The reference standards of curcumin, demethoxycurcumin, and bisdemethoxycurcumin (purity > 98%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Methanol and acetonitrile (Fisher, United States) were of high-performance liquid chromatography (HPLC) grade. Other reagents were of analytical purity. Water was glass distilled and filtered through a Milli-Q water purification system (Millipore, Bedford, MA, United States) prior to use.

SPF-grade Sprague-Dawley rats (200 ± 20 g) were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). Animals were housed in polypropylene cages and were sent to an animal room 5 days in advance for acclimatization (Chengdu University of Traditional Chinese Medicine, Chengdu, China). They were maintained under controlled conditions (a 12 h light-dark cycle at 22 ± 2°C) on a standard pellet diet and had free access to water. Animal experiments were approved by the Committee of Scientific Research and the Committee of Animal Care of Chengdu University of Traditional Chinese Medicine (Chengdu, China).

Medicinal materials were collected from the genuine producing areas and authenticated as the dried root tuber of Curcuma longa L. by Professor LI Min (Chengdu University of Traditional Chinese Medicine, Chengdu, China). The sample information is shown in Table 1.

Xuefu Zhuyu Pian (Lot:170409) was purchased from Weifang Zhongshi Pharmaceutical Co., Ltd. (Shandong, China). Epinephrine Hydrochloride Injection (Lot:170507) was purchased from Grand Pharma (China) Co., Ltd. (Wuhan, China).

Prepared slices of HSYJ was processed according to the Pharmacopeia of the People’s Republic of China (Vision 2015) (Chinese Pharmacopoeia Commission, 2015) and the Standard for Processing of Traditional Chinese Medicine in Sichuan Province (Vision 2015) (Administration, 2016).

The decoction was prepared according to the Standard for Management of TCM Decocting Room in Medical Institutions and Technical Requirements for Quality Control and Standard Formulation of TCM Granules.

HSYJ and its processed products were boiled for 30 min after soaking for 30 min with nine times the amount of water before being filtered. The dregs were boiled twice with seven times the amount of water for 30 min each time, and they were then filtered and mixed with the previous filtrate. The mixed decoction was concentrated by vacuum concentration (T ≤ 50°C). The dosage for rats was 0.9 g⋅kg^–1 according to the conversion of human clinical dosage.

A positive drug was prepared by grinding up Xuefu Zhuyu Pian and dissolving this in water. The dosage given to the rat was 0.432 g⋅kg^–1 according to the conversion of human clinical dosage.

We took 50 mL of decoction and put it in an evaporating dish that had been dried to a constant weight. We then steamed it in a water bath, dried it in an oven at 105°C for 3 h, and cooled it in a dryer for 30 min. We then weighed it quickly and precisely and calculated the extraction yield. The extraction yield (%) of the sample was calculated with the dried product.

A total of 36 animals were randomly and equally divided into six groups (male and female in half): a control group (CG), model group (MG), Raw HSYJ group (SHG), Stir-frying HSYJ with vinegar group (CHG), Stir-frying HSYJ with wine group (JHG), and positive group (PoG). Except for the CG, the rest of the groups replicated the blood stasis model due to liver-Qi depression by the referenced methods (Shuzhi, 2014). When the model was finished, the animals were fasted for more than 12 h, but water was given normally. The samples were taken under anesthesia. The experimental procedure and dosing treatment plan are shown in Figure 1.

We collected plasma using heparin as an anticoagulant and centrifuged it for 5 min at 3,000 rpm. The samples were stored in aliquot at −20°C for later use. We avoided repeated freeze-thaw cycles. Rat brain tissue samples were rinsed with normal saline and stored at −20°C. At the time of detection, rat liver tissue samples were thawed in 37°C water and weighed to the appropriate amount of tissue, ground with nine times the normal saline, and centrifuged for 10 min at 3,000 rpm. The supernatant was removed and assayed immediately. Rat 5-Hydroxytryptamine (5-HT) in plasma was detected with 5-HT ELISA Kit (Lot: C0167140192, Cusabio Biotech Co., Ltd., Wuhan, China). Rat beta-endorphin (β-EP) in plasma and brain were detected with β-EP ELISA Kit (Lot: C0167180196, Cusabio Biotech Co., Ltd., Wuhan, China).

The fixed brain was dehydrated by an automatic dehydrator before being embedded and sliced. The devoted slices were put in the dye vat and performed in 3% hydrogen peroxide (methanol and 30% H₂O₂ was 99:1) for 10 min at room temperature. Afterward, the slices were washed three times with PBS for 5 min each time. We then immersed them into 0.01M citrate buffer (PH 6.0), used high fire in a microwave oven to break off after boiling, and we repeated this one time after 5 min. After cooling, we washed them two times with PBS for 5 min each time. The slices were then incubated with normal goat serum (ZSGB-BIO, Beijing, China) at room temperature for 20 min. The sections were incubated with primary antibody for c-fos (rabbit polyclonal antibody, 1:100 dilution, Abcam, CA, United States) overnight at 4°C. The sections were then processed with the secondary antibody with goat anti-rabbit (ZSGB-BIO, Beijing, China) for 30 min at 37°C. We washed them three times with PBS for 5 min each time. Used DAB (ZSGB-BIO, Beijing, China) to develop color at room temperature, we controlled the reaction time under the microscope for 2 min and washed the sections with distilled water. Slices were slightly re-dyed with hematoxylin and sealed with neutral gum after dehydration and transparency.

Images were collected by a BA400Digital microscope (Motic China Group Co., Ltd.). The whole tissue was observed at 100 × magnification for each pathological section, and microscopic images were collected by selecting three fields of vision at 400 × magnification. Measured integrated optical density (IOD) and the area of all images by Image-Pro Plus 6.0 (Media Cybernetics, United States) and calculated mean density (MD) of each image.

Statistical analyses were performed by repeating one-way analysis of variance (ANOVA), and this was followed by LSD’s post hoc multiple-comparison test by using SPSS 19.0 software. The P < 0.05 was considered to represent statistical significance.

The extraction yield was significantly increased after being stir-fried with vinegar or wine, but there was no significant difference between the stir-frying HSYJ with vinegar and stir-frying HSYJ with wine, indicating that processing could increase the dissolution of chemical constituents (Table 2). Figure 2 shows the chromatogram of HPLC was well separated. After stir-frying with vinegar or wine, it was able to increase the dissolution of curcumins in water. The results are shown in Table 2. Similarly, there is no regularity in the content difference between stir-frying HSYJ with vinegar and stir-frying HSYJ with wine. The curcumins of H2 stir-frying HSYJ with vinegar were, however, much higher than those stir-frying with wine, which may be the source of the mechanism behind enhancing the analgesic effect of stir-frying with vinegar.

The results of changes to the 5-HT level are shown in Figure 3. Compared with the CG, the 5-HT level in plasma of MG was significantly increased (P < 0.01). After administration, HSYJ and its processed products could significantly improve 5-HT abnormality in plasma of model rats, but there was no difference between them.

The results of the changes to the β-EP level in plasma and brain are shown in Figure 4. Compared with the CG, the level of β-EP in plasma and brain of MG decreased significantly (P < 0.01). As an analgesic substance, the decrease of β-EP may be the mechanism behind Qi stagnation and blood stasis, causing pain. After administration, HSYJ and its processed products could improve β-EP abnormality in plasma and brain of model rats. Compared with MG, the β-EP in brain of HSYJ and its processed products increased significantly, and CHG and JHG were higher than those of GG. There was a significant difference between SHG and JHG. The results showed that HSYJ could enhance the analgesic effect by increasing the release of β-EP. The levels of β-EP in brain of drug groups were higher than that in CG, which may be due to the large release of β-EP in the brain in order to inhibit body pain. However, only the level of β-EP in plasma of CHG increased significantly, suggesting that stir-frying with vinegar not only increased the release of β-EP in brain but also promoted its entry into blood.

The results of the expression of c-fos in the brain of rats are shown in Figure 5. Compared with the CG, MG was significantly increased (P < 0.01). The indicator of the pain response is such that, the stronger the expression of c-fos, the stronger the pain the body experiences (Huang et al., 2010). After administration, the expression of c-fos in CHG and JHG decreased significantly (P < 0.01), but there was no significant difference in SHG, which indicated that HSYJ could enhance the analgesic effect of the body by stir-frying with vinegar or wine. There was, however, no significant difference in the administration groups.