Xue-Ying Yu ,Shi-Bo Zhao ,Hui-Man Yang ,Han-Jun Ma ,Xue-Wei Huang ,Peng-Yu Deng ,Shi-You Zhou,Suo-Yi Huang

1College of Clinical Medicine,Youjiang Medical University for Nationalities,Baise 533000,China.2College of Pharmacy,Youjiang Medical University for Nationalities,Baise 533000,China.3Key Laboratory of Guangxi Universities on National Medicine in Youjiang River,Youjiang Medical University for Nationalities,Baise 533000,China.

Abstract Objective: This study investigates the in vivo anti-inflammatory and analgesic pharmacological effects of white peony root and mucuna pruriens and their combinations.Methods: In this study,the corresponding solutions were administered in groups,and after 14 days of gavage,xylene-induced ear swelling test,acetic acid torsion test and hot plate pain test were performed.Results:It was found that both Paeonia lactiflora root and Kapok had anti-inflammatory and analgesic effects (P <0.05),with the 2:1 group having the best experimental effect (P <0.05).Conclusion:Paeonia lactiflora root and Kapok and its combination have good anti-inflammatory and analgesic effects in vivo,and the 2:1 group showed significant anti-inflammatory and analgesic effects compared with other groups.

Keywords:white peony root;papaya;aqueous extract;anti-inflammatory effect;analgesic effect

Highlight

The aim of this study was to investigate the in vivo anti-inflammatory and analgesic pharmacological effects of Paeonia lactiflora root and Kapok and their combination.In the study,it was found that the results of Paeonia lactiflora root and Kapok and their combination were highly significant in ear swelling test of xylene,acetic acid twisting test and hot plate pain test in mice,which proved that the combination of both was significantly better than the single drug in terms of in vivo anti-inflammatory and analgesic effects.

Background

White peony (Radix Paeonia Alba),also known as golden peony,is the dried root of peony (Paeonia Lactiflora Pall) in the buttercup family,which is bitter and cold in taste,with blood nourishing and pain relief,calming the liver Yang effects [1–2].It is commonly used in the treatment of headache and dizziness,abdominal pain and dysesthesia,painful contracture of the limbs [3].Modern pharmacological research on the root of Paeonia lactiflora has confirmed the diversity of its pharmacological effects,such as anti-inflammatory and analgesic,immune anticonvulsant,etc [4].The whole flower of Bombaxm alabaricum DC.is flat and pungent in nature,with the effects of clearing heat and cooling the blood,relieving dampness and toxicity,activating blood circulation and reducing swelling,mainly treating chronic gastritis and leg and knee pain [5–6].

In this experiment,the anti-inflammatory and analgesic effects of Paeonia lactiflora root and Kapok and their combination were investigated by auricular swelling test,acetic acid torsion test and hot plate pain-inducing method.By studying the anti-inflammatory and analgesic effects of Paeonia lactiflora root and Kapok and their combinations on mice,we aim to provide a research basis for the development of both.

Material

Experimental animal grouping and drug administration

Healthy Kunming breed (SPF) grade mice,weighing (18 ± 22) g,were provided by the Experimental Animal Center of the Right River School of Ethnic Medicine,license number:SCXK Gui 2012–0003.60 mice,half male and half female,were randomly divided into 6 groups in a 1:1 male to female ratio for each experiment.They were the control group,white peony root group,kapok group,white peony root to kapok volume ratio 1:1 group,white peony root to kapok volume ratio 1:2 group and white peony root to kapok volume ratio 2:1 group(hereafter referred to as 1:1 group,1:2 group and 2:1 group).Ethical approval was obtained from Youjiang Medical University for Nationalities,Number 201910599012.

The body weights of the mice were measured after 1 week of adaptive feeding.To ensure the accuracy of the gavage dose,the body weights were measured every 3 days during the experiment.In the experiment,each group was given the Chinese medicine extract according to 20 mL/kg of body weight,that is,a dose of 20 g/kg (the mass concentration of the drug solution was 1 g/mL,1mL of the drug solution contained 1g of Chinese medicine,and the gavage volume was 20 mL/kg,the dose was 20 g/kg).The control group was given 0.9% saline in the same volume,and the remaining groups were given the corresponding Chinese medicine extract by 24-hour gavage for a continuous period of 14 days.

Experimental equipment and consumables

RE-3000A rotary evaporator (Shanghai Yarong Biochemistry Instrument Factory);DLSB-5 low-temperature coolant circulation pump (Zhengzhou Greatwall Scientific Industrial and Trade Co.,Ltd.);HH-Z4 digital display constant temperature water bath (Jintan Chengdong Radiance Instrument Factory);RB-200 intelligent hot plate instrument (Chengdu Taimeng Software Co.,Ltd.).FA1204B electronic balance (Shanghai Jingke Tianmei Instrument Co.,Ltd.);TH-UP-10 laboratory special ultra-pure water machine (Shandong Tenghai Analytical Instrument Co.,Ltd.);clean grade laboratory rat maintenance feed (Jiangsu Synergy Pharmaceutical and Bioengineering Co.,Ltd.).

Herbs

Drug IdentificationWhite peony root and kapok were identified by experts and confirmed to be the dried root of Paeonia lactiflora Pall(buttercup family) and the dried whole flower of Bombaxm alabaricum.

Drug ExtractionPreparation of white peony root aqueous extract:taking 500 g of dried white peony root crushed,adding distilled water at a volume ratio of 1:8,decocting 3 times for 1 hour each time,combining all filtrates and filtering again,rotating and evaporating after filtration,concentrating into an infusion,and preparing a solution of 1 g/mL of raw drug with distilled water.After natural cooling,the solution was refrigerated at 4 °C,and configured into the desired concentration of white peony root aqueous extract before the start of the experiment.

Preparation of Kapok aqueous extract:taking 500 g of dried whole Kapok plant and pounding it,adding distilled water at a volume ratio of 1:10,soaking for 2 hours and then heating in a constant temperature water bath at 80 °C for 1 hour,filtering out the medicinal solution,combining all the filtrates after 2 extractions,concentrating by rotary evaporation to the form of an infusion,and preparing the solution with distilled water in the amount of 1 g/mL of the raw drug.After natural cooling,the solution was refrigerated at 4 °C and configured into the required concentration of water extract of mucuna pruriens before the experiment started.

Methodology

Experimental study on the effect of white peony root and mucuna pruriens and their combination on xylene-induced ear swelling

Sixty mice (male and female) of SPF grade were grouped,administered and their weight measured according to "1.1",and the auricular swelling test was performed after 14 days of continuous gavage administration.Xylene was applied to the left ear of the mice at a dosage of 50 μL/each,and the right ear was used as the control 1 hour after the last administration.After 15 minutes,the cervical vertebrae were dislocated and executed,and holes were punched along the same part of the left and right auricles respectively with an 8 mm diameter punch.

Swelling inhibition rate (%)=(mean value of model group -mean value of drug administration group)/mean value of model group ×100%

Experimental study on the analgesic effect of white peony root and mucuna pruriens and their combination

Effect of white peony root and mucuna pruriens and their combination on acetic acid torsion experimentSixty mice (male and female) of SPF grade were grouped,administered and their body weight was measured according to "1.1",and 0.2 mL of 0.6% acetic acid solution was injected intraperitoneally 30 minutes after the last dose.The inhibition rate [7] of writhing response was calculated according to the following formula.

Inhibition rate of torsional response (%)=(average number of torsions in the model group–the average number of torsions in the administration group)/average number of torsions in the model group× 100%

Effect of white peony root and mucuna pruriens and their combination on hot plate pain-causing experimentsThe mice were placed on the hot plate apparatus at (55 ± 0.5) °C,and the time(s) between contact with the hot plate and the mice licking the hind feet or jumping response was used as the pain threshold of the mice.The pain threshold of each mouse was remeasured twice and the average value was taken as the base pain threshold of the mouse before drug administration.After 14 days of continuous gavage administration,a hot plate pain test was performed and the time taken for the mice to lick their hind feet was recorded.At 1 hour after the last administration,the pain thresholds of the six groups of mice were measured by the same method,and the rate of improvement in pain thresholds was calculated according to the following formula.

Pain threshold improvement rate (%)=(average pain threshold after drug administration -average pain threshold before drug administration)/average pain threshold before drug administration ×100%

Statistical methods

The experimental data of each group were expressed as x±s,and statistical analysis was performed using the statistical software SPSS 17.0.Comparisons between the two experimental groups were performed using ANOVAt-test,and differences between multiple groups were made by one-way ANOVA with a test level of α=0.05.

Results and Analysis

Anti-inflammatory effect of white peony root and mucuna pruriens and their combination on a xylene-induced auricular swelling experiment in mice

The inhibition rate of ear swelling in mice caused by xylene was compounded by white peony root and mucuna pruriens and their combination.The three combination groups all had different degrees of inhibition on ear swelling in mice,among which the best inhibitory effect was observed in the combination 2:1 group,which had an obvious anti-inflammatory effect,as shown in Table 1.

Effect of white peony root and mucuna pruriens and their combination on the torsional response of mice to glacial acetic acid

A compound study was conducted on the inhibitory effect of white peony root and mucuna pruriens and their combinations on the inhibition rate of twisted body reaction in mice induced by glacial acetic acid,as shown in Table 2.The three combination groups had different degrees of inhibition on the twisted body reaction in mice,among which the 2:1 combination group showed the best inhibitory effect,with obvious analgesic effect,as shown in Table 2.

Effect of white peony root and mucuna pruriens and their combination on pain response in mice caused by hot plate

White peony root and kapok and their combination had a compound effect on the improvement rate of pain threshold in mice caused by the hot plate method.Compared with the control group,white peony root group and kapok group,the three combination groups all had different degrees of improvement on the pain threshold of mice,among which the combination 2:1 group had the best effect with an improvement rate of 66.81% in the pain domain and had obvious analgesic effects,as shown in Table 3.

Table 1 Effect of white peony root and mucuna pruriens and their combination on xylene-induced auricular swelling in mice (x±s,n=10)

Table 2 Effect of white peony root and mucuna pruriens and their combinations on glacial acetic acid-induced torsional response in mice (x±s,n=10)

Table 3 Effect of white peony root and mucuna pruriens and their combination on pain response in mice caused by a hot plate in mice (x±s,n=10)

Discussion and Outlook

Inflammation is a common pathological response of the body against damage when it is subjected to harmful stimuli and is a complex defense response involving multiple cells and factors,that is,damage caused by inflammatory factors,the body's defensive response and tissue damage recovery.It is clinically characterized by localized redness,swelling,heat,pain and loss of function [8-10].In the early stage of inflammation,inflammatory mediators secrete inflammatory factors such as interleukin 1β (IL-1β) and interleukin 6,causing capillary dilation,increased permeability,and leakage of fluid,proteins and leukocytes from the plasma into the tissue spaces and causing tissue swelling [11–13].Pain is an unpleasant sensation that occurs after tissue injury and is the most common clinical manifestation,and reducing pain is one [13–15] of the ways to improve the comfort of life.Both pain and inflammation are protective responses of the organism to injurious stimuli,while acetic acid-induced pain in the abdominal mucosa of mice is an acute pain[16–17].The anti-inflammatory mechanism of white peony root may be related to the upregulation of PTGS2,and that of mucuna pruriens may be related [18–19] to the regulation of miR-30b-3p/PDCD5 expression.Paeonia lactiflora root and its extracts TGP and PF can exert analgesic effects by regulating prostaglandin (PG) and receptors as well as related signal transduction pathways in vivo,and methanolic extract of kapok and mangiferin have good anti-inflammatory and analgesic activities [20–21].At present,clinically applied anti-inflammatory drugs are mainly divided into adrenocorticosteroid steroidal anti-inflammatory drugs,and non-steroidal anti-inflammatory drugs (NSAID),such as aspirin,celecoxib and other antipyretic and analgesic anti-inflammatory drugs.NSAIDs are mainly used to indirectly inhibit leukocyte aggregation by inhibiting prostaglandin synthesis,effectively reducing the formation of kinin substances,and thus achieving anti-inflammatory purposes [22] by inhibiting platelet agglutination.This study investigated the pharmacological effects of white peony root and mucuna pruriens and their combinations by using the classical inflammatory response model and the nociceptive response model,respectively.In this experiment,the anti-inflammatory effect was observed by the xylene ear swelling test;the analgesic effect was observed by the glacial acetic acid twisting test and the hot plate nociceptive test;all the experimental groups showed anti-inflammatory and analgesic effects compared with the control group,and the differences were statistically significant.The results showed that compared with the control group,the anti-inflammatory and analgesic effects were observed in the white peony root group,the mucuna pruriens group,the combination 1:1 group,the combination 1:2 group and the combination 2:1 group.Among them,the torsion inhibition rate and the pain threshold improvement rate of the combination 2:1 group were 86.76% and 66.81%,respectively,which were significantly different compared with other groups,and their anti-inflammatory and analgesic effects were the most significant.The characteristics and mechanisms of the anti-inflammatory and analgesic effects of Paeonia lactiflora root and Mucuna pruriens and their combinations were studied in depth so as to maximize their potential value and make them safer,more effective and more widely used in clinical practice.