Journal of Nanomaterials

Journal of Nanomaterials / 2015 / Article

Research Article | Open Access

Volume 2015 |Article ID 439540 | 12 pages | https://doi.org/10.1155/2015/439540

Preparation, Characterization, and Dissolution Rate In Vitro Evaluation of Total Panax Notoginsenoside Nanoparticles, Typical Multicomponent Extracts from Traditional Chinese Medicine, Using Supercritical Antisolvent Process

Academic Editor: Zafar Iqbal
Received04 Dec 2014
Revised12 Mar 2015
Accepted18 Mar 2015
Published14 Apr 2015

Abstract

Total Panax notoginsenosides nanoparticles, typical multicomponent extracts from traditional Chinese medicine, were prepared with a supercritical antisolvent (SAS) process using ethanol as solvent and carbon dioxide as antisolvent. The optimum conditions were determined to be as follows: TPNS solution concentration of 2.5 mg/mL, TPNS solution flow rate of 6.6 mL/min, precipitation temperature of 40°C, and precipitation pressure of 20 MPa. Under the optimum conditions, TPNS nanoparticles with a MPS of 141.5 ± 18.2 nm and total saponins amounts (TSA) of 78.9% were obtained. The TPNS nanoparticles obtained were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimeters (DSC), and high performance liquid chromatography (HPLC). The results showed that the chemical and crystal structure of the obtained TPNS nanoparticles has not changed. Dissolution in vitro studies showed that the solubility and dissolution rate of notoginsenosides R1 and ginsenoside Rb1 in TPNS nanoparticles are higher than these in raw TPNS, with no obvious difference in Rg1. These results suggest that TPNS nanoparticles can be helpful to the improvement of its bioavailability for the treatment of cardiovascular diseases.

1. Introduction

San-Chi, the root of Panax notoginseng (Burk.) Li et al. [1], is well known as an important component in various prescriptions in the traditional Chinese medicine that has been used for thousands of years. Total Panax notoginsenosides (TPNS) are the main active ingredients in San-Chi, which has been used to treat atherosclerosis. The pharmacological studies have proved that the active constituents of the TPNS are ginsenoside Rg1 (Figure 1(a)), ginsenoside Rb1 (Figure 1(b)), and notoginsenosides R1 (Figure 1(c)) [2, 3]. In previous pharmacological studies, TPNS showed various activities, for example, lowering blood cholesterol [4, 5], diminishing inflammation, relieving body pains [6, 7], antioxidant and hepatoprotective [8], immunomodulation [9, 10], and anticancer [11, 12] and antiatherosclerosis effects [13]. At present, TPNS has been widely used for the treatment of cardiovascular diseases in the view of their hemostatic and cardiovascular effects [14]. However, it has been reported that TPNS is poorly absorbed, which means that the oral absorption of ginsenosides Rb1 and Rg1 and of notoginsenosides R1 is poor [1519]. A self-micelle and lipid-based formulation was designed to enhance bioavailability [15].

Nanopowder technology by supercritical method is one of the ways to improve drug solubility, dissolution rate, and bioavailability. It is suitable to produce the nanocrystals of biological active substances because of low temperature and inertia [20]. Until now, many nanocrystals of materials including micromolecule compounds, macromolecule, and polymers have been successfully obtained with the help of inert gases, for example, griseofulvin, paclitaxel, insulin poly-(L-lactide), ethylcellulose, and hydroxypropyl-β-cyclodextrin [2126]. The nanopowder produced by supercritical antisolvent process (SAS) has high flowability, uniform morphology, and small and uniform particle size [27]. The use of inert gas, especially, makes the products uneasy to oxidize and the condition of low temperature makes SAS suitable to nanonize bioactive substances. However, to the best of our knowledge, there are very few reports on a nanocrystallization of multiple constituent traditional Chinese medicines using SAS method [2830].

Therefore, the aims of this work are to study the feasibility of TPNS nanocrystallization from ethanol by SAS process, to optimize SAS nanocrystallization process, and to evaluate powder characterization and dissolution rate and solubility in vitro. Moreover, TPNS nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimeters (DSC), and high performance liquid chromatography (HPLC) analyses.

2. Experimental

2.1. Materials

TPNS (purity ≥ 60%) was obtained from Yunnan Yunke Pharmaceuticals Co., Ltd. (Yunnan, China). The standard substances such as ginsenoside Rg1 (purity > 98.5%), ginsenoside Rb1 (purity > 98.5%), and notoginsenosides R1 (purity > 98.5%) were bought from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). High purity CO2 (99.99% purity) was purchased from Liming Gas Company of Harbin (Heilongjiang, China). Ethanol (99.5% purity) was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Trypsin (≥2,500 USP units/mg) was purchased from Sigma-Aldrich Company (Shanghai, China). Chromatography pure acetonitrile was purchased from Beijing J&K Chemical Technology Co. Ltd. (Beijing, China).

2.2. Apparatus and Procedure

TPNS nanoparticles powders were prepared by SAS process apparatus, which consists of a precipitation chamber and a gas-liquid separation chamber (Figure 2). The CO2 is cooled with a cooler (4) before being compressed by a liquid pump (8). The pressure is controlled by a backpressure-regulating valve. The CO2 is then preheated in a heat exchanger (13), after which it enters the precipitation chamber (18). Simultaneously, the TPNS ethanol solution is pumped, heated, and fed to the 1000 mL precipitation chamber through a stainless steel nozzle of 150 μm (16). A stainless steel frit vessel (17) of 200 nm is put into the precipitation chamber to collect the TPNS nanoparticles and to let the SC-CO2/ethanol mixture pass through. The flow rate of the mixture that leaves the precipitator is controlled by a valve (21) located between the precipitation chamber and the gas-liquid separation chamber (22). Further and detailed information of the apparatus can be referred to related literatures [31, 32]. The liquid pump (6) is stopped when the fixed quantity of TPNS ethanol solution is injected. Delivery of supercritical CO2 is continued for 30 minutes to wash the frit vessel of the residual content of liquid solubilized in the supercritical antisolvent. Finally, the samples of TPNS nanoparticles are taken from the frit vessel for further characterization analysis.

2.3. Orthogonal Design of Experiment

The pretest results showed that the main factors affecting the MPS and TSA of TPNS nanoparticles were TPNS concentration, precipitation temperature, precipitation pressure, and the flow rate of TPNS solution. So an orthogonal design OA16 (4)5 with 4 factors and 4 levels was selected for SAS process optimization. The horizontal gradients of each factor are as follows: TPNS concentrations of 2.5, 7.5, 12.5, and 17.5 mg/mL, precipitation temperatures 40, 50, 60, and 70°C, precipitation pressures 10, 15, 20, and 25 MPa, and the flow rates 3.3, 6.6, 9.9, and 13.2 mL/min. The levels and arranging of each factor were shown in Tables 1 and 2. The MPS and TSA of TPNS nanoparticles are the response variables. The CO2 flow rate is 8.5 Kg/h. The nozzle diameter is 150 μm. These two parameters were fixed in the experiments.


Factor
Concentration of TPNS solution (mg/mL)Precipitation temperature (°C)Precipitation pressure (MPa)TPNS solution flow rate (mL/min)

Levels
 12.540103.3
 27.550156.6
 312.560209.9
 417.5702513.2


Trial numbers
Concentration of TPNS solution (mg/mL)

Precipitation temperature (°C)

Precipitation pressure (MPa)

TPNS solution flow rate (mL/min)
CO2 flow rate
(kg/h)
MPS (nm) ± SD
()
TSA (R1, Rb1, Rg1) (%)

111110.998.5263.3 ± 28.266.43 (6.89, 35.03, 24.51)
212220.978.5185.0 ± 16.157.63 (6.55, 23.88, 27.21)
313330.968.5155.9 ± 14.496.04 (9.48, 45.61, 40.95)
414440.958.5238.8 ± 26.289.49 (9.86, 41.27, 38.36)
521230.968.5229.5 ± 22.566.54 (6.13, 29.12, 31.29)
622140.958.5271.2 ± 32.851.29 (3.95, 29.08, 18.26)
723410.998.5239.7 ± 25.056.16 (5.55, 27.94, 22.67)
824320.978.5265.0 ± 25.936.97 (2.65, 18.64, 15.68)
931340.958.5214.5 ± 23.166.02 (6.44, 34.12, 25.46)
1032430.968.5258.3 ± 24.771.78 (7.18, 37.47, 27.13)
1133120.978.5354.1 ± 38.466.91 (6.55, 33.94, 26.42)
1234210.998.5320.4 ± 21.571.81 (6.92, 36.85, 28.04)
1341420.978.5218.7 ± 22.665.60 (6.92, 38.50, 20.18)
1442310.998.5228.3 ± 20.557.02 (5.57, 29.12, 22.33)
1543240.958.5325.7 ± 28.159.48 (6.59, 24.93, 27.96)
1644130.968.5396.1 ± 40.045.99 (4.92, 22.11, 18.96)

MPS
210.75 ± 21.2231.50 ± 24.1321.18 ± 34.9262.93 ± 23.8
251.35 ± 26.6235.70 ± 23.5265.15 ± 22.1255.70 ± 25.8
286.83 ± 26.9268.85 ± 26.5215.93 ± 21.0259.95 ± 25.4
292.20 ± 27.8305.08 ± 28.4238.88 ± 24.6262.55 ± 27.6
81.4573.58105.257.23

Optimal level

TSA
77.39866.14857.65562.855
52.74059.43063.86556.777
69.13069.64764.01270.088
57.02361.06570.75766.570
24.65810.21713.10213.311

= (mean particle size at )/4, the mean values of mean particle size for a certain factor at each level with standard deviation.
.
is the mole fraction of the antisolvent CO2 in the mixture.
2.4. Powder Characterization
2.4.1. Scanning Electronic Microscopy (SEM)

The morphology of samples was determined by using SEM (Quanta 200, FEI, 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 (JFC-1100E, JEOL, Japan).

2.4.2. Dynamic Light Scattering (DLS)

The MPS of TPNS powder samples were measured with dynamic light scattering equipment (ZetaPALS, Brookhaven Instruments) with a He-Ne laser (632.8 nm, 35 mW) as light source. TPNS nanoparticles were suspended in acetone and special cares were taken to eliminate dust and to avoid the aggregation of particles. The acetone was presaturated with TPNS (5.2 μg/mL) to avoid dissolution of nanonize particles. All the measurements were repeated three times.

2.4.3. Fourier Transform Infrared (FTIR)

The unprocessed and processed TPNS samples were diluted with KBr mixing powder at 1% and pressed to self-supporting disks separately. The FTIR spectrum was obtained by IRAffinity-1 (SHIMADZU, Japan) and recorded in the wave number range of 4000–500 cm−1 at a resolution of 4 cm−1.

2.4.4. Differential Scanning Calorimetry (DSC)

Thermal analysis was carried out using DSC (TA instruments, model DSC 204) for processed and unprocessed TPNS. Analysis was performed for 5.0 mg samples at a temperature heating rate of 5°C/min and a temperature range of 30–300°C.

2.4.5. High Performance Liquid Chromatography (HPLC)

Chromatographic analyses were performed on a waters HPLC system consisting of a pump (Model 1525), an autosampler (Model 717 plus), and UV detector (Waters 2487 Dual λ Absorbance Detector). The C18 column (Diamonsil, 5 μm, 4.6 mm × 250 mm, Dikma technologies) was used at 25°C. The mobile phase consisted of acetonitrile delivered by pump A and water delivered by pump B at 1.0 mL/min. Specific procedures for gradient elution can be seen in Table 5. The injection volume was 10 μL. The signal was monitored at 203 nm. The data was expressed as a mean value ± S.D ().

2.5. Dissolution Studies In Vitro

In vitro drug dissolution studies were carried out in simulated intestinal fluid (SIF, pH 7.5) containing 1% trypsase. The 5 g trypsase was added to 500 mL dissolution medium (30.085 g Na2HPO4·12H2O, 2.496 g Na2HPO4·2H2O in 500 mL distilled water, pH 7.5) to form SIF. The unprocessed TPNS (1.04 g) and processed TPNS (1.04 g) were, respectively, added to 500 mL SIF to carry out dissolution experiments. Bath temperature and paddle speed were set at 37 ± 0.5°C and 100 rpm. After selected periods of 5, 10, 15, 30, 45, 60, 120, and 150 min, 3 mL aliquots was withdrawn for filtration. The same volume of fresh media (3 mL) was refilled to each original incubating sample. The samples were centrifuged at 3000 rpm for 10 minutes and notoginsenosides R1, ginsenoside Rg1, ginsenoside Rb1 were assayed for concentration by HPLC. The dissolution profiles were plotted as the cumulative dissolution concentration versus incubation time.

3. Results and Discussion

3.1. Orthogonal Study

The first step of preparation of TPNS nanoparticles by SAS process is to optimize the operational conditions to obtain the smallest MPS of powder. Preliminary experiments show that the main factors affecting the MPS during nanocrystallization process include drug concentration, precipitation temperature, precipitation pressure, and the flow rate of drug solution. The present study is analyzed by an orthogonal experiment with four factors and four levels. The orthogonal combination of the arrangements and the corresponding results were shown in Table 2. The results showed that the largest and the minimum diameter of TPNS nanocrystallization were 396.1 ± 40.0 nm and 155.9 ± 14.4 nm, respectively. According to value, we can see that the influence of the MPS of TPNS nanoparticles decreases in the following order: ; the best operating conditions are (2.5 mg/mL, 40°C, 20 MPa, and 6.6 mL/min). Through confirmatory test, smaller TPNS nanoparticles were got, with a minimum diameter of 141.5 ± 18.2 nm.

At the same time, the results showed that access to the highest amounts of TSA of TPNS nanoparticles was 96.04%, while the minimum amounts was 36.97%. We can see that the influence to the amounts of TSA of TPNS nanoparticles is as follows: according to value. Because TPNS is typical multicomponent extracts from traditional Chinese medicine, so it has a strict control over the content standards. In concern of this, HPLC was used to quantify the main components of TPNS nanoparticles obtained in the best operating conditions for , in which the minimum MPS was prepared. The HPLC spectra are obtained as shown in Figure 3. According to the corresponding standard curve, the content is calculated as follows: ginsenoside Rg1 39.4%, ginsenoside Rb1 31.3%, notoginsenosides R1 8.2%, and the TSA 78.9%. The contents of ginsenoside Rg1, ginsenoside Rb1, notoginsenosides R1, and TSA were regulated strictly in Chinese pharmacopoeia 2010 edition [33], which shall not be less than 25.0%, 30.0%, 5.0%, and 60.0%, respectively. So the quality standard of TPNS nanoparticles is consistent with rule of related part of Chinese pharmacopeia. And TPNS nanoparticles obtained in the trials numbers 3, 4, 9, 10, 11, and 12 can also meet the regulations.

3.2. Effect of Parameters on TPNS Nanoparticles
3.2.1. MPS of TPNS Nanoparticles

The relationships between the MPS of TPNS nanoparticles and different variables are shown in Figure 4. As indicated in Figure 4(a), the MPS of TPNS nanoparticles keep increasing when the TPNS solution concentration increased from 2.5 to 12.5 mg/mL. According to the atomization and droplet broken mechanism, the increase of TPNS solution concentration results in the increase of the viscosity and surface tension of the mixture in the precipitation chamber. Based on the concept of “one droplet one particle,” the large particles are formed at high concentration of TPNS solution.

Figure 4(b) shows the effect of precipitation temperature on the MPS of TPNS nanoparticles. As shown in the figure, the MPS increased with increasing precipitation temperature from 40 to 70°C. The increase of the temperature results in decreasing of CO2 content in the liquid solution. So, the large particles are formed at high precipitation temperature.

Figure 4(c) shows the effect of precipitation pressure on the MPS of TPNS nanoparticles. As shown in the figure, the MPS of TPNS nanoparticles decreased significantly when the precipitation pressure increased from 10 to 20 MPa and slightly increased from 20 to 25 MPa. With the increase of the pressure in the precipitation chamber, the content of CO2 in the liquid solution increases rapidly, while the viscosity and the surface tension of the mixture in the precipitation chamber reduce. The atomization becomes more violent and the primary droplets formed become smaller, which results in the precipitation of smaller particles. Therefore, when the pressure increases, the MPS becomes smaller.

Figure 4(d) shows the effect of drug solution flow rate on the MPS of TPNS nanoparticles. As shown in the figure, the MPS remained constant when the drug solution flow rate increased from 3.3 to 9.9 mL/min and increased significantly from 9.9 to 13.2 mL/min. With the increase of the drug solution flow rate, the mass flow rate of the liquid mixture increases; this leads to the increase in the thickness of the liquid film. Therefore, the droplet formed by the SAS process becomes large and the particles precipitated in the precipitator become large based on the mechanism of “one droplet one particle.”

The analysis of variance (ANOVA) was made in the use of Design Expert 7.0 software and the results were shown in Table 3. The ANOVA analysis revealed that each of the four factors exerted influence on the MPS of TPNS nanoparticles in the selected ranges, among which precipitation pressure was identified as the most important determinant based on ANOVA with 95% confidence.


SourceSum of squares (SS)Degrees of freedom (df)-ratioType of effect

() Concentration of TPNS solution17025.9636.3039.28
() Precipitation temperature14050.0035.0219.28
() Precipitation pressure24629.8339.1189.28Significant
() TPNS solution flow rate1044.27530.0729.28

3.2.2. TSA of TPNS Nanoparticles

The relationships between TSA of TPNS nanoparticles and different variables are shown in Figure 5. As indicated in Figure 5, TSA of TPNS nanoparticles showed similar variation, the irregular changes, with the change of the TPNS solution concentration, precipitation temperature, and TPNS solution flow rate in their respect fields. However, its TSA slightly increased with the increase of the precipitation pressure. The analysis of variance was made by Design Expert 7.0 software and the results are shown in Table 4. The ANOVA analysis revealed that each of the four factors exerted influence on the TSA of TPNS nanoparticles in the selected ranges, among which TPNS solution concentration was identified as the most important determinant based on ANOVA with 95% confidence.


SourceSum of squares (SS)Degrees of freedom (df)-ratioType of effect

() Concentration of TPNS solution1525.04832.4203.490
() Precipitation temperature263.93630.4193.490
() Precipitation pressure343.68130.5453.490
() TPNS solution flow rate388.46830.6163.490


Time Flow rate Acetonitrile Water
(min)(mL/min)(%, v/v)(%, v/v)

01.0020.080.0
201.0040.060.0
211.0020.080.0
301.0020.080.0
401.0020.080.0

3.3. Characterization of TPNS Nanoparticles
3.3.1. SEM and DLS

The preparation of TPNS nanoparticles is according to the orthogonal design in Table 2. The crystalline of unprocessed PNS was light yellow (Figure 6(a)), while the processed TPNS was loose white powder (Figure 6(c)). From Figure 6(b), we can see that the TPNS nanoparticles were evenly distributed in the precipitation chamber. The microstructure of unprocessed TPNS is irregular bulk crystal with diameter of 15.2 μm (DLS result) as shown in Figure 7.

The orthogonal experimental results show that TPNS nanoparticles had different MPS in different operating conditions (Table 2). The optimum nanocrystallization condition was that TPNS solution was 2.5 mg/mL, preparation temperature was 40°C, preparation pressure was 20 MPa, and the flow rate was 6.6 mL/min. Figure 8(a) is the SEM image of the TPNS nanoparticles prepared in the optimum nanocrystallization condition, which was also obviously spherical. From Figure 8(b), we can see that TPNS nanoparticles had a small particle size, about 150 nm.

3.3.2. FTIR Analysis

Some analyzing methods are applied on unprocessed and processed TPNS to obtain information of the change of chemical structure after SAS processing. FTIR spectra (see Figure 9) between unprocessed and processed TPNS show no significant differences. Because TPNS are a variety of similar mixtures of compounds, it is impossible to extract the spectrum of all the absorption peaks. The assignments of characteristic bands are as follows: 3403 cm−1 (O–H stretching vibration), 2935 cm−1 (C–H stretching vibrations), and 1644 cm−1 (C=C stretching vibration). But after learning that there existed a high-degree similarity between the raw and TPNS nanoparticles spectrum of FTIR, we can make a positive inference that the chemical structure of TPNS with various components has not changed.

3.3.3. DSC Analysis

In order to further investigate the crystalline type of TPNS nanoparticles, DSC was performed to carry out a further test. From Figure 10, it can be seen that unprocessed and processed TPNS have no distinct melting peaks, which indicated they are all amorphous state. These suggested that no change in crystal structure took place during SAS process.

3.4. Dissolution Studies In Vitro

The dissolution profiles of unprocessed and processed TPNS in SIF are shown in Figure 11. No obvious difference in dissolution rate of Rg1 in raw TPNS and TPNS nanoparticles could be found. However, dissolution rates of notoginsenosides R1, ginsenoside Rb1, and TSA in TPNS nanoparticles group are higher than these in raw TPNS. In the 90 min, notoginsenosides R1, ginsenoside Rb1, and TSA in TPNS nanoparticles achieving the maximum dissolution were about 99.56%, 99.57%, and 99.33%, respectively. Accordingly, these in raw TPNS are 64.86%, 45.51%, and 72.26%. According to Noyes-Whitney equation, with the particle size of micronization products decreasing, the specific surface area of the drugs increased accordingly, thereby increasing the contact area of the solid drugs and the dissolution medium; the dissolution of the drugs was also improved accordingly. These results suggested that the TPNS nanoparticles could have higher bioavailability when they are prepared for oral formulations such as tablet and capsule.

4. Conclusions

Spherical TPNS nanoparticles with MPS of 141.5 ± 18.2 nm and the TSA of 78.9% were obtained by SAS process. The optimum nanocrystallization conditions are determined as follows: TPNS solution concentration 2.5 mg/mL, TPNS solution flow rate 6.6 mL/min, precipitation temperature 40°C, and precipitation pressure 20 MPa. The MPS and TSA of TPNS nanoparticles are controllable by changing the process parameters. The quality standard of TPNS nanoparticles is consistent with rule of related part of Chinese pharmacopeia. The chemical and crystal structures of the obtained TPNS nanoparticles have not changed. The dissolution rates of notoginsenosides R1 and ginsenoside Rb1 in TPNS nanoparticles are higher than these in raw TPNS, while there was no obvious difference in Rg1. These results suggest that TPNS nanoparticles can be helpful to the improvement of its bioavailability for the treatment of cardiovascular diseases.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors are grateful for the precious comments and careful corrections made by anonymous reviewers. The authors would also like to acknowledge the financial support from the Special Fund for Forestry Scientific Research in the Public Interest (20140470102) and the National Natural Science Foundation of China (no. 21203018).

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Copyright © 2015 Ying Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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