2.1 Chemicals
All reagents were of analytical grade unless specified otherwise. Water (18.2 MΩ cm) was produced by a laboratory Milli-Q pure water meter.
trans-cinnamic acid, mesaconitine and hypoaconitine reference substance, Lot number: AA0806LB14, 110799–200505, 110798–201106 purity:≥ 98%). Chromatographic methanol (product batch number: Q/BSYZ01-2006) purchased in Tianjin Siyou Fine Chemicals Co., Ltd. Chromatographic acetonitrile (product batch number: R200813) purchased from Tanmo Quality Inspection Technology Co., Ltd. formic acid, 98% purity ≥, CAS NO. 64-18-6), Purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd. Ammonia, Lot No. 20091022, Purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd). Pure water (pure water produced by laboratory Milli-Q pure water meter). aconite (lot: 200901), cinnamon (lot: 181201), All purchased from Zhejiang University of Traditional Chinese Medicine Chinese Herbal Medicine Co., Ltd. Hydrocortisone injection (batch number: 1904021). Heparin sodium solution (0.1%, 125U/mL, LOT: L16N10G103237). saline (LOT༚L12A10G95086). Probe CMA 20 Elite 4mm 3/pkg (REF :8010435, LOT༚T54996), CMA 30Linear MD Probe 4/pkg (REF: 8010460, LOT༚T55153). ring reagent (NaCl( batch number: 8015031801), KCL( batch number: 20180110), MgSO4( batch number : 2015052501), KH2PO4( batch number: 2015082901), CaCL2( batch number: 2015050501). Pentobarbital Sodium( batch number: DW-PS-05)
2.2 Apparatus and software
Detection and quantification of the analytes were performed using a UPLC Acquity system from Waters (Waters, USA) equipped with a binary pump, vacuum membrane degasser, a thermostated column compartment, an autosampler, and an automatic injector. An Acquity ACQUITY UPLC BEH C8 column (100 mm×2.1 mm I.D., 1.7 µm particle size, Part No 186002878) from Waters were tested as chromatographic columns. Auxiliary apparatuses were: HC-3018 High-Speed Centrifuge (Anhui Zhongke Zhongjia Science Instrument Co., Ltd), WH-861 Vortex Mixer (Taicang Hualida Experimental Equipment Co., Ltd), Electronic scales (METTLER TOLEDO 1/100000 electronic scales), KQ-500B ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd), Liquid transfer gun (Eppendorf Research plus range 100–1000µl), SHZ-IIIB circulating water multi-purpose vacuum pump (Linhai Tan vacuum Equipment Co., Ltd).
2.3 Animals and microdialysis experiment
2.3.1 Animals
Sprague Dawley (SD) rats were purchased from the Animal Experimental Center of Zhejiang Chinese Medicine University. They were raised in the Animal Experimental Center of Zhejiang Chinese Medicine University (license number: 20180006004067). Before experimentation, the rats were allowed a 1-week acclimation period in the animal house using the independently isolated feeding cages with natural illumination. The rats were fed standard food and purified water. The environment temperature was kept at 25 ± 2°C, and the humidity was ≥ 40 %.
2.3.2 Establishment of pathological model of OP with kidney-yang deficiency
SD rats were randomly grouped according to their body weight. Anesthetized with ip10% chloral hydrate 3mL/kg, then fix the animal on the operating table. Through bilateral ophorectomy, the fallopian tubes and accompanying blood vessels are ligated first, and then the "mulberry-like" ovaries are separated with elbow surgical clamps, ligated, and cut off. The rats were injected with 40,000 U/mL penicillin every day after the operation, each injection was 1 mL/d, and the injection was continued for 3 days, and then the rats were routinely reared. Starting from the second week, the experimental group was injected with hydrocortisone injection (5 mg/mL) 20 mg/kg on the hind limbs every day. From the 4th week to the 8th week, the rats in the experimental group were injected with hydrocortisone injection twice a week to maintain the state of kidney-yang deficiency. Generally based on rat behavior indicators, serum E2, T, ACTH, cAMP, cGMP, etc. levels, bone density and bone mineral content and other tests. Judge whether the model of OP with kidney-yang deficiency type is successful.
2.3.3 microdialysis experiment
Ringer-HEPES buffer solution (precise weighing NaCl 1.78g, KCl 55.91mg, MgSO4 36.11 mg, KH2PO4 mg, NaHCO3 13.61 mg CaCl2 525.06 mg, and watering to 100 mL volume bottle) was used for the preparation of intermediate solutions and standard solutions for calibration purposes.
The rats were divided into Aconite group, Cinnamon group and the herb couple (1:1). The rats were a successful model and anesthetized. Rat body temperature was maintained at 37°C using a heating pad during the experiment, and their heads were fixed on the brain stereotaxic locator to fix the limbs of the rats. In the first step, to make an incision at the upper left of the left clavicle, then to find the jugular vein, insert the torn tube into the vein, then place the blood probe in the venous blood vessel, and suture the skin wound carefully. In the second step, insert the traction needle into the cartilage of the rat’s leg joint cavity, and insert the probe into the overlap of the traction needle and articular cartilage carefully, so that the probe membrane of the joint probe is partially placed subcutaneously.
To use Ringer's solution to prime and balance 30 ~ 60 min. Aconite and Cinnamon 1 mL / 100g were injected into the duodenum according to the weight of each rat. The samples of the jugular vein and joint dialysate were collected by cryopreservation sampler after administration at different times at 10 min、20nin、30min、40min、50min、60min、120min、180min、240min、300min、360min、480min, a total of 8 h.
After all the samples are collected, the last dose, half an hour after administration, to take out rat whole blood, to wait for it to stand and layer, to separate the supernatant and store it in -20℃ refrigerator.
2.4 Ultra-performance liquid chromatography (UPLC)
Chromatography was separation was carried out in an ACQUITY UPLC BEH C8 column (100 mm × 2.1 mm i.d., 1.7 µm ) from Waters Corp., USA. The column temperature was maintained at 30℃. The analysis was achieved with gradient elution using (mobile phase A) acetonitrile and (mobile phase B) water (containing 0.1% formic acid). The gradient elution program started with 0–3 min, 21% A; 3–11 min, 21%-25% A; 11–18 min, 25% A; 18-18.1 min, 25%-21% A; 18.1–20 min, 21% A.The total analysis time including the washing and equilibrium steps was 20 min. The flow rate was maintained at 0.3 mL/min. The injection volumes of the samples or standard solutions were 5 µL.
2.4.1 Preparation of standard and unmeasured solutions and Determination
Preparation of standard solutions
Precision weighing standard mesaconitine 4.60 mg, hypoaconitine 4.70 mg, and trans-cinnamic acid standard 2.26 mg, with Chromatography Acetonitrile and methanol at 10 mL capacity bottle, to dilute ten times and store at 4°C.
Preparation of blood samples group
To precision absorption the blood 60 uL of different components, precision addition 20 uL ammonia water, vortex 2 min, precision addition 520 uL Chromatography Acetonitrile and methanol to precipitation protein, and vortex 3 min, 15000 r/min centrifuges for 10 minutes. Take supernatant, use 0.22 um organic filter membrane to filter.
And to analyze them according to the chromatographic conditions respectively, and record the peak area data. Calculate the content value of each group.
2.4.2 Method validation and Determination
There is the linearity of the standard solution, preparation of reference standard solution method was 0.2 mL、0.4 mL、0.6 mL、0.8 mL、1 mL、2 mL、4 mL、5 mL in 5 mL capacity bottle, Volume to scale with Chromatographic methanol and Chromatography Acetonitrile (1:1) solution, different concentrations of mixed reference solution were obtained and filtered into the injection bottle with a disposable syringe to use 0.22 um organic filter membrane to filter. According to chromatographic conditions to inject the sample, determine the peak area. With the peak area as the ordinate and the concentration of each group of samples as the abscissa to draw a standard curve. The specificity of the method was determined by comparing the chromatograms of blanks with those corresponding to the samples. The precision of the method was determined by taking each group of blood samples, according to the chromatographic conditions for 8 times, and to determine the peak area of blood samples. To take the same batch of the test solution and analyze it at 0, 2, 4, 6, 8, 12, 16, 18, 24 h according to the chromatographic conditions to determine the stability of the method, and record the peak area data. The repeatability of the method was determined by preparing the same batch of blood for 8 groups, then the peak area of the peak plasma sample was determined according to the chromatographic conditions.
2.5 Freeze-dried powder production and determination
Preparation of freeze-dried powder of Aconiti or Cinnamon: To use 240g Aconiti or 80g Cinnamon with water as a solvent to condense and reflux for extraction twice respectively. Put it in a freeze dryer to freeze-drying, quickly weigh and transfer the powder to a plastic bag, and store at 4℃ in the refrigerator.
To weigh freeze-dried powder of Aconiti 1.0085g, freeze-dried powder of Cinnamon 0.5081g and 0.1096g freeze-dried powder of the herb couple precisely, To draw 20 ul formic acid to add, and fix to 20 mL volumetric flask with methanol, to vortex for 5min, after standing for 1h, to ultrasound for 30min, and to centrifugation (10000r/min, 10min) to separate the supernatant, use 0.22 um organic filter membrane to filter. And analyze them according to the chromatographic conditions respectively, and record the peak area data.
2.6 Statistical analysis
All statistical analyses were performed using the SPSS software (Version 13.0). P-values less than 0.05 were considered statistically significant.