Chemicals and materials
The P. notoginseng root was purchased from Anhui Boyao Qiancao Traditional Chinese Medicine Co. Ltd. (Anhui, China) and authenticated by Professor Peng Huasheng (Anhui University of Traditional Chinese Medicine). Pepsin from porcine gastric mucosa was supplied by Yuanye Co. Ltd. (Shanghai, China). The water used was distilled and passed through a Milli-Q water purification system (Millipore, Bedford, USA). All other reagents were of analytical grade or pharmaceutical grade.
Animals
All Sprague Dawley (SD) rats (weight, 250 ± 20 g) were purchased from the Experimental Animal Center of Jinan Pengyue Co. Ltd. (Jinan, China). All animal welfare and experimental procedures were approved by the Animal Ethical Committee of Anhui University of Chinese Medicine (Approval No. 1107261911002543) and were in accordance with the guide for the care and use of laboratory animals. The rats were acclimated for approximately 7 d with a 12 h light and dark cycle throughout the experiment. The rats were housed, given free access to food and water, and the temperature was maintained at room temperature (25°C).
PNP preparation
The PNP was crushed by a highly efficient pulverizer, and the particle size was analyzed using a laser particle size analyzer (BT-9300HT, BAITE, Dandong, China) using water as the dispersion medium.
Characterization of PNP
Morphological appearance
The morphological appearance of PNP was observed by scanning electron microscopy (SEM, S3400N HITACHI). The hydrothermal treated samples (Powder 4) were soaked in water for 5 min at 20, 30, 40, 50, 60, 70, 80, 90, or 100°C, respectively,and then the PNP was prepared by freeze drying. The powders were placed on double-sided adhesive tape on SEM stubs with and coated with gold [20].
Polarized light microscopy (PLM)
The prepared PNP were characterized by polarization microscope (CK-500, Caikon, China) at room temperature [21]. 500 mg of PNP in a tube was diluted with distilled water (2 mL). The hydrothermal treated samples (Powder 4) were steeped in water for 5 min at 20, 30, 40, 50, 60, 70, 80, 90, or 100°C, respectively.
Differential scanning calorimetry (DSC)
The DSC analysis of the PNP was measured using differential scanning calorimetry (DSC200F3, NETZSCH, Germany) under an ultrapure nitrogen atmosphere [22, 23]. The PNP (3 mg) were mixed with 12 μL of distilled water, hermetically sealed in an aluminum pan, and equilibrated at 4°C for 24 h. The scanning temperature was then heated from 20°C to 220°C at a heating rate was 10°C/min.
Analysis of notoginsenoside R1 and ginsenosides Rg1 and Rb1 by HPLC
The content of notoginsenoside R1 and ginsenosides Rg1 and Rb1 in the samples were determined using HPLC and the method reported in the Chinese Pharmacopoeia (2015). HPLC analyses were performed on an Ultimate 3000 series system (Thermo Fisher Scientific, China) consisting of a quaternary pump, DAD detector, and autosampler, and the data were analyzed using Chromeleon 7 software. A Thermo Syncronis C18 column (250 × 4.6 mm, 5.0 µm) was adopted for the separation. The mobile phase consisted of A (pure water) and B (acetonitrile). The gradient mode was as follows: 0 – 12 min, 19% A; 12 – 60 min, 19% ~ 36% A. The flow rate was 1.0 mL/min, and the detection wavelength was set to 203 nm. The column temperature was 25°C, and the sample injection volume was set to 20 µL.
In vitro dissolution studies
The effects of particle size and hydrothermal treatment (Powder 4 were treated at 30, 40, 50, and 100°C) on the dissolution behavior of the PNP were investigated. Dissolution studies were carried out on a dissolution apparatus (ZRS-8L, Tianda Tianfa, China) with a rotation speed of 75 rpm according to the China Pharmacopoeia apparatus Ⅲ paddle method [24, 25]. The tests were performed in 150 mL of freshly prepared aqueous solution and simulated gastric fluid (10 g/L pepsin, 0.1 M HCL, pH = 1.2, ChP 2015). Maintaining a temperature of 37 ± 0.5°C, samples of 4 mL each were withdrawn and replaced with fresh medium of equal volume at fixed time points of 0, 5, 10, 15, 20, 25, 30, 60, 90, and 180 min, respectively. The content of notoginsenoside R1 and ginsenosides Rg1 and Rb1 in the medium were determined by HPLC as described above. Because the dissolution of ingredients was affected by the particle size, the dissolution rate calculated using the revised equation is as follow:
![](https://myfiles.space/user_files/58853_cdc0f79cc190fa60/58853_custom_files/img1597860997.png)
In this formula, Ve means 4 mL, Ci means the sample concentration of release medium from the ith permutation, V0 means the total volume of release medium, n means the number of replacement release medium, mPNP means the weight of PNP.
In vivo pharmacokinetic study
Before administration, sixty rats were fasted for 12 h, but free access to water. The rats were randomly divided into ten groups, six rats in each group. Rats were pretreated with different particle sizes of PNP (Powders 1 ~ 6, 540 mg/kg, i.g.) or the PNP (Powder 4) treated at different temperatures (30, 40, 50, and 100°C, 540 mg/kg, i.g.). After intragastric administration of PNP, 200 µL of blood was collected into heparinized tubes, at 0.08, 0.17, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 12, 24, 48 h from the ophthalmic veins and centrifuged at 3000 rpm for 10 min. Plasma was separated and frozen at -20°C for analysis. 10 μL of digoxin (1.0 μg/mL) as an internal standard (IS) was added to each blood sample (100 μL), which was pretreated according to the previously published method [26], and the concentrations of notoginsenosides were analyzed using a validated UHPLC-MS/MS method.
The notoginsenosides in the biological samples were analyzed using a validated UHPLC-MS/MS method. The UHPLC-MS/MS system (UHPLC-MS/MS-5500 system, AB Sciex Instrument, USA) contained an ExionLC AD UHPLC system and a QTRAP 5500 triple quadrupole mass spectrometry instrument equipped with an electron spray ionization (ESI) source. The data acquisition and analysis were performed using MultiQuant software (AB Sciex Instrument). Liquid chromatographic separation was carried out at 25°C using a 1.8 µm, 100 × 2.1 mm Epic C18 column. The mobile phase was composed of acetonitrile (solvent A) and water containing 0.1 mM ammonium chloride (solvent B). An optimized gradient elution condition was set as 10% A (0 ~ 0.02 min), 10% ~ 75% A (0.02 ~ 5 min), 75% A (5 ~ 5.5 min), 75% ~ 10% A (5.5 ~ 6 min), and 10% A (6 ~ 9 min). A sample volume of 2 µL was injected, and a flow rate of 0.2 mL/min was employed. The ESI source was operated in negative ionization mode, and the MS conditions were set as follows: desolvation temperature, 250°C; heat block temperature, 550°C; nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; and interface voltage, 3.5 kV. Data were acquired by multiple reaction monitoring (MRM). Table 1 lists the quantitatively optimized parameters, including the declustering potential (DP), collision energy (CE), etc.
Statistical analysis
All data are expressed as the mean ± SD. Differences between groups were compared by SPSS Statistics 24 using the independent-samples t-test or paired-samples t-test. Statistically significant differences were determined at P < 0.05 and P < 0.01.