LI Fa-jie, GU Jin-yu, ZHANG-Yue, SHEN Shuo, YAO Yao, WAN Tian-hao, XIA Di, ZHANG Qing?

1. Wang Jing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China

2. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China

3. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

Keywords:Hai Tong-pi decoction ointment Anti-inflammation Analgesia interleukin-1β Tumor necrosis factor

ABSTRACT Objectve: To investigate the effect and mechanism of Hai Tongpi decoction ointment on analgesic and anti-inflammatory effects in mice after transdermal administration. Methods: The hot plate method and the acetic acid writhing experiment were used to verify the analgesic effect of Hai tong-pi extract on pain model mice; the xylene-induced auricular swelling model and the carrageenan-induced right foot swelling model were used, combined with the ELISA method to determine the analgesic effect. The levels of TNF-α, IL-1β and IL-6 in the serum of mice were observed. Anti-inflammatory effect of Haitongpi Tang decoction ointment on inflammation model mice. Results: In terms of analgesia, compared with the model group, both the treatment group 1 and the treatment group 2 can significantly prolong the hot plate pain threshold and the latency of the writhing response (P＜0.05), and significantly reduce the writhing in the mice. The number of times (P＜0.001), and the effect of process 1 is more obvious (P＜0.05); In terms of antiinflammatory, compared with the blank group, the swelling degree and swelling rate of the right hind paw of the mice in the model group were significantly increased (P＜0.01), indicating that the acute inflammation model was successfully established in the mice. Compared with the model group, both the process group 1 and the process group 2 can significantly reduce the swelling degree and swelling rate of the mice feet (P＜0.05), and reduce the auricle swelling degree of the mice, and increase the swelling inhibition rate (P＜0.05), and the effective effect of process 1 group was more significant (P＜0.05). At the same time, compared with the blank group, the contents of TNF-α, IL-1β and IL-6 in the model group were significantly increased (P＜0.01). Compared with the process group 2, the process group 1 had a more significant decrease in the content of IL-1β (P＜0.05), and IL-6 content of the two had no statistical difference (P＞0.05). Conclusion: The Hai tong-pi decoction ointment prepared by two different processes have good analgesic and antiinflammatory effects on mice after transdermal administration. inflammatory cytokine expression.The anti-inflammatory and analgesic effect of process group 1 was better than that of process group 2, which may be related to the separate extraction and storage of volatile oil of traditional Chinese medicine, which ensured the long-term pharmacological activity of volatile oil.

1. I ntrod uction

7-248 Persistent pain and inflammation caused by injurious stimuli are the most common clinical symptoms and one of the important global health problems, which can lead to individual pain,deterioration of quality of life, heavy family burden and huge social and economic burden[1]. Opioid analgesics and non-steroidal antiinflammatory drugs are commonly used as first-line drugs for acute and chronic pain and inflammation after orthopedic surgery and fall injury. However, these drugs can cause gastrointestinal toxicity such as nausea and vomiting, drug resistance, addiction, lethargy,increased bone fragility and other side effects[2]. Transdermal administration of Traditional Chinese medicine is a promising complementary and alternative therapy with definite curative effect and less toxic and side effects on pain prevention and treatment and inflammation reduction[3]. Hai Tong-pi decoction ointme first in the Yi Zong Jin Jian, consist of the haitongpi, tougucao, ruxiang,moyao, danggui, chuanjiao, honghua, chuanxiong, fangfeng, baizhi,weilingxian, gancao, which has the promoting blood circulation and reducing swelling and relieve pain[4].Clinically, Haitongpi decoction is commonly used to fumigate and wash the affected area, and to promote the healing of patients after fracture through percutaneous administration, and to treat osteoarthritis, calcaneodynia, fall injury and other orthopaedic diseases[5-7]. Basic research has shown that Haitongpi decoction can reduce the apoptosis rate of chondrocytes and the expression of interleukin-1β and interleukin-6[8,9].However,the original prescription has many flavors and the dosage form is decoction, which is inconvenient to carry, needs to be decocted,and the drug is not fully utilized. Therefore, according to the classic original formula of Haitongpi decoction, our team prepared the extract of Chenghaitongpi decoction, in order to provide an antiinflammatory and analgesic external traditional Chinese medicine ointment for clinical diseases such as orthopedics.

In this study, the extract of Haitongpi decoction was used for percutaneous administration of mice. The hot plate method, foot swelling caused by carrageenan and acetic acid writhing reaction were used, and the levels of Tumor Necrosis Factor-α (TNF-α),IL-1β, IL-6 and other inflammatory cytokines in plasma of mice were measured. To explain the anti-inflammatory and analgesic effects of haitongpi formula extract and its possible mechanism.

2. Materials and methods

2.1 Animals

SPF KM mouse, male, body weight (20±2) g, provided by China National Institute for Food and Drug Control (Beijing Daxing),License Number: SCXK (Beijing)-2017-0005. The rats were routinely fed with ventilation and light for 12/12h, the temperature was (21±2)℃C, the relative humidity was 50%±10%, and the rats were free to eat and drink. This experiment was reviewed by the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences. The application for ethical review NO.:Zhongke Zhilun D2022-04-11. The experiment was conducted in strict accordance with the "3R" principle and the relevant provisions of the "Guiding Opinions on The Treatment of Experimental Animals" issued by the Ministry of Science and Technology.

2.2 Experimental drugs and preparation technology

2.2.1 Medicine

Chinese medicine composition of the extract of Haitongpi decoction: Haitongpi (Batch Number: 20211116, Beijing Materia Medica Fangyuan Pharmaceutical Group Co., LTD.), Safflower,vinegar myrrh, vinegar frankincense, Weilingxian, parsnip (batch number: 21072701, 21032201, 21062301, 21040601, 2108098,Beijing Sifang Traditional Chinese Medicine Decoction Pieces Co., LTD.), angelica, licorice, Sichuan pepper, Turbidum turbidum(batch numbers are: 211035, 211099, 211033, 211121, Beijing Shengshilong Pharmaceutical Co., LTD.), Angelica dahurica and Ligusticum chuanxiong (batch numbers are: B9051511, B8291511,Beijing Jinsogo Pharmaceutical Co., LTD.), the above-mentioned drugs were identified as genuine by Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and all were in line with the inspection standards of The 2020 edition of Chinese Pharmacopoeia. Diclofenac diethylamine emulsion (batch NO.:8P3U, specification: 20g/piece, Sino-American Tianjin Smithkline Pharmaceutical Co., LTD.).

2.2.2 The preparation technology

Technology 1: On the basis of pittosporum skin soup original party proportion according to proper prescription of frankincense,myrrh, angelica sinensis, rhizoma ligustici wallichii, sichuan pepper,angelica dahurica, windproof, 10 times the amount of water in the extraction of volatile oil by steam distillation, and save the essential oil and water decoction, medical and pittosporum skin, tougucao,clematis root, safflower, liquorice 5 kinds of combined water,two volatile oil after water decoction combined decompression enrichment, beaten, A traditional Chinese medicine preparation containing 1.512 g crude drug (1.512 g·g-1) per gram was prepared and stored in a refrigerator at 4 ℃ for future use. It was thawed at room temperature before use and added into hydrogel matrix to adjust the crude drug concentration to 2.0 g/mL.

Technology 2: The whole formula was extracted with water, the residue was discarded, and a TCM preparation containing 1.712 g(1.712 g·g-1) crude drug per gram was prepared with concentrated water decoction. The preparation was stored in a refrigerator at 4℃for reserve, thawed at room temperature before use, and added into hydrogel matrix to adjust the crude drug concentration to 2.0 g/mL.

2.3 Main reagents and instruments

2.3.1 Main reagents

TNF-α enzyme-linked immunosorption (ELISA) kit (Batch number: Cat.#ELS286, Shanghai Alysa Biotechnology Co., LTD.),IL-1β, IL-6 ELISA kit (batch number: AB197742, AB222503,Abcam, UK, respectively); Carrageenan (Batch No.: 20170912,Beijing Solaibao Technology Co., LTD.); Formaldehyde and glacial acetic acid are analytically pure, provided by Sinopharm group.

2.3.2 InstrumentsPaw swelling tester (Ahlborn Almemo, 2450), hot and cold plate(Ugo Basile, 35150), microelectronic balance (Mettler Toledo,ML204), and high-speed refrigerated Centrifuge (Eppendorf Centrifuge). 5430R), hole punch (diameter 6mm, ZEYA science education, ZEYA/EP-1).

2.4 The dosage

One day before the experiment, hair removal was performed in the abdominal area of about 3 cm2. According to literature [10-11], dose conversion between mice and human body: (1.44×10000 cm2×0.05 mL/cm2×0.003)÷67 cm2=0.03 mL/cm2, where 1.44×10000 cm2is the body surface area of human (50 kg), 0.05 mL/cm2is the common dose of topical drugs in human body, 0.003 is the equivalent dose conversion coefficient converted from mouse to human according to body surface area, and 67 cm2is mouse body surface area. The dose was 0.03 g/cm2×3 cm2=0.09 g/mouse.

2.5 Observation indeices and detection methods

2.5.1 The analgesia

(1) The hot plate

Before grouping, SPF normal male KM mice were screened for pain threshold, and the temperature of the hot and cold plate pain meter was adjusted at (55±0.5) ℃. Male KM mice were placed on the hot plate respectively, and the time from the back foot to the hot plate was measured, namely, the pain threshold. The time was measured every 5 min for 3 times, and the average value was 5～30 s. Male mice eligible for pain threshold screening were randomly divided into model group (blank gel, 0.09 g/mouse), process group 1(process 1 extract, 0.09 g/mouse), process group 2 (process 2 extract,0.09 g/ mouse) and butaline group (diclofenac sodium diethylamine emulsion, 0.09 g/mouse) according to body weight, with 10 mice in each group. The corresponding dose of ointment was evenly applied to the abdomen depilation of the corresponding mice, once a day,and the intervention lasted for 14 days. The pain thresholds were determined by the above method before the first administration and 30, 60, 90 and 120 min after the last administration. If there is no afterfoot reaction after 60 seconds, stop observing immediately and calculate according to 60 seconds[12].

(2) The body torsion reaction

A total of 40 SPF normal male KM mice were divided into the same groups, treated with the same medication and treated with the same intervention method as 1.5.1 (1) once per day for 14 consecutive days. Half an hour after the last administration, mice in each group were intraperitoneally injected glacial acetic acid (0.6%,10 mL/kg), and the time of the first twist (namely, the twist latency)and the number of twists within 15min were observed and recorded.The main manifestations are abdominal indentation, trunk distortion,hindlimbs extension and hip elevation[13].

2.5.2 The anti-inflammatory effects

(1) Foot swelling test

Fifty SPF normal male KM mice were randomly divided into 5 groups according to body weight. Except blank group, the other groups and intervention methods were the same as 1.5.1 (1), once a day, continuous intervention for 14 days. Half an hour after the last administration, 30 μL 1% carrageenan was subcutaneously injected into the right hind foot to induce inflammation. Right rear foot volume of mice in each group was measured successively before (0 h) and 1, 2, 3 and 4 h after inflammation with paw swelling meter[14],and right rear foot swelling degree (mL)= right rear foot volume after inflammation (mL)- right rear foot volume before inflammation(mL) was calculated. Swelling rate (%)= degree of swelling/volume of right hind foot before inflammation ×100%.

(2) Ear swelling test

Ear swelling experiment induced by xylene was used to verify the anti-inflammatory effect of the drug. Forty SPF normal male KM mice were divided into the same groups and treated with the same intervention method as 1.5.1 (1), once a day, and treated continuously for 14 days. One hour after the last administration,30 μL of xylene was applied inside and outside the right auricle of mice to induce inflammation, and no drug was applied to the left ear as the control group. The mice were sacrificed for 1h after xylene intervention, and the ears of the mice were cut off along the baseline of the auricle, and the circular ear pieces were made at the same part of both ears with the help of a hole punch with a diameter of 6mm.After weighing with the help of a microelectronic scale, the swelling degree and swelling inhibition rate[15] were calculated, swelling degree (mg)= the mass of the right ear piece (mg)- the mass of the left ear piece (mg). Swelling inhibition rate (%)=[(swelling degree of mice in control group - swelling degree of mice in administration group)/ swelling degree of mice in control group]×100%

(3) ELISA

After the foot swelling experiment, the blood was removed from the eyeballs and centrifuged for storage. After standing at room temperature for 20 min, the blood was centrifuged for 15 min at 3500 r/min at 4 ℃, and the supernatant was stored in a refrigerator at-80 ℃. The contents of TNF-α, IL-1β and IL-6 were determined by ELISA kit.

(4) HE

After the foot swelling experiment, the right rear fully plantarized subcutaneous tissue was taken and placed in 4% paraformaldehyde solution, fixed at 4 ℃ for 48 h, then dehydrated in 25% sucrose solution for 24 h. The dehydrated tissue was removed, embedded in paraffin, and sectioned continuously with a paraffin slicer with a thickness of 4um, followed by HE staining. The pathological changes of subcutaneous tissues were observed under a microscope.

2.6 Statistics

SPSS26.0 statistical software was used for statistics. All values were expressed as mean ± standard deviation (±s). Normal distribution, homogeneity of variance test and one-way anOVA were used to judge the differences between groups. Use GraphPad 8.0.1 to do the graphs.

3. Results

3.1 Comparison of pain thresholds in the hot plate test of mice in each group

There was no significant difference in baseline pain threshold before administration (P＞0.05). 30 min after the last administration,compared with model group, the pain threshold of the other three groups was significantly increased (P＜0.05 or P＜0.01); Compared with process 1 and 2 groups, the pain threshold in futalin group was significantly increased (P＜0.05). 60 min after the last administration,compared with model group, the pain threshold of process 1 group and futalin group was increased (P＜0.01); Compared with process 1,the pain threshold in futalin group was increased more significantly(P＜0.05). 90 min after the last administration, compared with the model group, the pain threshold in butaline group was still significantly increased (P＜0.01), while there was an upward trend in process groups 1 and 2, but no statistical difference was found.Compared with futalin group, the pain threshold in process groups 1 and 2 was decreased (P＜0.05). 120 min after the last administration,compared with the model group, the pain threshold of futalin group was significantly increased (P＜0.01), and the pain threshold of process group had an increasing trend, but there was no statistical difference (P＞0.05). (Table 1).

Table 1 Comparison of pain thresholds in the hot plate test of mice in each group (n=10,±s)

Table 1 Comparison of pain thresholds in the hot plate test of mice in each group (n=10,±s)

Note:Compared with the model group, *P＜0.05, **P＜0.01; Compared with the Process 1 group, #P＜0.05; Compared with the Process 2 group, △P＜0.05.

Groups baseline 30 min 60 min 90 min 120 min Model 11.82±3.57 8.24±2.64 11.26±2.34 11.62±2.39 12.97±5.79 Process 1 11.89±3.50 13.33±4.38** 16.78±4.84** 15.33±5.20 15.88±5.86 Process 2 11.44±3.78 10.87±2.25* 12.89±4.39 14.30±5.68 13.87±2.72 Voltaren 11.41±3.50 15.92±4.81**# 18.44±5.25**# 20.59±8.25**# 18.90±5.43*F 0.048 7.972 5.866 4.234 2.595 P 0.986 0.000 0.002 0.012 0.067

3.2 Comparison of the latency period and number of torsional reactions of mice in various groups

The results showed that after 14 days of administration, compared with model group, the incubation period of twisting body of mice in process 1, Process 2 and futaline group was significantly prolonged,and the number of twisting body was significantly decreased (P＜0.01 or P＜0.001). Compared with process 1, the number of writhing body in process 2 group increased (P＜0.05) and the latency of writhing body decreased, but there was no statistical difference. Compared with futalin group, the incubation period of twisting body in process group 2 was shortened more obviously, and the number of twisting body in process group 2 was significantly increased (P＜0.01). (Table 2).

Table 2 Comparison of the latency period and number of torsional reactions of mice in various groups(n=10,±s)

Table 2 Comparison of the latency period and number of torsional reactions of mice in various groups(n=10,±s)

Note:Compared with the model group, *P＜0.05, **P＜0.01; Compared with the Process 1 group, #P＜0.05; Compared with the Process 2 group, △P＜0.05.

Groups the latency period/(s) number of torsional reactions/(times)Model 179.80±18.20 41.50±10.51 Process 1 329.00±64.38*** 22.20±5.71**Process 2 275.50±53.42** 29.20±7.15**#Voltaren 374.30±47.54*** 19.20±5.96***F 29.172 17.110 P 0.000 0.000

3.3 Comparison of swelling degree of right hind paw of mice in each group

Compared with the blank group, the swelling degree and swelling rate of the model group were significantly increased after the injection of carrageenan into the right hind foot of the mice (P＜0.01),indicating that the model of acute inflammation was successful.Compared with model group, process group 1, process group 2 and futalin group significantly reduced right hind foot swelling degree and swelling rate at each time point (P＜0.01 or P＜0.05); Compared with process group 1, the swelling degree and swelling rate of toes of mice in process group 2 increased 1～3 h after inflammation(P＜0.05), and the swelling degree of toes of mice in butalin group decreased more significantly 1～2 h after inflammation (P＜0.05).Compared with process group 2, foot swelling degree and swelling rate of mice in process group 1 and futalin group 1 and 1-4 hours after inflammation were decreased (P＜0.05). (Table 3).

3.4 Comparison of ear swelling and swelling inhibition rate of mice in each group

Compared with model group, the other three groups had significantinhibitory effect on the auricle swelling (P＜0.05 or P＜0.01). The inhibitory rate of auricle swelling in process group 1 was 43.86%,that in process group 2 was 25.79%, and that in butalin group was 61.27%. Compared with process group 1, the auricle swelling degree in process group 2 was increased, while that in butaline group was decreased (P＜0.05 or P＜0.01). Compared with the butaline group,the auricle swelling degree of process 2 increased (P＜0.05), while the auricle swelling degree of process 1 group increased, but there was no statistical difference. (Table 4).

Table 3 Comparison of swelling degree of right hind paw of mice in each group (n=10,±s)

Table 3 Comparison of swelling degree of right hind paw of mice in each group (n=10,±s)

Note:Compared with the model group, &P＜0.05, &&P＜0.01; Compared with the Process 1 group, *P＜0.05, **P＜0.05; Compared with the Process 2 group,△P＜0.05.

1 h 2 h 3 h 4 h Swelling (mL) Swelling rate(%) Swelling(mL) Swelling rate(%) Swelling(mL) Swelling rate(%) Swelling(mL) Swelling rate(%)Control - 0.263±0.028 0.022±0.018 7.9 0.015±0.012 5.2 0.010±0.011 3.5 0.012±0.009 4.3 Model 0.09 0.286±0.033 0.110±0.031&&& 39.5 0.118±0.037&& 41.3 0.096±0.028&& 34.5 0.071±0.016&& 25.3 Process 1 0.09 0.262±0.034 0.054±0.027*** 19.7 0.048±0.012** 17.8 0.039±0.019**Δ 14.6 0.037±0.020* 13.9 Process 2 0.09 0.259±0.032 0.062±0.029** 22.3 0.056±0.023* 21.5 0.049±0.018* 19.8 0.046±0.023* 17.6 Voltaren 0.09 0.263±0.028 0.049±0.026***Δ# 18.9 0.042±0.014**Δ# 17.1 0.034±0.013**Δ 13.9 0.030±0.010**Δ 25.3 F 1.298 15.569 32.951 29.468 18.641 P 0.285 0.000 0.000 0.000 0.000 Groups Dose(g)baseline volume(mL)

Table 4 Comparison of ear swelling and swelling inhibition rate of mice in each group (n=10,±s)

Table 4 Comparison of ear swelling and swelling inhibition rate of mice in each group (n=10,±s)

Note:Compared with the model group, *P＜0.05, **P＜0.01; Compared with the Process 1 group, #P＜0.05, ##P＜0.01; Compared with the Process 2 group,△P＜0.05.

Groups Swelling(mg) Swelling rate(%)Model 13.3±2.0 Process 1 7.5±1.9**△ 43.86 Process 2 9.9±2.9*# 25.79 Voltaren 5.1±1.4**##△ 61.27 F 27.914 ---P 0.000 ---

3.5 Comparison of TNF-α, IL-1β, IL-6 content of mice in each group

Compared with blank group, the contents of TNF-α, IL-1β and IL-6 in serum of model group were significantly increased (P＜0.01 or P＜0.001); Compared with model group, the content of IL-1β in process group 1, process group 2 and futalin group was significantly decreased (P＜0.05 or P＜0.01), the content of TNF-α in process group 1 and futalin group was significantly decreased (P＜0.01),and the content of IL-6 in futalin group was significantly decreased(P＜0.05). Il-6 in process group 1 and process group 2 showed a decreasing trend, but there was no statistical difference (P＞0.05).Compared with process 1 group, the TNF-α in futaline group was significantly decreased (P＜0.05); Compared with process 2 group,TNF-α in futalin group was decreased (P＜0.01), Il-1 β in process 1 and futalin group was decreased (P＜0.05 or P＜0.01). (Table 5)

Table 5 Comparison of TNF-α、IL-1β、IL-6 content of mice in each group (pg/mg,n=6,±s)

Table 5 Comparison of TNF-α、IL-1β、IL-6 content of mice in each group (pg/mg,n=6,±s)

Note:Compared with the control group, &&P＜0.01, &&&P＜0.001; Compared with the model group, *P＜0.05, **P＜0.01, ***P＜0.001; Compared with the Process 1 group, #P＜0.05; Compared with the Process 2 group, △P＜0.05,△△P＜0.01.

Groups TNF -α IL -1β IL-6 Control 49.10±8.12 30.12±5.56 68.00±19.29 Model 71.21±12.86&& 62.21±8.86&&& 95.08±26.36&&Process 1 60.55±15.00** 36.00±11.25** 82.15±23.55 Process 2 66.79±7.80 51.86±10.58*# 90.13±17.85 Voltaren 52.12±7.94**#△△ 34.12±6.01**△△ 73.21±27.29*F 4.552 14.349 1.464 P 0.007 0.001 0.243

3.6 Comparison of HE of Subcutaneous tissue of the right foot

The results showed that the expression of inflammatory cells such as neutrophils in subcutaneous tissues of the blank group was less,and no obvious inflammatory reaction was observed (FIG. 1C). The subcutaneous tissues in model group were swollen and thickened after inflammation, and the space was loose and widened, diffuse neutrophil infiltration, lymphocyte proliferation and aggregation,and vascular endothelial cell proliferation were observed under microscope (Figure 1M). The number of neutrophils in process 1 group and process 2 group was increased with scattered distribution,but the tendency of tissue cell aggregation in process 2 was worse than that in process 1, and vascular dilation was obvious (FIG.1A-b). After inflammation, the swelling degree of subcutaneous tissue in the futaline group was relatively mild, and a large number of myofibroblasts were infiltrated with neutrophils and lymphocytes scattered (Figure 1F). The results showed that process 1, 2 and futalin group could inhibit the paw swelling induced by xylene, and futalin group had the most obvious effect, and process 1 was superior to process 2.

Figure 1 Comparison of HE of Subcutaneous tissue of the right foot(×20, bar=100 um)Notes:C-Control, M-Model, A-Process 1, B-Process 2, F-Voltaren

4. Discussion

Hai Tong-pi decoction ointme is made by the haitongpi, tougucao,ruxiang, moyao, danggui, chuanjiao, honghua, chuanxiong,fangfeng, baizhi, weilingxian, gancao, which has the volatile oil,polysaccharides and flavonoids active ingredients. Hai Tongpi decoction ointme has the effects of antioxidation, antipyretic analgesia and anti-inflammatory bacteriostasis, in order to prevention and treatment the painful diseases, such as the neck,shoulder, waist and leg pain[16-26]. Li Lun Pian Wen said that where the effective decoction pills, can be boiled paste[27]. In order to solve the party carry inconvenience, need decoction, inadequate drug use of disadvantages, such as the team in accordance with the classic original party of the bear's skin proportion according to take drugs,prepared by two different technology into paste, including 1 process first of all drugs containing volatile oil of volatile oil and water decoction extraction save, and then mixed with water extraction solution concentration of lofty; Process 2 is complete water extraction, direct water decoction concentrated into clear cream.Valtaren is commonly used in clinical treatment of osteoarthritis diseases, which can be anti-inflammatory and analgesic, and improve the pain threshold of patients[28-30]. Therefore, futalin ointment was selected as a positive control drug in this study, and the antiinflammatory and analgesic effects of haitongpi decoction extract prepared by different technologies and its possible mechanism were preliminarily-discussed through hot plate pain, writhing reaction,foot swelling and auricle swelling experiments.

Anti-inflammatory drugs need to have analgesic activity at the same time. Hot plate pain and writhing reaction are two commonly used experiments to evaluate the analgesic potential of drugs. The results of this experiment showed that the pain threshold of process group 1, process group 2 and butalin group were significantly higher than that of model group 30min, 60min and 90min after the last administration, and the pain threshold of process group 1 was higher than that of process group 2, and there was no statistical difference between process group 1 and butalin group after 60min and 120min after the last administration. Studies have shown that intraperitoneal injection of acetic acid can cause peritoneal tension in mice, and activate the synthesis and release of inflammatory mediators such as serotonin, histamine, bradykinin and prostaglandin, and induce abdominal contraction, body distortion, back arch and reduced activity[31]. The experiment showed that haitongpi decoction extract obtained by two different processes could prolong the incubation period of writhing in mice and reduce the number of writhing in mice, and the effect of process group 1 was similar to that of butalin group. The results showed that the extract of Haitongpi decoction had peripheral analgesic effect, and the preparation method of extraction and storage of volatile oil and water decoctions of drugs containing volatile oil was better.

Xylene induced ear swelling in mice and carrageenan induced foot swelling in mice are acute inflammatory models, which have been widely used to evaluate the efficacy of anti-inflammatory drugs and screen the anti-inflammatory activity of compounds[32]. Carrageeneinduced foot edema in mice was mainly exudative edema at the early stage of inflammation. The exudative edema caused by carrageeneinduced foot edema could be divided into two stages according to the different release of inflammatory mediators. The first stage was the injection of carrageene-induced foot edema within 2 hours, and the inflammatory mediators were histamine, serotonin and bradykinin.In the second stage, 2 hours after injection of carrageenan,inflammation is mediated by the release of inflammatory substances such as prostaglandin and cyclooxygenase. In addition, the second stage is associated with local and systemic inflammation and is also a target of nsaids[33-34]. This experiment proved that the extraction cream of Haitongpi decoction prepared by process 1 and Process 2 could significantly inhibit the two stages of exudative edema induced by carrageenan and reduce the swelling degree and swelling rate of mice feet, and process 1 was superior to process 2. According to the experimental results, it is speculated that the anti-inflammatory effect of haitongpi decoction extract is related to the down-regulation of histamine, serotonin, bradykinin and prostaglandin expression and the inhibition of cytase activity.

It has been proved that TNF-α, IL-1β and IL-6 are important cytokines involved in immune response, immune regulation and inflammatory response[35]. At the same time, it has been found that TNF-α regulates cellular and humoral immunity through paracrine and autocrine forms. Excessive TNF-α can abnormally activate nuclear transcription factor (NF-κB) and other signaling pathways,which can further lead to inflammation and autoimmune diseases.In addition, a large number of pro-inflammatory cytokines including IL-1β and IL-6 can be induced[36]. In short, the excessive presence of the above cytokines will increase inflammatory mediators and form inflammatory cascade effects, so inhibiting the excessive release of cytokines is also an important indicator to evaluate the anti-inflammatory potential of drugs[37]. Clinical trials have shown that anti-TNF -α and anti-IL-1β treatments have a definite effect on rheumatoid arthritis[38]. The results of this experiment confirmed that the extract of Haitongpi decoction prepared by process 1 and process 2 can reduce the contents of TNF-α and IL-1β in serum of carrageenan-induced foot swelling model mice, and the effect of process 1 on reducing the overexpression of IL-1β was better than that of process 2.

Compared with the existing osmotic enhancers such as azone and dimethyl sulfoxide, volatile oil of Traditional Chinese medicine has strong promoting effect of transdermal absorption, little skin irritation and toxic side effects, and can also have synergistic effect with drugs absorbed through the skin. It has become a research hotspot of absorption enhancers of transdermal delivery[39]. 34 volatile oils of TCM has been used as transdermative infiltration enhancers, including danggui, chuanjiao, honghua, baizhi, fangfeng,etc[40]. The experimental results indicate that the anti-inflammatory and analgesic effect of haitongpi decoction extract prepared by process 1 is better than that of process 2, and the biggest difference between process 1 and process 2 lies in the extraction and storage of volatile oil of Traditional Chinese medicine. Therefore, it is inferred that the single extraction and storage of volatile oil of traditional Chinese medicine is the main reason why process 1 is better than process 2.

In conclusion, the extracts of Haitongpi decoction prepared by two different processes have certain anti-inflammatory and analgesic effects, which may be caused by inhibiting the release of proinflammatory cytokines such as TNF-α, IL-1β and IL-6, especially IL-1β. In terms of process preparation, the anti-inflammatory and analgesic effect of process 1 is better than that of process 2, which may be related to the separate extraction and storage of volatile oil of Traditional Chinese medicine, and this process ensures that the volatile oil has a long aging pharmacological activity.

Statement of Conflict of interest

All authors declare no conflict of interest.

Author contribution

The experiment was designed by LI Fa-jie and GU Jin-yu. LI Fajie, GU Jin-yu, WAN Tian-hao, XIA Di responsible for animal modeling, drug intervention, sampling, index detection; SHEN Shuo and YAO Yao prepared the extract of Haitongpi by different techniques. ZHANG Yue conducted statistical analysis of data; LI Fa-jie wrote the article, and ZHANG Qing was reviewed by the corresponding author.