Preparation and characterization of betulin nanoparticles for oral hypoglycemic drug by antisolvent precipitation.

http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–13 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.881438

Preparation and characterization of betulin nanoparticles for oral hypoglycemic drug by antisolvent precipitation Xiuhua Zhao, Weiguo Wang, Yuangang Zu, Ying Zhang, Yong Li, Wei Sun, Chang Shan, and Yunlong Ge

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Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang, China

Abstract

Keywords

Betulin, a kind of small molecular compound, was reported that has hypoglycemic effect. Due to its low aqueous solubility and high permeability, betulin has low and variable oral bioavailability. In this work, betulin nanoparticles were thus prepared by antisolvent precipitation for accelerating dissolution of this kind of poorly water-soluble drugs. Ethanol was used as solvent and deionized water was used as antisolvent. The effects of various experimental parameters on the mean particle size (MPS) of nanocrystallization betulin were investigated. The MPS of betulin nanoparticles suspension basically remain unchanged when precipitation time was within 60 min and then increased from 304 nm to 505 nm later. However, the MPS of betulin nanoparticles suspension decreased with increased betulin solution concentration. On the contrary, the MPS of betulin nanoparticles suspension decreased along with the increase of temperature. Stirring intensity and the speed ratio of solvent adding into antisolvent had no significant influences on the MPS of betulin nanoparticles suspension. Betulin nanoparticles suspension with a MPS of approximately 110 nm was achieved under the optimal precipitation conditions. FTIR, Liquid chromatography coupled with tandem mass spectrometry (LC-MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to analyze the characteristic of betulin nanoparticles powder. These results show that betulin nanoparticles powder has the same chemical structure as raw drug, but a smaller size and lower crystallinity. The dissolution rate and solubility of betulin nanoparticles powder were separately 3.12 and 1.54 times of raw drug. The bioavailability of betulin nanoparticles powder increased 1.21 times compared with raw betulin. The result of in vivo evaluation on diabetic animals demonstrates that the betulin nanoparticles powder show an excellent hypoglycemic effect compared with raw betulin. In addition, the residual ethanol is less than the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human) limit for class 3 solvents of 5000 ppm or 0.5% for solvents.

Absorption enhancer, bioavailability, in vitro/in vivo correlations, nanotechnology, oral absorption

Introduction Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and glucose intolerance, either due to insulin deficiency or impaired effectiveness of insulin’s action, or to a combination of both. It is regarded as a non-curable but controllable disease (Deutschla¨nder et al., 2009). In our body, the only protein hormone that decreases glucose is insulin, which is, accordingly, commonly used in hospitals to treat diabetes mellitus. Insulin has been administered to type-2 diabetic patients through parenteral routes because it is easily metabolized in the gastrointestinal tract. Subcutaneous administration by self-injection is the most common route for insulin delivery. However, the pain associated with the injection and the risk of infection at the injection site cannot be completely avoided. Furthermore, the costs of treatment are too high for poor patients by insulin injection. Hence, a cheap oral drug has long been desired. Address for correspondence: Prof. Yuangang Zu, Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, Heilongjiang, China. Tel: +86-45182191517. Fax: +86-451-82102082. E-mail: [email protected]

History Received 19 November 2013 Revised 6 January 2014 Accepted 6 January 2014

Betulin, a kind of small molecular compound which is abundant in birch bark, could be a leading compound for the development of drugs for diabetes mellitus (Tang et al., 2011). It is a compound of pentacyclic triterpenes (Figure 1) that could be extracted from the bark of white birch which is widely distributed in northeast China. So, the raw material of betulin can be easily obtained. However, because of its limited bioavailability due to poor solubility in aqueous media, betulin failed to be utilized generally. Among many factors that influence oral drug absorption, solubility in water and physiological media as well as intestinal permeability are keys for determining the fraction of dose absorbed (Viçosa et al., 2012). Dissolution is considered as the rate limiting step to drug absorption for some poorly soluble drugs which may be increased by reducing the particle size to increase the surface area (Perrut et al., 2005) by many methods such as coating drug particles with hydrophilic surfactants to enhance wetting and solvation by intestinal fluids (Raghavan et al., 2001a, b; Chen et al., 2004), formation of solid dispersions (Leuner & Dressman, 2000; Vasconcelos et al., 2007), and the transformation of crystalline drug to amorphous state. Various drug formulation development strategies have been

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Figure 1. Chemical structure of betulin.

extensively studied to improve the dissolution rates of these drugs of poor solubility over the past few decades. One of the newest formulation strategies is to prepare nanomicroparticles of these drugs. Decrease in particle size increases the surface area to volume ratio which increases the solubility and dissolution rate of poorly water-soluble molecules, and hence bioavailability (Merisko-Liversidge & Liversidge, 2008). Top–down methods and down–top methods are two kinds of methods which are used generally to produce nanoparticles. The top–down methods start with larger solid particles, and break them down into nanomicroparticles mechanically (Keck & Mu¨ller, 2006). These methods are capable of producing fine particles and are reliable for industrial scale-up (Patravale & Kulkarni, 2004). However, breaking drug particles to nanoparticles with size below 100 nm is extremely difficult with these methods (Shah, 2006). Moreover, these methods are very time consuming and require significant energy. What’s more, these methods may induce contamination from milling media or homogenization chamber (Mu¨ller et al., 2001; Patravale & Kulkarni, 2004; Chow et al., 2007). The down– top approaches on the other hand begin with atomic level. These methods give better control over particle properties such as size, morphology and crystallinity as compared to top–down approaches (Chow et al., 2007). Rapid expansion of supercritical solution (RESS), gas antisolvent precipitation (GAS) and liquid antisolvent precipitation (LAS) are three kinds of down–top methods which are commonly used to produce nanoparticles. Supercritical fluid technique is believed to be an attractive method for size reduction, especially providing particles with narrow size distribution. However, it also has the limitations of low yield and high equipment cost (Viçosa et al., 2012). LAS is reported as a simple and cost-effective approach with scale-up potential (Hu et al., 2011). And LAS has been used to precipitate nano-microparticles of various active pharmaceutical ingredients (APIs) by many researchers. The basic principle of LAS is that the solvent in which the drug dissolved mixed with an antisolvent in which the drug is insoluble. The drug precipitates as a result of a consequence of the change of supersaturation caused by mixing the solution and the antisolvent. The key to produce ultrafine particles by antisolvent precipitation is to create conditions that favor very rapid particle formation and little or no particle growth. In this work, to our knowledge, we prepared betulin nanoparticles suspension using antisolvent precipitation process, which has not been reported in literature up to now.

Drug Deliv, Early Online: 1–13

Therefore, the purpose of this work is to prepare betulin nanoparticles powder for improving the dissolution rate and solubility. And the antisolvent precipitation process was optimized and the factors which influence the MPS of betulin nanoparticles suspension by single factor design were analyzed. Because of its simple and useful feature, single factor design method is widely used in many engineering fields. The effects of various experimental parameters on particle size and morphology such as concentration of betulin solution, the volume ratio of antisolvent to solvent, stirring intensity, precipitation time and temperature were investigated severally by a single factor design. And the betulin nanoparticles powder obtained were characterized using transmission electron microscope (TEM), Fourier-transform infrared (FT-IR) spectroscopy, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dissolution test and solvent residual. Bioavailability and in vivo evaluation on diabetic animals taking betulin nanoparticles powder were also performed.

Materials and methods Materials Betulin was obtained from Xi’an Xiaocao Botanical Development Co. Ltd. (Xi’an, China). Deionized water was purified by a Milli-Q water purification system from Millipore (Bedford, MA, USA). Tween-80 was purchased from Tianjin Bodi Chemical Co. Ltd. (Tianjin, China). Octanol was purchased from Tianjin Bodi Chemical Co. Ltd. Pepsase was obtained from Sinopharm Chemical Reagent Co. Ltd. (Jiangsu, China). Acetonitrile was of chromatographic grade. Other reagents were all of analytical grade. Octanol/water partition coefficient of betulin To determine the permeability of betulin, its octanol/water partition coefficient was tested in this part. 20 ml of three different concentrations of betulin octanol solution, i.e. 0.4, 2 and 4 mg/ml was added severally into three erlenmeyer flasks contained 180 ml deionized water. The erlenmeyer flasks were put into a constant temperature vibrator and oscillated for 72 h at 37 C under a rotation speed of 150 r/ min. Then 1 ml of sample was taken respectively from the water in the erlenmeyer flasks for betutin concentration analysis by HPLC method described in the section ‘‘HPLC conditions’’. The octanol/water partition coefficient was calculated with Equation (1) P ¼ ðC0 V0 Cw Vw Þ=Cw V0

ð1Þ

where C0 is the initial concentration of betulin octanol solution, Cw is the concentration of betulin in deionized water when the system reached equilibrium, V0 is the volume of octanol, Vw is the volume of deionized water. Preparation of betulin nanoparticles The betulin nanoparticles suspension was prepared by antisolvent precipitation technique. In brief, a certain amount of raw betulin was dissolved in 100 ml ethanol

Betulin nanoparticles for oral hypoglycemic drug

DOI: 10.3109/10717544.2014.881438

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Table 1. Summary of the experiments performed designed by the single factor method.


Experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Concentration of betulin solvent (mg/ml)

Temperature ( C)

Stirring intensity (r/min)

The volume ratio of antisolvent to solvent

Precipitation time (min)

The speed ratio solvent add into antisolvent (ml/min)

MPS (nm)

2 3 4 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

20 20 20 20 20 0 10 20 30 40 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 500 1000 1500 2000 2500 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000

3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 10 5 3.3 2.5 2 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3

12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 3 12 30 60 90 12 12 12 12 12

5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 0.5 1.0 2.0 3.5 5.5

358.8 338.4 304.2 287.6 154.4 717.1 638.5 398.6 380.2 292.5 320.1 326.1 343.5 343.6 347.3 117.8 225.2 154.4 398.8 401.7 269.0 285.0 322.1 306.0 507.0 346.0 334.0 317.0 306.0 257.0

solution. The obtained drug solution using the dual channel injection pump at a certain flow rate was then injected into certain multiple antisolvent aqueous solution with 0.5% (w/w) tween-80 under stirring with certain speed intensity. Bath temperature was set at certain pre-determined value. Precipitation of solid drug particles occurred immediately upon mixing. After a certain time, the suspension was centrifuged at 10 000rpm for 5 min. And then the precipitate was washed using deionized water via ultrasound. The washing process was repeated three times. After washing process the precipitate was dispersed in the deionized water. The milk-like suspension (batulin nanoparticles suspension) obtained was then lyophilized at 50 C for 48 h. The powder after lyophilization was named betulin nanoparticles powder. Every experiment was repeated at least three times. Optimization of antisolvent precipitation process The single factor method was used to investigate the effects of experimental parameters on antisolvent precipitation process. The summary of the experiments performed designed by the single factor method was shown in Table 1. In this method, only one factor was changed while the other factors remained constant. When concentration was the factor studied, the other factors were set under the following conditions: the precipitation temperature, stirring intensity, volume ratio of antisolvent to solvent, precipitation time, speed ratio of solvent adding into antisolvent were set at

20 C, 1000 r/min, 3.3, 12 min 5.5 ml/min, respectively. Laser diffraction particle size analyzer was used to detect the MPS of betulin nanoparticles suspension in order to optimize conditions of the antisolvent precipitation process. The same method as above was done to investigate the other factors. The other factors were varied. The concentration of betulin solvent was studied at 2.0, 3.0, 4.0, 5.0 and 6.0 mg/ml. The stirring speed was studied at 500, 1000, 1500, 2000 and 2500 r/min. The volume ratio antisolvent to solvent was studied at 2, 2.5, 3.3, 5 and 10. Precipitation time was studied at 3, 12, 30, 60 and 90 min. The speed ratio of solvent adding into antisolvent was studied at 0.5, 1.0, 2.0, 3.5 and 5.5 ml/min. Characterization of betulin nanoparticles Analysis of morphology and particle diameter Morphology of raw betulin particles was detected using SEM (Quanta 200, FEI, The Netherlands). The samples were prepared by direct deposition of the powders onto a carbon tape placed on the surface of an aluminium stub. Before analysis, the samples were coated with gold for 4 min using a sputter coater. The produced betulin nanoparticles were imaged using TEM H-7650 transmission electron microscope at 100 kV. The sample for TEM characterization was prepared by placing 8 ml of betulin nanoparticles suspension which was composed of betulin nanoparticles and deionized water on a carbon-coated copper grid and drying at room temperature.

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The diameter distribution of betulin nanoparticle before and after frozen-dried process was measured by dynamic light scattering analyzer (ZetaPALS, Brookharen Instrument Corporation, USA). Three microliters of betulin nanoparticles suspension was placed in a cuvette and measured at room temperature. A certain quality of betulin nanoparticles powder was redispersed in 3 ml artificial gastric juice. Then the artificial gastric juice was measured by dynamic light scattering analyzer.


Peak areas were used for obtaining quantitative data. The conditions of GC analysis of ethanol were as follows: oven temperature was maintained at 40 C for 12 min initially, and then raised at the rate of 10 C/min to 240 C which was maintained for 10 min at last. The injector and the detector temperatures were set 200 and 280 C, respectively. Nitrogen was used as carrier gas at a flow rate of 25 ml/min, and 5 ml samples were injected manually in the split mode with a split ratio 20:1. Hydrogen gas and air flow rate were 30 and 400 ml/min.


FTIR and LC-MS analysis The chemical components of the raw betulin and betulin nanoparticles powder were analyzed through FT-IR spectroscopy using IRAffinity-1 spectroscope (Shimadzu Corporation, Kyoto, Japan). Betulin samples were diluted with KBr mixing powder at 1% and pressed to self-supporting disks separately. The spectra, in transmission mode, were recorded at room temperature in the 4000–400 cm1 range at a resolution of 4 cm1. The raw betulin and betulin nanoparticles powder were dissolved separately in methanol. LC-MS/MS spectra were obtained by analyst 1.4 of API 3000 (AB SCIEX, Framingham, MA, USA). The mass spectrometer was operated in positive ion mode. XRD analysis Crystal forms of betulin were evaluated using an X-ray powder diffractometer (Philips, Xpert-Pro. PANalytical BV, Lelyweg 1 7602 Ea Almelo, The Netherlands). Ten milligrams samples of betulin particles, formed a weighted dispersion on a glass slide. Test conditions were as follows: the samples were irradiated using a Cu-Ka1 radiation at 30 mA and 40 kV. The samples were filled to the same depth inside the sample holder by leveling with a spatula. The XRD patterns were 2y. The scanning rate (5 /min) was constant for all XRD analysis. The scanning ranged from 5 to 60 with a step size of 0.02 . DSC and TG analysis DSC (TA instruments, model, DSC 204, Woodland, CA, USA) was conducted for betulin powders. 5.0 mg samples were weighed and analyzed from 25 to 330 C at a rate of 10 C/min. Thermal gravimetric of the betulin nanoparticles powder and raw betulin were analyzed by Thermo gravimetrical Analyzer (NETZSCH TG 209 F3, PerkinElmer, Waltham, MA, USA). Test conditions were as follows: approximately 3 mg samples were weighed in open aluminum pans. The experiment was performed with a heating rate of 10 C/min, nitrogen flow rate of 50 ml/min. The percentage weight loss of the samples was monitored from 30 to 500 C. GC analysis The residual ethanol in the betulin nanoparticles powder were analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA) with HP-5 5% phenyl methyl siloxane capillary column (30.0 m 320 mm 0.25 mm, nominal) equipped with an G1540N-210 FID detector.

HPLC conditions HPLC was carried out by a Waters chromatograph (Waters Co, USA). The chromatographic column was Diamonsil C18 reverse-phase column (250 mm 4.6 mm, 5 mm, China). The mobile phase consisted of 86:14 (v/v) mixtures of acetonitrile and water. The column temperature was at room temperature. The injection volume was 10 ml. The flow rate was 1.0 ml/min. The effluent was monitored at 210 nm. Drug purity test Drug purity test of raw betulin and betulin nanoparticles powder was investigated by HPLC. 10 mg of betulin powders were separately dissolved in 10 ml ethanol solution. The drug solution was shaken for 60 min by ultrasonic agitation at 20 C. And then samples were diluted 10 times with ethanol solution. The drug solution was centrifuged at 12 000rpm for 10 min. 10 ml of the supernatant was directly injected into the HPLC system. The analysis conditions were the same as described in the above section. The experiment was conducted in triplicate. The method for determination of tween 80 in betulin nanoparticles was established in this part. It was performed by HPLC–ELSD with Diamonsil C18 reverse-phase column (250 mm 4.6 mm, 5 mm, China). Acetonitrile and 20 mmol/l ammonium acetate (10:90) was used as mobile phase. The flow rate was 0.6 ml/min and the temperature was set at 30 C. The evaporated light scattering detector was adopted. The drift tube temperature was 100 C, and the nitrogen pressure was 55 psi. The reference substance was prepared by putting 10 mg tween 80 into deionized water. 20 mg betulin nanoparticles were weighed and redispersed in 1 ml deionized water and then processed by ultrasonic wave for 30 min. Then, the suspension was centrifuged at 12 000rpm for 10 min. 0.8 ml of supernate was finally used as sample for determination. The experiment was conducted in triplicate. Dissolution study Dissolution study of raw betulin and betulin nanoparticles powder was determined by HPLC. The paddle speed and solution temperature were set at 100 r/min and 37.0 ± 0.5 C, respectively. Artificial gastric juice with 0.5% (w/w) tween-80 was used as the dissolution medium. 10 mg of raw betulin and betulin nanoparticles powder was severally added to a vessel containing 200 ml dissolution medium. The samples (5 ml) were withdrawn each time at 5, 10, 15, 30, 45, 60, 80, 100, 120, 150 and 180 min, and filtered using a 0.22 mm filter. And then the same volume dissolution medium was supplemented. The filtrate was directly injected into HPLC system

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and assayed for betulin concentration by HPLC. The analysis conditions were the same as described in the section ‘‘HPLC conditions’’. The experiment was conducted in triplicate. Bioavailability study


Animals and treatment Six male Sprague–Dawley rats weighing 200 g to 250 g were used in the study. Rats were randomly divided into two groups of three. Animals were housed under standard conditions of temperature, humidity, and light with food and water provided ad libitum and allowed to acclimatize in the laboratory for at least 1 week prior to the experiment. Before administration, the animals were fasted overnight with free access of water. About 15 mg raw betulin and betulin nanoparticles powder were dispersed in 3 ml deionized water containing 0.5% (v/v) tween-80, respectively. For the bioavailability study, two groups of male rats (n ¼ 3) were administered with an oral dose (20 mg/kg by gavage). Blood samples by puncture of the orbital venous sinus were collected into heparinized tubes before and at 0.17, 0.34, 0.5, 1, 3, 4, 6, 8, 10, 12, 24 after oral administration. The samples were immediately centrifuged at 3000 rpm for 5 min and aliquots of plasma were stored at 40 C until additional extraction and analysis. Calibration standards and quality control samples A standard stock solution of betulin (0.618 mg/ml) was prepared in ethanol and then diluted to get eight working solutions containing betulin with concentration ranging from 2 to 100 mg/ml. All the solutions were stored at 4 C. Calibration standards were prepared daily by spiking 10 ml of standard working solutions into 50 ml of blank rat plasma. Final concentration of betulin in calibration samples were 0.33, 0.66, 1, 2, 4, 8.3, 12 and 16.67 mg/ml. Preparation of plasma sample Frozen samples were thawed at room temperature and treated as follows. Each 200 ml aliquot of plasma sample was mixed with 1000 ml of ethanol and vortexed for 3 min. Followed by centrifugation at 3000 rpm for 10 min, 800 ml aliquot of supernatant was transferred and the residue was mixed with 800 ml of ethanol for an another same extraction procedure. Finally, both of the supernatants were combined and evaporated to dryness at 40 C under a gentle stream of nitrogen. The dried residue was then reconstituted in 800 ml ethanol. After being vortexed for 1 min, the content was centrifuged at 12 000rpm for 10 min. The supernatant was then transferred to 2 ml glass vials and an aliquot of 10 ml was injected for the HPLC analysis. The analysis conditions were same as described in the section ‘‘HPLC conditions’’. The oral bioavailability (F) of betulin nanoparticles was calculated by comparing the corresponding values of AUC from the two groups. F¼

AUCba 100% AUCrb

where AUCba is the area under the plasma concentration–time curve of betulin nanoparticles AUCrb is the area under the plasma concentration–time curve of raw betulin.


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In vivo evaluation on diabetic animals Male Sprague–Dawley rats weighing 200 g to 250 g were used in the study. The streptozotocin (STZ) -induced diabetic rats were prepared by administering injections of STZ (60 mg/kg i.v.) in a citrate buffer of pH 4.5. The rats with blood glucose (GLU) level higher than 16.7 mmol/l were selected as the diabetic model. These diabetic rats were fasted for 12 h prior to each blood collection. The following formulations were intragastrically administered to rats (four rats per group) by a single oral gavage: (1) distilled water (control group), (2) raw betulin in distilled water, (3) betulin nanoparticle powder in distilled water. The blood samples were collected from the eye-ground venous plexus at predetermined time points (after 0.5, 1.5, 3, 4.5 and 6 h). The blood GLU concentration was measured using blood GLU assay kits (Glucose GODPAD kit, Biosino Bio-Technology and Science Inc., Beijing, China). These plasma GLU levels were plotted against time after administration.

Results and discussion Result of octanol/water partition coefficient The average value of the log P was calculated to be 2.28 (in Table 2). The result illustrates that betulin belongs to the compound of high permeability. The solubility of betulin in water detected in this part by HPLC analysis was 20.13 mg/ml was quite low. The result shown in Table 2 demonstrates that betulin belongs to the compound characterized with low solubility and high permeability. So, it is possible to increase the bioavailability of betulin by preparing nanocrystals that can improve dissolution rate. Effect of operating conditions on the MPS of betulin nanoparticles and optimization study Stirring intensity, precipitation time and the speed ratio solvent adding into antisolvent Figure 2(a and c) as a result of single factor experimental design demonstrates that stirring intensity ranged from 500 to 2500 r/min and the speed ratio solvent adding into antisolvent ranged from 0.5 to 5.5 ml/min make no difference in the preparation of betulin nanoparticles suspension by antisolvent precipitation. The result illustrates that the crystallization rate keeps dynamic balance with the dissolution rate when stirring intensity ranged from 500 to 2500 r/min and the speed ratio solvent adding into antisolvent ranged from 0.5 to 5.5 ml/min. The MPS of betulin nanoparticles suspension had not significant changed when the precipitation time was within 60 min. However, the MPS of betulin nanoparticles suspension increased from 304 to 505 nm when the precipitation time increased from 60 to 90 min. The growth rate of betulin particles is shown in Figure 2(b). Due to the Ostwald ripening effect the MPS of betulin nanoparticle suspension increased obviously after 90 min preparation. Concentration of betulin solution Precipitation experiments were severally carried at a concentration of betulin solution (C) of 2, 3, 4, 5 and 6 mg/ml. At the moment, the temperature, stirring intensity, the volume ratio

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Table 2. Summary of the experiments performed of octanol/water partition coefficient of betulin. C0 (mg/ml)

V0 (ml)

Vw (ml)

P

Log P

Average of log P

Solubility of betulin in water (mg/ml)

2.29 10.56 18.34

20 20 20

180 180 180

175 189 209

2.24 2.27 2.32

2.28

20.13


400 2000 4000

Cw (mg/ml)

Figure 2. The effect of each parameter on the MPS of betulin nanoparticles suspension: (a) stirring intensity; (b) precipitation time; (c) the speed ratio solvent adding into antisolvent; (d) concentration of betulin solution; (e) temperature; (f) the volume ratio of antisolvent to solvent.

of antisolvent to solvent, precipitation time and the speed ratio solvent adding into antisolvent were fixed separately at 6 mg/ml, 1000 r/min, 3.3, 12 min and 5.5 ml/min, respectively. Figure 2(d) shows the MPS of betulin nanoparticles suspension changed along with concentration of betulin solvent. Betulin nanoparticles suspension with MPS of 358.8, 338.4, 304.2, 287.6 and 175.4 nm were obtained at a concentration

of betulin solution of 2, 3, 4, 5 and 6 mg/ml, respectively. In Figure 2(d), the MPS of betulin nanoparticle suspension decreases with its increasing concentration. This can be explained by that high concentration can lead to high supersaturation when solvent mixed with antisolvent. According to Equation (2), the nucleating in precipitation systems with higher supersaturation was reduced and the

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crystal number was increased correspondingly. Therefore the particle size decreased. ! 16sl3 v2 0 B ¼ Ahom exp ð2Þ 3k3 T 3 ðlnð1 þ sÞÞ2 where B0 is the nucleation rate, Ahom is the pre-exponential factor, sl is the interfacial tension at solid–liquid interface, v is the molar volume and T is the temperature.


Precipitation temperature Precipitation experiments were carried at precipitation temperature (T) of 0, 10, 20, 30 and 40 C when the concentration of betulin solvent, stirring intensity, the volume ratio of antisolvent to solvent, precipitation time and the speed ratio solvent adding into antisolvent at 6 mg/ml, 1000 r/min, 3.3, 12 min and 5.5 ml/min, respectively. Figure 2(e) shows the MPS of betulin nanoparticles suspension changed along with temperature. The MPS of betulin nanoparticles suspension obtained at T of 0, 10, 20, 30 and 40 C were 717.1, 638.3, 398.6, 380.2 and 292.5 nm, respectively. It illustrates that the MPS of precipitate is smaller when temperature is higher. The temperature influenced the size in two ways. For one thing the high temperature can lead to the increase of solubility and further the decrease of supersaturation. Thus, the nucleating was reduced and number of crystals was increased correspondingly. Therefore, the particle size decreased. However, for the other thing, Equation (2) indicated that higher temperature can lead to higher nucleating and number of crystal number was increased correspondingly. Thus, the particle size decreased. In this work, the supersaturation ranged from 41.56 to 830.17 when temperature ranged from 0 to 40 C. According to Equation (2), the variation of crystal number brought on by temperature is greater than supersaturation. Therefore, the MPS of betulin nanoparticles suspension decreased while precipitation temperature increased.

Figure 3. The relationship between supersaturation and MPS.

supersaturation (x) can be fitted as Y ¼ 1.5763x + 452.41 (R2 ¼ 0.6533). Higher supersaturation results in smaller size of precipitate, and basically in accordance with the linear relation. Optimization study In this study, we choose a volume ratio of antisolvent to solvent of 3.3 in the single factor design in consideration of yield as an important factor. So the minimum MPS of betulin nanoparticles suspension was obtained when the concentration of betulin solution, the volume ratio of antisolvent to solvent, precipitation temperature, stirring intensity, precipitation time and the speed ratio solvent adding into antisolvent were 6.0 mg/ml, 3.3, 40 C, 1000 r/min, 12 min and 5.5 min, respectively. Through a confirmatory test, betulin nanoparticles suspension with a MPS of about 110 nm was obtained. The subsequent characteristics of the optimum sample were obtained under this condition. Characterization of betulin nanoparticles powder

The volume ratio of antisolvent to solvent Precipitation experiments were carried separately at a volume ratio of antisolvent to solvent of 10, 5, 3.3, 2.5 and 2. The concentrations of betulin solution were at 6.6, 3.6, 2.6, 2.1 and 1.8 mg/ml, respectively. Other conditions such as precipitation temperature, stirring intensity, precipitation time and the speed ratio solvent adding into antisolvent were kept constant at 20 C, 1000 r/min, 12 min and 5.5 ml/min, respectively. In this section, the concentration of betulin solution was controlled to keep the same concentration of betulin in the mixture of solvent and antisolvent. Figure 2(f) shows the MPS of betulin nanoparticles suspension changed along with the volume ratio of antisolvent to solvent. HPLC was used to investigate the solubility of betulin in the mixture of solvent and antisolvent. As the volume ratio of antisolvent to solvent decreased from 10 to 2, the solubility of betulin increased from 7.22 to 102.52 mg/ml correspondingly. Therefore, the decreased supersaturation brought on the increased MPS from 117.8 nm to 401.7 nm (Figure 2f). The curve fitting in Figure 3 shows the relationship between supersaturation and MPS. A regression equation between MPS (Y) and

Morphology and particle diameter of betulin Figure 4(a) shows a SEM picture of unprocessed betulin. It can be seen that raw betulin particles are cuboid structure crystals with length varying from 1 to 60 mm. The TEM picture of betulin nanoparticles powder shows it has a nearly spherical shape (Figure 4b). Figure 4(c and d) show the diameter distribution of betulin nanoparticles suspension obtained by antisolvent precipitation and redispersion in artificial gastric juice of betulin nanoparticles powder. As seen from Figure 4(c and d), the MPS of the betulin nanoparticle increased from 110 to 563 nm after lyophilization owing to the conglomeration of particles during the lyophilization. It can be seen that the betulin nanoparticles suspension obtained by the antisolvent precipitation process under optimum condition have a mean particle size of 110 nm. FTIR and LC-MS analysis We performed some analysis on raw betulin and betulin nanoparticles powder to obtain information on the chemical structure of the material after antisolvent precipitation


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Figure 4. Morphology and particle diameter of betulin, (a) SEM images of raw betulin particles; (b) TEM images of betulin nanoparticles; (c) the size distribution and mean diameter of betulin nanoparticles suspension measured by laser light scattering technique; (d) the size distribution and mean diameter of betulin nanoparticles suspension (redispersion in artificial gastric juice of betulin nanoparticle powder) measured by laser light scattering technique.

Figure 6 shows LC-MS spectra of raw betulin and betulin nanoparticles powder. It can be seen that no modification occurred in molecular weight. The protonated molecule was detected at m/z 443.7 for [M + H]+. This mass agrees with the published structure C30H50O2 of betulin. The two forms of betulin exhibited the same molecular weight (442.7). Combining the results of FTIR and LC-MS, we can determine that the chemical structure has not changed during the antisolvent precipitation process XRD analysis

Figure 5. FTIR spectrum of raw betulin and betulin nanoparticles powder.

process. Both of raw betulin and betulin nanoparticles powder present the same FTIR spectrum as shown in Figure 5, which does not demonstrate any differences. The assignments of bands are as follows: 3383.20 cm1 (Associate O–H stretching vibration) and 1643.39 cm1 (C ¼ C stretching vibration).

In order to further investigate the occurrence of eventual structural changes at the crystal level, XRD analyses were performed. Figure 7(a) shows the XRD results for raw betulin and betulin nanoparticles powder. Several distinct peaks of raw betulin at the diffraction angles of 2y ¼ 3.78 , 5.23 , 5.97 , 7.42 , 9.04 , 10.40 , 11.63 , 12.37 , 13.83 , 14.31 , 15.01 , 16.46 , 18.66 , 19.31 , 24.14 , 25.81 , 31.03 , 37.21 and 43.71 reveal that the drug is present as a crystalline form. On the contrary, betulin nanoparticles powder show only several weak peaks at 2y ¼ 3.78 , 7.33 , 9.04 , 10.36 , 11.59 , 14.31 , 15.01 and 18.78 compared to raw betulin. The disappearance of diffraction peak is attributed to the reduced particle. Crystallinity was calculated on the basis of peak area at 2y ¼ 14.31 according to Equation (3) (Majerik et al.,



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Figure 6. The LC-MS spectra of (a) raw betulin and (b) betulin nanoparticles powder.

2007), and the results show that betulin nanoparticles powder has an 42% crystallinity in comparison with raw betulin. Crystallinity ¼

Peak area ðbetulin nanaoparticleÞ 100% Peak area ðraw betulinÞ ð3Þ

This fact suggests that betulin particles after the antisolvent precipitation process has smaller particle size and is partially amorphous. The reduced crystallinity would lead to a higher surface disorder, resulting in higher saturation solubility and enhanced dissolution rates than crystalline materials (Kim et al., 2012). Figure 7 also shows that the betulin nanoparticles could be polymorphy for the distinct peaks which are different from raw betulin not only in the intensity of diffraction peak but also in diffraction angles. DSC analysis DSC was performed to further confirm the physical state of the betulin nanoparticles. As seen in Figure 7(b), thermogram of the raw betulin showed a narrow endothermic peak around 250 C, corresponding to its melting point which implied its crystalline form. However, betulin nanoparticles powder presented two endothermic melting peaks at 203 and 223 C, respectively. The result shows that the melting point of betulin

nanoparticles powder is lower than raw betulin. These facts imply betulin nanoparticles are less crystalline and present polymorphism, which is consistent with the results of XRD. TG analysis The TG results, used to examine the thermal property of betulin nanoparticles powder, are shown in Figure 7(c). Raw betulin was observed to barely lose weight from 30 to 240 C. And then its weight decreased quickly from about 240 C. Betulin nanoparticles powder lost weight immediately from about 223 C. This fact demonstrates the melting point of raw betulin is higher than betulin nanoparticles powder which is consistent with the DSC. Moreover, betulin nanoparticles powder had higher weight loss percentage than the raw betulin at the same temperature before carbonization. This result was consistent with the previous DSC observations since the betulin nanoparticles powder had smaller particle size and thus had higher specific surface energy, which subsequently led to an easier vaporization and earlier decomposition. Solvent residue analysis The problem of solvent residues is also under consideration in pharmaceutical products. In this work, betulin

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Figure 8. Gas chromatograms of samples: (a) ethanol standard solution and (b) betulin nanoparticles powder.

purity using the HPLC method, the product has been purified from 95.8 to 99.8% during the recrystallization. Enhancement of purity may lead to the color change of betulin. The change of crystal structure present in DSC and XRD maybe is another reason brought on the change of color. Figure 10 shows the determination result of tween 80. According to the regression equation, the concentration of tween 80 in sample (a) was approximately 0.0216 mg/ml and the reference substance (b) was 10 mg/ml. The content of tween 80 was calculated to be 0.108%. The content was very low. Therefore, tween 80 in betulin nanoparticles had no effect on the dissolution and bioavailability test. Dissolution results

Figure 7. Results of (a) XRD, (b) DSC and (c) TG of the raw betulin and betulin nanoparticles powder.

nanoparticles powder was prepared by antisolvent precipitation process using deionized water and ethanol with low toxicity. Figure 8(a and b) shows the results of ethanol residue using the GC method. From the chromatograms of ethanol standard solution, a regression equation between peak area (Y) and ethanol concentration (x) can be fitted as Y ¼ 371.94x + 91.803 (R2 ¼ 0.9997). The linear range of ethanol was 0.0015625–0.1 mg/ml. According to the regression equation, the residual ethanol content in betulin nanoparticles is 2085 ppm. Since the ICH limit for ethanol in class 3 solvents is 5000 ppm or 0.5%, the betulin nanoparticles powder met ICH requirements and is suitable for pharmaceutical use.

The dissolution test is widely used for formulation optimization and quality control in manufacturing. The dissolution profiles of the raw betulin and betulin nanoparticles are shown in Figure 11. The result demonstrates that the betulin nanoparticles powder showed a more rapid dissolution rates and higher solubility than the raw betulin. The maximum solubility (approximately 4.18 mg/ml) was observed in betulin nanoparticles powder at 240 min. However, this of raw betulin was only 2.87 mg/ml. According to the Noyes–Whitney equation, the dissolution rate can be proportionally increased by reducing the particle size to increase the interfacial surface area (Mosharraf & Nystro¨m, 2003). In addition, the solubility of betulin nanoparticles powder can be as well accordingly increased by reducing the particle size based on the Oswald–Freundlich equation (Grau et al., 2000). Therefore, the partially amorphous and smaller drug particles have a higher dissolution rate and better bioavailability than crystals.

Comparison of drug appearance and purity Figure 9 shows the appearance photos of raw betulin and betulin nanoparticles powder. As shown in Figure 9, betulin nanoparticles powder obtained by antisolvent precipitation process is white fine powder, while raw betulin is yellow powder. The result can be explained by both purity and crystal structure of betulin nanoparticles powder. Analyzed by the

Bioavailability analysis The result of bioavailability is shown in Figure 12. It can be seen that the betulin concentration in rat plasma of betulin nanoparticles powder group is always higher than that of the raw betulin group. The betulin concentration in rat plasma of the betulin nanoparticles powder and the raw betulin group


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Figure 9. Images of (a) betulin nanoparticles powder and (b) raw betulin.

Figure 10. The determination result of tween 80: (a) sample and (b) reference substance (10 mg/ml).

reached the maximum of 6.5 and 3.6 mg/ml after 4 and 6 h of taking drugs, respectively. The oral relative bioavailability (F) of betulin nanoparticles powder was calculated by comparing the corresponding AUC values of the two groups, and the results demonstrate that oral administration of betulin nanoparticules powder leads to 121% increase in bioavailability. The significant enhancement of oral bioavailability is also in accordance with the result of the dissolution test and octanol/water partition coefficient of betulin. Oral administration of betulin nanoparticles in diabetic rats Figure 13 shows the variation of glycemia in diabetic rats after oral administration of betulin-containing formulation. The blood GLU of the blank control group maintained the level from 20.44 mmol/l to 26.67 mmol/l after 6 h. In the first 0.5 h, the GLU level of rates in the raw betulin group and the betulin nanoparticles powder group increased from 15.94 mmol/l to 19.98 mmol/l and 14.81 mmol/l to 17.10 mmol/l, respectively. From 0.5 h to 1.5 h the GLU level of rates in the raw betulin group did not significantly drop and then from 1.5 h to 6 h basically maintain invariable at a mean GLU of 15.49 mmol/l. However, the GLU level of the betulin nanoparticles powder group keeps decreasing from 17.10 mmol/l to 10.23 mmol/l from 0.5 h to 6 h. The result illustrates that the betulin has anti-hypoglycemic effect in

diabetic mice. Insulin is a kind of traditional drug which is used to treat diabetes. However, the defect of insulin is that it is not suitable for oral administration. In this part, the Ins-SiO2-HP55 (insulin-loaded silica coating HP55) group (in our previous work) (Zhao et al., 2013) in Figure 12 shows the hypoglycemic effect after oral administration. The GLU change rule of the Ins-SiO2-HP55 group and the betulin nanoparticles powder group are nearly identical. The growth rate of GLU of betulin nanoparticles powder group was significantly less than that of the Ins-SiO2-HP55 group from 0 to 0.5 h. The result indicates that betulin can effectively slow the growth rate of GLU in diabetic mice. The GLU level of rats in betulin nanoparticles powder group was always lower than that of the raw betulin group when they reach maximum after 0.5 h and minimum after 6 h. This result was due to the high dissolution rate and high solubility of the betulin nanoparticles powder compared with raw betulin, which was consistent with dissolution test and bioavailability test. The analysis shows that betulin nanoparticles powder can effectively slow the growth rate of GLU compared with InsSiO2-HP55 and decreased the GLU in comparison with raw betulin in diabetic mice. Betulin is a compound that could be extracted from the bark of white birch, which is widely distributed in northeast China. So, betulin may have a potential application foreground on oral antidiabetic therapy due to its abundant source and convenience in oral administration.

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Figure 13. The result of in vivo evaluation on diabetic animals. Figure 11. Dissolution profiles of the raw betulin and betulin nanoparticles powder.

In addition, solvent residual of the betulin nanoparticles powder are suitable for pharmaceutical use. The result of in vivo evaluation on diabetic animals demonstrates that the betulin nanoparticles powder can effectively slow the growth rate of GLU compared with Ins-SiO2-HP55 and decreased the GLU in comparison with raw betulin in diabetic mice.

Acknowledgements The authors are grateful for the precious comments and careful corrections made by anonymous reviewers.

Declaration of interest

Figure 12. The bioavailability result of betulin nanoparticles powder and raw betulin.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors would like to acknowledge the financial support from the Fundamental Research Funds for the Central Universities (DL13CB08), and the National Natural Science Foundation of China (No. 21203018).

Conclusions In this study, antisolvent precipitation technique was successfully applied to prepare betulin nanoparticles powder for the enhancement of solubility and dissolution rate. In the process, ethanol as solvent, water was found to be the suitable antisolvent, respectively. The minimum MPS of betulin nanoparticles suspension was obtained when the concentration of betulin solution, the volume ratio of antisolvent to solvent, precipitation temperature, stirring intensity, precipitation time and the speed ratio solvent adding into antisolvent were 6.0 mg/ml, 3.3, 40 C, 1000 r/min, 12 min and 5.5 min, respectively. Under the above conditions, betulin nanoparticles suspension was obtained with a MPS of 110 nm. According to FTIR, XRD, DSC and TG data, the betulin nanoparticles powder had the same chemical structure as raw drug, but its crystallinity was reduced. The dissolution rate of betulin nanoparticles powder was 3.12 times faster than raw drug. Furthermore, maximum solubility was 1.54 times larger than raw drug. The oral bioavailability of the betulin nanoparticles powder was 2.21 times of raw betulin.

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