Experimental Study on Analgesia and Hemostasis of Paris polyphylla, Scutellaria baicalensis and Their Compatibility

Shibo ZHAO, Suoyi HUANG, Zhenjing SI, Lanlan GAN, Shusheng HUANG, Ye FU, Jianjie LAN, Xueying YU, Yuekong CEN

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

Abstract [Objectives] The analgesic and hemostatic effects of Paris polyphylla, Scutellaria baicalensis, and their compatibility were studied. [Methods] (i) In the experiment on the writhing of mice induced by acetic acid, 60 Kunming SPF mice were randomly selected, and were randomly divided into 6 groups according to the male-to-female ratio of 1∶1, including blank control group, P. polyphylla group, S. baicalensis group, and three P. polyphylla and S. baicalensis compatibility groups (with the ratio of 1∶2, 1∶1 and 1∶2). There were 10 mice in each group. In the experimental groups, the mice were given 1 g/mL Chinese medicine extract according to a dose of 20 mL/kg. In the control group, the mice were given 0.9% normal saline in equal volumes. Gavage was performed one time every 24 h and lasted for 14 d. 1 h after the gavage on the 14th day, they were given 0.5% glacial acetic acid solution via intraperitoneal injection. (ii) In the hot plate experiment, 60 Kunming SPF mice were selected, and the ratio of male to female, grouping, administration and gavage were the same as those of the glacial acetic acid-induced writhing experiment. Gavage was lasted for 14 d. 1 h after the gavage on the 14th day, the mice were placed on a hot plate apparatus at (55±0.5) ℃, and the time of licking hindfoot was measured. (iii) The hemostatic effect was explored through three experimental methods of tail hemostasis, femoral artery hemostasis and liver hemostasis. 60 Kunming SPF mice were taken in each of the three experiments, and the male-female ratio and grouping were the same as above. The powder was covered on the surface of the wound sites, and the bleeding was observed. The hemostatic time was recorded, and the hemostatic time was recorded as 3 min if it exceeded 3 min. [Results] (i) In the acetic acid-induced writhing experiment, compared with the blank control group, the pain of mice in the experimental groups was inhibited. Among them, P. polyphylla group, S. baicalensis group, compatibility 1∶2 group, compatibility 1∶1 group, and compatibility 2∶1 group had significant effects (P<0.05), and the inhibition rate of writhing was 20.43%, 28.32%, 52.30%, 32.79 %, and 39.02%, respectively. The analgesic effect of the compatibility 1∶2 group was the most obvious. (ii) In the hot plate experiment, compared with the blank control group, the experimental groups had analgesic effect. Among them, P. polyphylla group, S. baicalensis group, compatibility 1∶2 group, compatibility 1∶1 group, and compatibility 2∶1 group had significant effects (P<0.05), and the increase rate of pain threshold was 29.17%, 47.83%, 61.54%, and 50.61%, and 53.83%, respectively. The analgesic effect of the compatibility 2∶1 group was the most significant. (iii) In the hemostatic experiment, there was a significant difference in hemostatic time between the compatibility 1∶2 group and the blank control group, P. polyphylla group, and S. baicalensis group (P<0.05). The hemostatic effect of the compatibility 1∶2 group was the best. [Conclusions] P. polyphylla, S. baicalensis, and its compatibility had good analgesic and hemostatic effects, and the best compatibility ratio was 1∶2.

Key words Paris polyphylla, Scutellaria baicalensis, Compatibility, Analgesic effect, Hemostatic effect

1 Introduction

Parispolyphyllais dried rhizomes of the Liliaceae plantParispolyphyllaSmith var. yunnanensis (Franch.) Hand.-Mazz. orParispolyphyllaSmith var. chinensis (Franch.) Hara[1]. It is slightly cold and poisonous, has a bitter taste, and belongs to the liver meridian; it has a variety of pharmacological activities such as relieving cough and asthma, anti-inflammation and analgesia, antibacterial and antiviral effects, hemostasis and protecting the liver and kidney[2-3]. Yao Qinetal.[4]found that the alcohol extract ofP.polyphyllahas a significant analgesic effect. Modern pharmacological studies have shown that it can significantly inhibit the edema of a mouse’s feet induced by carrageenan and shorten the bleeding time and coagulation time of the mouse[5].Scutellariabaicalensisis a perennial herb of the Labiatae, and its roots are mainly used as medicine. It is cold and bitter, and mainly has the effects of tocolysis, hemostasis and cooling blood, detoxification and reducing fire, clearing heat and eliminating dampness. It occupies an important position in traditional Chinese medicine, the preparations of dried roots ofS.baicalensisare used to treat liver and lung diseases and in the adjuvant treatment for cancer[6-8].

As traditional Chinese medicine,P.polyphyllaandS.baicalensis, which are also edible, have been used for thousands of years, and are eaten in the form of medicated diet, soup, and tea. A single drug has been studied and applied very extensively, but there is no related report on the analgesic and hemostatic effects of the compatibility ofP.polyphyllaandS.baicalensis. Based on the research of many scholars and modern pharmacological theory, Kunming mice were as the research object to investigate the analgesic and hemostatic effects ofP.polyphylla,S.baicalensisand their compatibility, and to study whether the compatibility of the two had good synergy in this study. Moreover, the optimal ratio of the two in the body after being eaten was studied to provide theoretical basis for in-depth research of edible development and clinical application ofP.polyphyllaandS.baicalensis.

2 Materials and Methods

2.1 MaterialsThe main instruments included RB-200 intelligent hot plate meter (Chengdu Taimeng Software Co., Ltd.), TWCL-T 5000 magnetic stirrer (Shanghai Biaohe Instrument Co., Ltd.), DLSB-5 low-temperature coolant circulation pump (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.), JSC electronic balance CN-BH (Fuzhou Kedi Electronic Technology Co., Ltd.), FA1204B electronic balance (Shanghai Jingke Tianmei Instrument Co., Ltd.), and JMF-320G multi-stage flash evaporator (Henan Zhijing Biological Technology Co., Ltd.

P.polyphylla(batch No.170801) andS.baicalensis(batch No.170301) were from Chinese Medicine Drink Factory of Guangxi Guigang Luzhiyuan Breeding Development Co., Ltd.; the clean feed of experimental mice was from Jiangsu Province Synergy Pharmaceutical Bioengineering Co., Ltd.

2.2 Experimental animals and their groupingThere were 300 SPF-class Kunming mice, with the male-to-female ratio of 1∶1 and the body weight of 18-22 g. The age was unknown. They were provided by the Experimental Animal Center of Youjiang Medical University for Nationalities in Baise, Guangxi, and the license number is SCXK Gui 2012-0003.

There were 60 mice in each experiment. They were randomly grouped according to the male-to-female ratio of 1∶1, and there were 10 mice in each group. They were marked with 5% picric acid and divided into 6 groups by random number table method, including blank control group,P.polyphyllagroup,S.baicalensisgroup,P.polyphyllaandS.baicalensiscompatibility group (1∶2),P.polyphyllaandS.baicalensiscompatibility group (1∶1), andP.polyphyllaandS.baicalensiscompatibility group (2∶1) (hereinafter referred to as compatibility 1∶1 group, compatibility 1∶2 group, and compatibility 2∶1 group).

2.3 Identification of medicinal materialsThe traditional Chinese medicineP.polyphyllaandS.baicalensiswere identified by Professor Huang Suoyi, director of the Scientific Experimental Center of Youjiang Medical University for Nationalities, and identified as the dried rhizomes of the Liliaceae plantP.polyphyllaSmith var. yunnanensis (Franch.) Hand.-Mazz. orP.polyphyllaSmith var. chinensis (Franch.) Hara and the dried roots of the Labiatae plantS.baicalensis.

2.4 Extraction of drugsAccording to the method to extractP.polyphyllawith alcohol[9], 500 g ofP.polyphyllapowder was weighed and ultrasonically extracted 3 times with methanol. The filtrates were merged, concentrated, frozen, and dried to obtain dry drug powder. Distilled water was used to make a solution with crude drug concentration of 1 g/mL.

Firstly, 500 g ofS.baicalensispowder was taken and decocted for 1 h after 10-time water was added. Secondly, it was cook for 1 h after 8-time water was added. The two extractions were merged and filtered. It was concentrated to a decoction with crude drug concentration of 1 g/mL in a water bath. After autoclaved sterilization, the two extracts were stored in a refrigerator at 4 ℃ for later use.

2.5 Animal grouping and administration of drugsThe body weight of the experimental animals was measured after one week of adaptive feeding. In order to ensure the accuracy of intragastric administration, the body weight was measured every 3 d during the experiment. In the experiment, each group was given a corresponding medicinal solution according to a dose of 20 mL/kg (the mass concentration of the medicinal solution was 1 g/mL, that is, 1 mL of the medicinal solution contained 1 g of the traditional Chinese medicine; the volume was 20 mL/kg, and the dose was 20 g/kg). The blank control group, namely the normal saline group, was given 0.9% normal saline in equal volumes. Gavage was performed one time every 24 h and lasted for 14 d. Each animal experiment complied with the requirements of animal experiment ethics.

2.6 Experiment on the effect ofP.polyphylla,S.baicalensisand their compatibility on the writhing reactions of mice induced by glacial acetic acidSixty mice were taken, and the ratio of males to females, grouping, administration, and body weight determination were the same as those in Section2.5. 30 min after the last administration, each mouse was given 0.5% glacial acetic acid solution via intraperitoneal injection according to the dose of 20 mL/kg. The first writhing time of the mice and the frequency of their writhing reactions were recorded within 30 min after the injection (note: the mice had a writhing reaction when they had concave abdomen, extending trunk and hind legs, and rising buttocks). The inhibition rate of the writhing reactions was calculated according to the following formula: inhibition rate of the writhing reactions (%) = (average number of writhing times in the control group-average number of writhing times in the administration groups)/average number of writhing times in the control group×100%.

2.7 Experiment on the effect ofP.polyphylla,S.baicalensisand their compatibility on the pain reactions of mice induced by a hot plateSixty mice were taken, and the ratio of males to females, grouping, administration, and body weight determination were the same as above. Before the start of the experiment and 1 h after the last administration, the mice were placed on a hot plate [temperature was (55±0.5) ℃], and the time required for the mice from contacting the instrument to licking the hind feet (s) was recorded immediately and as the pain threshold of the mice. Moreover, the increase rate of the pain threshold was calculated according to the formula as follows: increase rate of the pain threshold (%) = (average pain threshold after the administration-average pain threshold before the administration)/average pain threshold before administration×100%.

2.8 Experiments on the hemostatic effect of P. polyphylla, S. baicalensis and their compatibility on mice

2.8.1Experiment on the hemostasis of tail-broken mice. 30 male mice and 30 female mice were taken, and the tail was cut off 2 mm from the tip of the tail. After the first drop of blood flowed out, the tails of mice in the experimental groups were put in the corresponding medicine powder, and the powder was in full contact with the tail tips of the mice. Meanwhile, the timing was started. The tails were taken out to observe the bleeding, and the bleeding was stopped when the blood no longer overflowed. At the same time, the hemostasis time of the mice was recorded. In the blank control group, the tail-broken mice bled freely until the hemostasis, and the hemostasis time of the mice was recorded. The hemostasis time of the mice that bled for more than 3 min was recorded as 3 min.

2.8.2Experiment on the hemostasis of femoral artery of mice. Firstly, 30 male mice and 30 female mice were anesthetized with 10% urethane (1 300 mL/kg). Afterwards, the skin on the inside of the right lower limb of the anesthetized mice was cut, and the tissue was cut by blunt dissection to expose the femoral artery. The femoral artery was cut off, and an appropriate amount of powder was applied quickly to the artery to stop bleeding in the experimental groups. The timing was started after the application, and the hemostatic effect of the drugs within 3 min was observed. The bleeding mice in the control group were allowed to bleed naturally. The hemostasis time of all the mice that bled for more than 3 min was recorded as 3 min.

2.8.3Experiment on the hemostasis of the liver of mice. At first, 30 male mice and 30 female mice were anesthetized with 10% urethane (1 300 mL/kg). Afterwards, the abdomen of the anesthetized mice was dissected to fully expose the liver, and a piece of gauze was placed under the liver. The large and small leaves of the liver were gently pull out and spread on the gauze. A scalpel was used to cut a wound of about 0.4 mm in the right large lobe of the liver. After it bled for 10 s, an appropriate amount of medicine powder was sprinkled on the wound in the experimental groups. Meanwhile, the timing was started, and the bleeding was observed. When the powder was no longer infiltrated by the blood and crusts were formed on the surface of the powder, the blood had been stopped, and the hemostasis time was recorded. In the control group, after the wound was cut on the large lobe of the liver, and no treatment was performed. The hemostasis time of the mice that bled for more than 3 min was recorded as 3 min.

3 Results and Analysis

3.1 Effects ofP.polyphylla,S.baicalensisand their compatibility on the writhing reactions of mice induced by glacial acetic acidP.polyphylla,S.baicalensisand their compatibility had a synergistic effect on the first writhing time, the number of writhing times within 30 min, and the inhibition rate of writhing reactions of mice induced by glacial acetic acid (Table 1). The compatibility 1∶2 group had an obvious inhibition effect, and the inhibition rate of writhing reactions was 52.30%. It had a good inhibition effect on the writhing reactions of mice induced by acetic acid.

Note: a.P<0.05 (compared with blank control group); b.P<0.05 (compared withP.olyphyllagroup); c.P<0.05 (compared withS.baicalensisgroup).

3.2 Effects of P. polyphylla, S. baicalensis and their compatibility on the pain reactions of mice induced by a hot plate

P.polyphylla,S.baicalensisand their compatibility had a synergistic effect on the time of licking hind feet and the increase rate of pain threshold of pain reactions of mice induced by a hot plate. The compatibility 1∶2 group had a significant effect (P<0.05), and the increase rate of pain threshold was 61.54% (Table 2).

Note: a.P<0.05 (compared with blank control group); b.P<0.05 (compared withP.olyphyllagroup); c.P<0.05 (compared withS.baicalensisgroup).

3.3 Hemostatic effect ofP.polyphylla,S.baicalensisand their compatibility on miceBothP.polyphyllaandS.baicalensishad a good hemostatic effect, and the hemostatic effect of the two mixed in proportion had a synergistic effect in time. Table 3 showed that the bleeding of mice in the three compatibility groups was inhibited to a certain degree. The hemostatic effect of the compatibility 1∶2 group was more significant than that of the other two groups (P<0.05).

GroupHemostasis timeof broken tail Hemostasis timeof femoral arteryHemostasis timeof the liverBlack control 162.076±21.013145.109±30.296131.666±39.417P. polyphylla114.300±32.301122.045±40.790105.084±35.213aS. baicalensis120.572±31.047134.687±45.353120.274±26.204Compatibility 1∶282.285±22.719abc87.679±43.712abc73.700±22.469abcCompatibility 1∶199.093±25.476a110.829±36.388ac87.709±34.341abCompatibility 2∶194.898±26.182ac98.406±43.015ac81.662±19.826a

Note: a.P<0.05 (compared with blank control group); b.P<0.05 (compared withP.olyphyllagroup); c.P<0.05 (compared withS.baicalensisgroup).

4 Discussion and Outlook

The main ingredients ofP.olyphyllainclude steroidal saponin, ecdysteroid, sterol, polysaccharide, flavone, fatty acid ester,etc. They are also rich in β-aminoisobutyric acid, alanine, γ-aminobutyric acid, serine, aspartic acid, and trace elements such as Mg and Ca[10]. Liu Fangetal.[11]mentioned that the main active ingredient ofP.olyphyllaused for analgesia and hemostasis is steroid saponin, which accounts for more than 80% of total compounds.S.baicalensishas many components, and flavonoid is its main active ingredient[12-13]. Wu Tingetal.[14]mentioned the hemostatic and analgesic effects ofS.baicalensisin the comparative study of its main components before and after being processed. The pain of mucous membranes in the abdominal cavity of mice induced by acetic acid is similar to acute sharp pain[15], and the pain induced by a hot plate belongs to an acute heat pain model of mice[16]. The primary center reflexed by the two models is located in the spinal cord, and the reflection arc is relatively simple; the correspondence between stimulus and response is good, so the above two methods are used[17]. Previous studies confirmed thatP.polyphyllaandS.baicalensishad anti-inflammatory and analgesic effects. This experiment also confirmed thatP.polyphyllaandS.baicalensishad anti-inflammatory and analgesic effects in single use, and their compatibility had an obvious synergistic effect. The results of this experiment showed that the number of writhing and the time of licking hind feet of mice in theP.polyphyllaandS.baicalensisgroups reduced compared with the control group. The analgesic and hemostatic data of the compatibility 1∶2, 1∶1, and 2∶1 groups were significantly lower than those of the control group. The data of the three compatibility groups indicated that the compatibility groups had obvious analgesic and hemostatic effects than the control group. In the analgesic experiment, the compatibility groups had significant analgesic and hemostatic effects compared with the control group, and the effect of the compatibility 1∶2 group was the best.

Compared with the control group,P.polyphyllaandS.baicalensisgroups, the inhibition rate of writhing and the increase rate of pain threshold were 52.30% and 61.54%, respectively. The analgesic effect of the compatibility groups was the best, showing a synergistic effect. In the hemostatic experiment, the administration groups had a certain inhibition effect on the bleeding of mice. The hemostatic effect of the compatibility 1∶2 group was more obvious than that of the other experimental groups,P.polyphyllaandS.baicalensisshowed a better synergistic effect in the hemostatic experiment. Compared with the use of a single drug, the compatibility 1∶1 and 2∶1 groups did not show a more significant synergistic effect. PGE2, NO, and TNF-α are important inflammatory mediators. According to the content of PGE2, NO, and TNF-α, the severity of inflammation can be clearly determined[18]. Clinically, it is often treated with non-steroidal antipyretic and analgesics,e.g. aspirin.

As a potent analgesic and hemostatic substance, the steroidal saponin inP.polyphyllahas no clear mechanism of analgesia and hemostasis, which may be related to the inhibition of the expression of inflammatory factors. The analgesic effect ofS.baicalensisis related to its inhibition of PGE2, LTB4, and LTC4 synthesis as well as cytokine secretion[19], so the analgesic mechanism of their compatibility may be related to the inhibition of the expression of multiple inflammatory factors by two drugs. The exact mechanism of analgesia needs further study.

In summary, the experiments verified that the alcohol extract ofP.polyphylla, the water extract ofS.baicalensis, and their compatibility medicinal liquids had good analgesic and anti-inflammatory effects, andP.polyphylla,S.baicalensis, and their compatibility were effective for acute neurogenic pain, inflammatory pain and acute inflammation, and the effects were better than that of a single drug. The development of the mechanism and medicinal value ofP.polyphylla,S.baicalensis, and their compatibility still needs in-depth research.