmer GX Spectrometer by using potassium bromide pellet. The size and morphology of the sample was investigated by using Transmission Electron Microscope (TEM) Philips CM12 model.
The Ultra-violet spectra were determined by Shimadzu UV–vis Spectrophotometer (UV 2400PC series). The sample was dissolved in ethanol and the solution was scanned from 200 to 450 nm. Ethanol was used as the background for α-mangostin sample while a mixture of ethanol and pnipam-co-2VP microgel was used for α-mangostin and microgel mixture.
The 1H and 13C nuclear magnetic resonance spectra were measured with Jeol JNM-ECP 400 NMR Spectrometer. Samples were dissolved in dimethylsulfoxide (DMSO)-d 6 and chemical shifts were given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard.
DLS and zeta potential characterisation
Microgel particle sizes and polydispersities index (PDI) were determined by dynamic light scattering (DLS) using a Zetasizer Nano-S (Malvern,PA). The electrophoretic mobility (μe) for α-mangostin and microgel is determined as a function of pH for dispersions at 25°C. The α-mangostin μe values remain negative across the entire pH range from 2 to 10, whereas the PNIPAM-co-2VP microgel μe values remain positive which are consistent with the cationic polymer remaining with positive charge.
Extraction of α-mangostin
Dried mangosteen rind samples were collected from Terengganu, Malaysia and the extraction of α-mangostin was carried out by following the normal procedure of isolating natural products as previously reported . The grinded mangosteen rind was extracted with methanol for three weeks and then separated by column chromatography eluted with the mixture of dichloromethane-hexane (6:4) giving a fine yellow powder.
Synthesis of PNIPAM-co-2VP microgel
The PNIPAM-co-2VP microgels were synthesized by a surfactant free polymerization technique as previously reported . Briefly, 800 ml of purified water was purged with nitrogen for 30 minutes in a 1 L, five-neck round bottom flask fitted with a mechanical stirrer, which operated at 150 rpm. About 0.50 g cationic initiator 2, 2’-azobis (2-methylpropionamidine) dihydrochloride (V50, Waco) was then added to the reaction flask and stirred. In a beaker 200 ml of purified water (milli-Q standard) (PureLab, Elga), 3.75 g of NIPAM and 0.55 g of BA together with 1.25 g of 2-vinylpyridine (2VP) were added together and stirred for 15 minutes. This solution was then added to the reaction vessel with the temperature raised to 70°C. The polymerization reaction was left to proceed for 6 hours with continuous stirring (~150 rpm). The outcome of the dispersion was filtered through glass wool followed by extensive dialysis against milli-Q water for one week with two changes of water per day.
The authors would like to thank the Faculty of Science and Technology, Universiti Kebangsaan Malaysia for the provision of laboratory facilities and technical assistance. MA also gratefully acknowledges the scholarship from the National Science Fellowship (NSF), MOSTI. AML acknowledge funding from DIP 2012–11 and FRGS/1/2011/SG/UKM/01/25.
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