Micronization of Curcuma xanthorrhiza Extract with Addition of PVP Using Supercritical CO2 as Anti-solvent

Curcuma xanthorrhiza Roxb, known as Temulawak or Javanese ginger, is a plant species. Its rhizomes are used as a medicinal herb. It contains curcumin as an active compound and ethereal oils mainly consisted of sesquiterpenes. In this work, Curcuma xanthorrhiza Roxb ethanolic extract was micronized with an addition of PVP using supercritical antisolvent (SAS) method. The ethanolic extract was obtained from dried Curcuma xanthorrhiza Roxb using soxhletation. For the micronization, the extracted compound solvent was a mixture of acetone and ethanol (90:10 (v/v)), while the supercritical CO2 was used as an antisolvent. The effect of operating conditions on the particle size and morphology was evaluated. Through this method, spherical Curcuma xanthorrhiza/PVP particles with mean diameter ranging from 191 ± 70 nm to 178 ± 57 nm were successfully formed. The particle size not significantly decreased as the pressure increased from 8 12 MPa. The addition of PVP is very effective to reduce the particle size, increase the solubility, and enhance the bioavailability of Curcuma xanthorrhiza extract. This work has the potential to improve the use of Curcuma xanthorrhiza in pharmaceutical and nutraceutical applications.


Introduction
For several years an increasing number of herbal medicines has appeared in worldwide marketplace. There are many varieties of plant that can be studied in deliver its function for herbal medicine. Natural products are known as more drug-likely and biological friendly than synthetic molecule product. Many of them have been proven to have better compatibility with biological system and lesser side effect (Oon et al., 2015). Nowadays, the studies for modern pharmaceutical industry has been more focused on medicine from natural product. One of the most known used on plant-based-medication is the Curcuma genus, which consist of more than eighty species. Curcuma xanthorrhiza Roxb, known as Temulawak or Javanese ginger, is an original plant from Indonesia that belongs to Zingiberaceae family. Today, the cultivation of this plant is grown wild in Thailand, Indochina, The Malay Archipelago, India, Japan, Korea, and Northern Australia (Jantan et al., 2012).
Curcuma xanthorrhiza Roxb has been traditionally used as herbal medicine in Indonesia. There are more than fifty recipes of herbal medicine using C. xanthorrhiza. Its rhizome is a major material in the herbal medicine production. C. xanthorrhiza known as remedy for many diseases such as stomach diseases, liver disorders, rheumatism, and high cholesterol (Yasni et al., 1994). From previous study showed that extract from C. xanthorrhiza proven to have antioxidant, anti-microbial, anti-inflammatory, analgesic, immunostimulant, antifungal, antitumor, and anti-diuretic activity. Beside for medical needs, its rhizome is also used as spice on foods, giving a yellow colour on some cuisine, and for cosmetics (Jae et al., 2008;Kim et al., 2007a;Mary et al., 2012;Ozaki, 1990). All the benefits were support by the active compounds in C. xanthorrhiza extract such as curcumin, xanthorrhizol, and few volatile compounds (Devaraj et al., 2010;Hwang et al., 2000). The phytochemical studies showed that Curcuma rhizomes contain two important bioactive compounds, namely diarylheptanoid (curcuminoid) and terpenoid (mainly sesquiterpenes) (Diastuti et al., 2019). The micronization of C. xanthorrhiza extract into microparticles is needed to prevent the rapid oxidation of bioactive compounds.
The particle mironization of bioactive compound with biopolymer addition has been found to be particular interest because it can improve drug delivery system by preventing the rapid degradation of bioactive compounds, increasing the bioavailability in the body, and enhancing the solubility in the water (Chhouk et al., 2018;Nuchuchua et al., 2017). Polyvinylpyrrolidones (PVP), water-soluble carriers with high molecular weight, have been the most used biopolymer for particle micronization. According to previous study, the dissolution of drug in physical mixtures were higher with the present of PVP. The ability to form good solubility in water and various solvent had become a suitable reason to use PVP as a carrier. The C. xanthorrhiza extract has low solubility in water. Therefore, in this work, the addition of PVP was expected to give a great influence in increasing the solubility of C. xanthorrhiza extract. The formation of hydrogen bonds between PVP and the phenolic compound in the extract plays an important role in particle formation (De Almeida et al., 2018).
The size of particle holds an important effect in the drug delivery system and its bioavailability. Decreasing the particle size will increase the surface area and enhance the solubility itself. There are many conventional techniques used to produce coprecipitates, such as solvent evaporation, coacervation, and spray drying. However, these techniques have some shortage such as the particle size obtained relatively large, the thermal-degradation of bioactive compounds, and the large amount of solvent needed that can affect residue in final product (Kwon et al., 2011). Supercritical Fluids (SCFs) based techniques has been used to overcome the shortage in conventional methods. Supercritical fluid with the antisolvent (SAS), one of supercritical assisted micronization method, is widely used for microparticles formation from natural products (Boonnoun et al., 2013;Chhouk et al., 2018;Kim et al., 2007b;Lestari et al., 2019;Xia et al., 2012;Yoon et al., 2016). This method suitable for bioactive compounds that difficult to dissolve in supercritical fluid. In SAS method, an organic solution of solute is streamed through a nozzle and is conjugated with a supercritical fluid in a chamber simultaneously. The phenomenon of mass transfer occurs between solution and supercritical fluid, which plays role as an anti-solvent, leading to supersaturation state and then resulting in the formation of solute particles. SAS method generates smaller particle size, better morphology and even more equitable particle distribution. Carbon dioxide (CO2) is the most commonly used as supercritical fluid due to it is non-inflammable, non-toxic, inexpensive, available in abundant amount, eco-friendly, and its properties are controlled easily.
In this study, the SAS method using carbon dioxide as the anti-solvent was done for particle micronization of C. xanthorrhiza extract with the addition of PVP as a biopolymer. The effect of operating conditions on the particle size and morphology was examined.

Plant materials
Curcuma xanthorrhiza Roxb were purchased from local suppliers in Surabaya, East Java, Indonesia. The sample preparation from C. xanthorrhiza were dried at room temperature to decrease its moisture content. Then the dried sample were grounded into fine powder.

Extraction of C. xanthorrizha
Fine powder of C. xanthorrhiza were extracted using soxhletation with ethanol as the solvent for 15 hr. Then the ethanol was removed using rotary vacuum evaporator (RE-1000VN, B-ONE, Indonesia). The crude from C. xanthorrhiza extract were obtained. This method was done to achieve C. xanthorrhiza extract in higher yield than using another extraction method (Leblebici et al., 2012;Tambun et al., 2017).

Micronization with SAS method
The particles micronization process started by heating the oven drying chamber into the desired temperature. Then using a coil, the supercritical CO2 was streamed through a chiller to maintain the liquefaction of CO2 and prevent cavitation in pump. With a high-pressure pump (PU-1586 Intelligent prep. pump, JASCO, Japan), the liquid CO2 was pumped into the system at a constant flow rate of 15 mL/min. The operating pressure in the system was maintained by using a back-pressure regulator (BPR, Tescom, U.S.). It was equipped with a heater to prevent the CO2 that passing through the BPR from freezing and clog the precipitator tube. After the operating pressure and temperature were achieved, another pump (PU-980 Intelligent HPLC pump, JASCO, Japan) was used to inject the crude extract and PVP solution with ratio varied from 0 to 1:10 (w/w) into the system at a constant flow rate of 0.25 mL/min. The concentration of feed solution about 2 mg/mL with a mixture from acetone and ethanol ((90:10, (v/v)) as organic solvent to dissolve the extract and PVP. The supercritical CO2 with a constant flowrate of 15 mL/min and the solution were contacted through a precipitator tube inside the heating chamber for 90 min. This process leads SC-CO2 dissolved into organic solvent and causing supersaturation condition, thus resulting in the formation of fine particles. After the micronization process was completed, the SC-CO2 was continuously pumped for 90 min to wash out the remaining solvent in precipitator. The obtaining particles were collected inside a collector with 0.5 µm filter (Swagelok, USA). Then the micronized particles were kept in desiccator to prevent degradation of the particle due to light until further analysis.

Product analysis
The Scanning Electron Microscopy (SEM, S-4300, Hitachi) was used to determine the surface morphology of the particles. Before conducting SEM analysis, the sample of particles was dispersed on a carbon tab in the aluminium holder and coated with thin layer of gold at high pressure evaporator. The mean particle size and distribution were calculated using Image J analysis software from approximately ± 250 particles observed in SEM image.
Fourier Transform Infrared (FTIR) spectrophotometry was used to determine chemical structure and the existence of remaining solvent after micronization process in C. xanthorrhiza extract, PVP, and C. xanthorrhiza/PVP particles. Data were obtained using wavelength range of 4000-400 cm -1 .

Dissolution studies
The dissolution rate of C. xanthorrhiza extract and C. xanthorrhiza/PVP particles were analysed using UV spectrophotometer (Genesys 10-S, Thermo Fisher Scientific, US) at wavelength of 425 nm as the highest absorbance peak. Sample from C. xanthorrhiza particles and C. xanthorrhiza/PVP particles were tested in 10 mL of ultra-pure water with equal concentration as dissolution medium. Through a disposable filter, aliquots of sample were filtered and then measured the absorbance with interval time up to 24 hr. The solubility of particles was compared with particles solubility in ethanol as a complete dissolution (Lestari et al., 2019).

SAS micronization of C. xanthorrhiza/PVP
In supercritical fluid with antisolvent method, knowing the thermal properties of CO2 and its organic solvents is important to assure the micronization process conducted in the right condition. There are many studies done for determine the phase diagram of CO2 in a mixture with acetone/ethanol. Acetone and ethanol as organic solvent have a higher solubility in CO2 at high-pressure than lowpressure system. Their binary mixture achieves the critical state of CO2 at an enough high-pressure system (Day et al., 2002;Hsieh and Vrabec, 2015). Hence, based on the P-T diagram reported by Ziegler et al. (1995), it can conclude that the operating condition in this study were conducted in the mixture critical point (MCP). When the operating condition above the MCP, interfacial tension between C. xanthorrhiza/PVP solution and supercritical CO2 disappear then the precipitate particles were formed as the result (Yoon et al., 2016).
In order to observe the morphology, particle of PVP, C. xanthorrhiza extract, and C. xanthorrhiza/PVP that obtained from SAS method were analysed using SEM. As shown in Figure 1, the particle form of C. xanthorrhiza extract were irregular with agglomeration and PVP particle form also irregular but almost spherical. Whereas the morphology for particle of C. xanthorrhiza extract with the addition of PVP were spherical more evenly. The range particle size of C. xanthorrhiza extract from 307 ± 87 nm to 349 ± 68 nm while C. xanthorrhiza/PVP was ranged from 178 ± 57 nm to 191 ± 70 nm. From the result, the existence of PVP in C. xanthorrhiza extract solution exhibit a completely different morphologies at the same operating condition. The C. xanthorrhiza extract were completely micronized or encapsulated in biopolymer microspheres. PVP was simply attached on the surface of the C. xanthorrhiza extract and resulting an improvement in the particle morphology. Smaller and better spherical particle were formed.

Effect of pressure
The micronization of C. xanthorrhiza/PVP using SAS method was carried out at various pressure in the range 8 MPa to 12 MPa. At a temperature of 40ºC, CO2 constant flow rate of 15 mL/min, C. xanthorrhiza/PVP ratio of 1:10, the feed concentration of 2 mg/mL, the morphology and particle distribution were examined. When the pressure raised from 8 MPa to 12 MPa, the morphology of particle was less agglomeration as shown in Figure 2. The formation of spherical particle was triggered by raising the operating pressure. When the pressure was increased, the density difference in CO2 and organic solvent (acetone/ethanol) were decreased and generate a higher mass transfer between them, faster supersaturation, then resulting smaller mean particle size with better spherical (Kim et al., 2007b). From SEM image, the mean particle size and the distribution of particles can be determine using Image J analysis software. As pressure increased from 8 MPa to 12 MPa, the mean particle size was slightly decrease from 191 nm to 178 nm. Figure 3 shown a change in pressure has an insignificant effect on particle size distribution.

FTIR analysis
FTIR analysis were conducted to determine the modification of the particle chemical structures. From Figure 4, the particle produces from C. xanthorrhiza extract represent a characteristic absorption band at 3328 cm -1 that related to hydroxyl O-H group. Another peak was appeared at 2922, 1514, and 1031 cm -1 which attribute to the stretching of C-H aliphatic, C=O stretching, and C-O-C stretching, respectively. The FTIR spectra of PVP represent a characteristic absorption band around 1649 cm -1 corresponded to C=O stretching band. Absorption band at 2952 and 3419 cm -1 were corresponded to C-H and O-H stretching. Meanwhile for particles of C. xanthorrhiza/PVP shows a similar peak with PVP alone at 1649 cm -1 with slightly difference in their intensity. This FTIR result indicates the presence of PVP gave an interaction in the C. xanthorrhiza/PVP particles.

Dissolution studies
Dissolution studies were conducted by dissolving an equal concentration of C. xanthorrhiza extract particles and C. xanthorrhiza/PVP particles into 10 mL ultra-pure water for 24 hr. The sample mixture was stirred and heated at 100 rpm and 37ºC. The dissolution rate was examined by comparing the absorbance of micronized particle in aqueous solution with the absorbance of C. xanthorrhiza extract particles in ethanol. The highly solubilities of C. xanthorrhiza extract particles in ethanol was define as complete dissolution of the particles. After 24 hr, the C. xanthorrhiza/PVP particles were approximately 71% dissolve in aqueous solution while the C. xanthorrhiza extract particles only 40% dissolved. The UV spectra scanning result from C. xanthorrhiza extract and C. xanthorrhiza/PVP particle in aqueous solution have a different peak absorbance. As shown in Figure 5, the peak absorbance was higher than without the addition of PVP. The significant increase in solubility of C. xanthorrhiza by PVP can be explained due to the formation of soluble complexes between water-soluble biopolymer and low-soluble active ingredients (Sethia and Squillante, 2004). The reduction of particle size in C. xanthorrhiza extract particles with PVP also contribute in

Conclusions
Supercritical antisolvent (SAS) micronization using acetone was successfully produce fine particle of C.
xanthorrhiza extract with addition of PVP. The variety in operating pressure induced an insignificant effect on the morphology and the particle size distribution. The mean size of particle slightly reduced in line with increased pressure. The FTIR analysis shows that C. xanthorrhiza extract exist inside the particle and coated by PVP. The addition of PVP produced a smaller and better spherical particle. From the dissolution study, micronized particle of C. xanthorrhiza/PVP has a higher ability to dissolve in aqueous solution and enhance bioavailability. Therefore, this experiment will improve the use of C. xanthorrhiza in the pharmaceutical and nutraceutical applications.