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Abstract ［Objectives］ This study was conducted to investigate the antitussive and anti-inflammatory effects of Miao medicine Aspidistra caespitosa C. Pei.
［Methods］A mouse cough model was made by the SO2 cough induction method, and the antitussive effects of different extraction fractions of A. caespitosa were observed. The inflammation models of acute inflammatory ear swelling in mice caused by xylene, granuloma in mice caused by cotton balls, and footpad swelling in mice caused by carrageenan were made, and the different extraction fractions were used to observe the effects of different extraction fractions on mouse ear swelling, granuloma and footpad swelling. And the levels of interleukin 6 (IL-6) and tumor necrosis factor (TNF-a) in the serum of different groups of mice were detected to screen out the extraction fractions of A. caespitosa with good antitussive and anti-inflammatory activity.
［Results］ Antitussive experiments on mice showed that the water layer extraction fraction, ethyl acetate extraction fraction and petroleum ether extraction fraction of Miao medicine A. caespitosa could prolong the incubation period of cough in mice and reduce the number of coughs within 2 min to varying degrees, and its different extraction fractions all had different inhibitory effects on mouse ear swelling, granuloma and footpad swelling. Compared with the blank control group, the different extraction fractions could reduce IL-6, TNF-a and other indicators to different degrees.
［Conclusions］Ethyl acetate extraction fraction of A. caespitosa has good antitussive and anti-inflammatory effects.
Key words Aspidistra caespitosa; Antitussive effect; Anti-inflammatory effect; Screening
Received: June 10, 2021 Accepted: August 15, 2021
Supported by Scientific and Technological Research Project of Traditional Chinese Medicine and Ethnic Medicine of Guizhou Administration of Traditional Chinese Medicine (QZYY2017-116); Scientific Research Project of Guizhou University of Traditional Chinese Medicine (GZYKYN［2017］28).
Yongyue GAO (1986-), male, P. R. China, associate chief pharmacist, master, devoted to research about ethnic medicine.
*Corresponding author. E-mail: 125193312@qq.com.
With the acceleration of the pace of life and the aggravation of environmental pollution, climate change and population aging, the incidence and mortality of cough and respiratory system inflammation are increasing year by year, and the harm to the human body cannot be ignored［1］. A large number of studies have proved that Chinese medicine can significantly improve various lung diseases［2］. Miao medicine Aspidistra caespitosa C. Pei is a species included in Guizhou Chinese Herbal Medicine Resources. It refers to the whole plant of Aspidistra in Liliaceae. It is widely distributed in Guizhou, Yunnan, Guangxi and other places. A. caespitosa is mainly used for cough, acute and chronic bronchitis, and chronic pharyngitis in Guizhou Miao medicine［3］, and thus has high medicinal value and development prospects. Therefore, in order to further study its mechanism of action and lay a foundation for follow-up research and new drug development, its anti-tussive and anti-inflammatory effects were investigated in this study conducted studies. Materials and Methods
Materials
Experimental animals
KM mice, SPF grade, half male and half male, weighing (20±2) g, animal quality certificate number: No: 00200406, purchased from the Experimental Animal Center of Guizhou Medical University, license number: SCXK-(Q) 2012-001; Feed: full-price pellet feed provided by the Experimental Animal Center of Guizhou Medical University; feeding conditions: clean and dry litter, temperature: 16-22 ℃, humidity: 45%-75%, ventilation 10-18 times, lighting: 8-12 h.
Control drug
Yan Guo Zhi Ke Ye (Guang Zheng Pharma Co., Ltd., National Medicine Standard: Z20013188, product batch number: HAF0416), adult dosage: 20 ml once, 3 times a day, which is equivalent to 1 ml/kg.
Experimental reagents and instruments
99% sulfuric acid, Chengdu Kelong Chemical Test Site, lot number: 20120704; anhydrous sodium sulfite, Chongqing Jiyuan Chemical Reagent Co., Ltd., lot number: 20170326; xylene, Shanghai Qiangshun Chemical Reagent Co., Ltd., lot number: 20120122; sodium chloride injection, Sichuan Kelun Pharmaceutical Co., Ltd., lot number: 20180115; carrageenan, imported from Fluka, Switzerland, lot number: CH-9826; electronic balance, Shanghai Jinghai Instrument Co., Ltd.; table centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; electric thermostatic drying oven, Tianjin Taisite Instrument Co., Ltd.; vernier caliper, Shandong Meike Tools Co., Ltd.
Methods
Experimental drug preparation
A. caespitosa was accurately identified as the dry rhizome and aboveground part of A. caespitosa in Aspidistra of Liliaceae by Professor Sun from Guizhou University of Traditional Chinese Medicine. First, 10 kg of A. caespitosa was reflux-extracted with 95% ethanol for 4, 3, and 3 h respectively. Ethanol was recovered, concentrated, and then extracted with ethyl acetate and petroleum ether, respectively, obtaining 336.25 g of ethyl acetate extraction fraction, 39.12 g of petroleum ether extraction fraction, and 135.32 g of water layer extraction fraction, which were stored in a refrigerator at 4 ℃ for later use.
Grouping and model preparation
Mice, half male and half male, weighing (20±2) g, 12 mice in each group, were divided into 11 groups according to body weight, namely blank group, and high, medium and low doses of ethyl acetate, petroleum ether and water extraction fraction groups. Cough model: The mice were quickly put into a beaker filled with SO2 at 1 h after the last administration, and a cough model was made by inducing cough with SO2. Inflammation models: Each mouse in the xylene group was coated with xylene on the front and back of the right ear 1 h after the last administration, with a dosage of 0.03 ml/mouse, and the left ear was used as a control; the mice in each group were anesthetized with ether, and a 5 mg of autoclaved cotton ball was placed under the skin of the mouse’s right armpit which was then sutured; for each mouse in the foodpad group, 20 μl of 1% carrageenan was subcutaneously injected to the foodpad of the right foot of the hind limb, and the same amount of normal saline was subcutaneously injected to the foodpad of the left foot of the hind limb; and for the inflammatory factor detection group, each mouse in various groups was subcutaneously injected with 20 μl of 1% freshly-prepared carrageenan to the foodpad of the right foot of the hind limb, and the mice were decapitated for blood 3 h later. Method of administration
The mice in each group were given intragastric administration and fasted overnight before the administration. The dose of each group of mice was 0.1 ml/10 g, that is, 10 ml/kg, once a day for 10 d consecutively. For the experimental groups, according to the average dosage of A. caespitosa for adults in Mao medicine, 20 g/day, high-, medium- and low-dose groups of 20, 10, and 5 times the adult dosage were prepared respectively for administration. The mice in the control group were given a dose of 10 ml/kg, which was 10 times the adult dose, and in the experiment, the medicine was prepared to 1.55% with distilled water and administrated to mice at 20 ml/kg. The normal group was given the same amount of normal saline.
Observation methods and indicators
Antitussive experiment
The incubation period of cough and the number of coughs within 2 min were recorded from the first cough after the mice entered in beakers filled with SO2, and the differences between different groups were compared. Cough of mice was judged based on the contraction of the abdominal muscles, chest contraction, or opening of mouth. Cough relieving rate was calculated according to Cough relieving rate (%)=Cough reaction time in the experimental group/Cough reaction time in the control group×100%.
Effects on xylene-induced ear swelling in mice
Xylene was applied on both sides of the right ear of each mouse, and the mice were decapitated 4 h later. Then, the left and right ears of each mouse were cut along the auricles, and a hole punch with a diameter of 8 mm was used to take the ear pieces from the same part of the ears. The obtained pieces were accurately weighed, and the swelling degree, swelling rate and swelling inhibition rate were calculated according to following formulas: Swelling degree=Weight of ear piece from the right ear-Weight of ear piece from the left ear, Swelling rate (%)=Weight of ear piece from the right ear-Weight of ear piece from the left ear/Weight of ear piece from the right ear×100%, and Swelling inhibition rate (%)=Average swelling rate of the normal group-average swelling rate of the experimental group/Average swelling rate of the normal group×100%.
Effects on cotton ball-induced granulomas in mice
One hour after the last administration, the mice were killed by dislocation of cervical vertebra. The cotton ball and the surrounding connective tissue were taken out together, and the fat tissue was removed. The tissues were placed in an oven at 70 ℃, continuously dried for 18 h to constant weight, and weighed accurately. The degree of granulation swelling in mice and the inhibition rate of granulation swelling were calculated according to following formulas: Degree of granulation swelling=Weight of the cotton ball-Weight of the original cotton ball; Inhibition rate of granulation swelling (%)=Average swelling rate of the normal group-Average swelling rate of the experimental group/Average swelling rate of the normal group×100%. Effects on carrageenan-induced footpad swelling in mice
After injecting 20 μl of 1% carrageenan into mouse footpads, the thickness of footpads was measured with a vernier caliper at 0.5, 1, 2, 4, and 6 h after the administration, and the swelling rate was calculated: Swelling rate (%)=(Swelling degree of normal group-Swelling degree of administration group)/Swelling degree of normal group×100%.
Effects on inflammatory factors
The collected blood of each group of mice was spontaneously coagulated at room temperature for 15-20 min and then centrifuged for 15 min at a rotation speed of 3 000 r/min. The serum was collected and stored in liquid nitrogen at minus 20 ℃ for later use. ELISA kits were used to detect the levels of IL-6, TNF-a and other indicators, and differences between different groups were compared.
Statistical methods
SPSS 2.0 statistical software was used to analyze the data, which were expressed as mean±standard deviation (x±s). Differences between groups and with the control group were compared for significance.
Results and Analysis
Antitussive effect on mice［4-5］
Compared with the normal group and the control group, the ethyl acetate, petroleum ether and water extraction fraction groups of A. caespitosa had lower cough inoculation periods and cough frequencies, but the antitussive effect of the ethyl acetate extraction fraction was significant (P<0.01). The details are shown in Table 1.
Inhibition of xylene-induced ear swelling in mice［6-7］
Different concentrations of ethyl acetate extraction fraction, petroleum ether extraction fraction and water layer extraction fraction of A. caespitosa all had obvious anti-inflammatory effects (P<0.01), and compared with the normal group and the control group, the inhibitory rates of different extraction fractions on swelling degree were all greater than 30%, indicating that different extracts of A. caespitosa all had an inhibitory effect on the acute inflammation model caused by xylene. The details are shown in Table 2.
Inhibition of cotton ball-induced granulomas in mice［8］
Compared with the normal group, different extracts of A. caespitosa had significant inhibitory effects on cotton ball-induced granulomas in mice (P<0.01). The inhibitory effects of the ethyl acetate groups with different doses were higher than that of the control group, and the petroleum ether groups also had strong inhibitory effects. The details are shown in Table 3. Inhibition of carrageenan-induced foodpad swelling in mice［8］
Compared with the normal group, the A. caespitosa groups from different extraction fractions all inhibited carrageenan-induced footpad swelling in mice, but the ethyl acetate groups had a significant inhibitory effect on carrageenan-induced inflammatory footpad swelling in mice (P<0.01). The details are shown in Table 4.
Agricultural Biotechnology2021
Effects on the contents of tumor necrosis factors alpha and interleukin 6［9］
The results showed that the TNF-α and IL-6 indexes of the groups of different A. caespitosa extraction fractions decreased compared with the normal group; and compared with the control group, the high- and medium-dose groups of the ethyl acetate extraction fraction had significant inhibitory effects on TNF-α and IL-6 (P<0.01). The details are shown in Table 5.
Conclusions and Discussion
Coughing is a protective reflex of the respiratory system, but pathological coughing can lead to increased pressure in lungs, and severe coughing can also weaken the elasticity of the alveolar wall and rupture alveoli［10］. Inflammation refers to a series of pathological reactions or pathological processes caused by the body’s own defense after biological tissues are infected by pathogens after being wounded or infected［10］. Early inflammation is mainly characterized by increased permeability, telangiectasia, and inflammation and swelling, and is often accompanied by inflammation and swelling, chemotaxis and migration of leukocytes, etc.; and late inflammation is mainly characterized by hoof tissue hyperplasia and granuloma formation. It usually manifests as redness, swelling, heat, and pain. Some autoimmune diseases and even cancers are related to long-term inflammation.
A. caespitosa belongs to the whole plant of Aspidistra in Liliaceae and is a species included in Guizhou Chinese Herbal Medicine Resources. It is mainly used for lung deficiency cough, acute and chronic bronchitis, pneumonia recovery period and chronic pharyngitis in Guizhou Miao People’s habitual medication, and has the advantages of definite curative effect, uneasy relapse, and less toxic and side effects. It can be seen that A. caespitosa has a long history of antitussive and anti-inflammatory treatment, and the research on its antitussive and anti-inflammatory effect is of great value for the further research and development of this medicinal plant［11-12］. To study the antitussive and anti-inflammatory effects of drugs, animal models of cough and inflammation must be constructed. In this study, we used the SO2 cough-inducing method to make a mouse cough model, and induced acute inflammatory ear swelling in mice with xylene, granulomas in mice with cotton balls, and footpad swelling in mice with carrageenan. The models have the characteristics of small differences and good reproducibility, and are the most commonly used for the study of antitussive and anti-inflammatory drugs. This study explored the antitussive and anti-inflammatory effects of different extracts from Miao medicine A. caespitosa in mice. The results showed that compared with the normal group, the ethyl acetate extraction fraction had significant inhibitory effects on SO2-induced mouse cough, xylene-induced mouse ear swelling, cotton ball-induced mouse granulomas, and carrageenan-induced mouse footpad swelling, and could significantly reduce the levels of TNF-α and IL-6 in the serum of inflammation model mice. In summary, we verified the antitussive and anti-inflammatory effects of Miao medicine A. caespitosa from a scientific point of view, and through a series of experiments found that its antitussive and anti-inflammatory active ingredients were mainly present in the ethyl acetate extraction fraction, providing a reliable scientific basis for the clinical application of A. caespitosa, and laying a foundation for the rational development and utilization of A. caespitosa.
References
［1］ LAHOUSSE L, SEYS LJM, JOOS GF, et al. Epidemiology and impact of chronic bronchitis in chronic obstructive pulmonary disease［J］. Eur Respir J, 2017, 50(20): 160.
［2］ KIM V, CRINER GJ. Chronic bronchitis and chronic obstructive pulmonary disease［J］. Am J Respir Crit Care Med, 2013, 187(3): 228-237.
［3］ HE SZ, XU WF. Congsheng Chinese herbal medicine resources［M］. Congsheng: Congsheng Technology Press, 2007: 734. (in Chinese)
［4］ CHEN Q. Research methods of pharmacology of traditional Chinese medicine［M］. Beijing: People’s Medical Publishing House, 1993: 6361. (in Chinese)
［5］ HE XY, LI Q, ZHOU Y, et al. Experimental study on anti-inflammatory, antitussive and expectorant effects of Viburnum burejaeticum Regel et Herd.［J］. Lishizhen Medicine and Materia Medica Research, 2015, 26(2): 306-308. (in Chinese)
［6］ MA SB, LI KB, SUI YH. Experimental study on the effects of San’ao Capsule in relieving cough, resolving phlegm and relieving asthma［J］. Journal of Guiyang College of Traditional Chinese Medicine, 2008, 30(5): 74-76. (in Chinese)
［7］ SHI YJ, SI LJ, LIU Y, et al. Effects of total flavonoids from Artemisia Scoparia on anti-inflammatory, relieving cough and antipyretic［J］. Journal of Xinjiang Medical University，2015,38(5): 574-577. (in Chinese)
［8］ LIANG SL, HUANG FH, ZHONG XH, et al. Screening of anti-inflammatory and analgesic effective fractions from Cayratia japonica［J］. Chinese Traditional and Herbal Drugs, 2016, 47(4): 634-638. (in Chinese)
［9］ WANG ZM, DONG LR, JIA P, et al. Observation on antitussive, expectorant and anti inflammation effects of Sonchus oleraceus water extract on mice［J］. World Chinese Medicine，2018,13(10): 2604-2612. (in Chinese)
［10］ MA SB, SUI YH, WANG SH, et al. Screening effective parts of antitussive and anti-inflammatory from Miao medicine Guoshangye［J］. Journal of Guiyang College of Traditional Chinese Medicine, 2016, 38(1): 1-5. (in Chinese)
［11］ LYU HZ, HUANG BY, PAN CL, et al. Research review of Aspidistra (Asparagaceae)［J］. Guangdong Agricultural Sciences, 2016, 43(9): 17-25. (in Chinese)
［12］ ZHANG JL, ZHU H, PENG ZJ, et al. Investigation of Aspidistra plants in Guizhou Province［J］. Guizhou Agricultural Sciences, 2012, 40(12): 23-26. (in Chinese)
［Methods］A mouse cough model was made by the SO2 cough induction method, and the antitussive effects of different extraction fractions of A. caespitosa were observed. The inflammation models of acute inflammatory ear swelling in mice caused by xylene, granuloma in mice caused by cotton balls, and footpad swelling in mice caused by carrageenan were made, and the different extraction fractions were used to observe the effects of different extraction fractions on mouse ear swelling, granuloma and footpad swelling. And the levels of interleukin 6 (IL-6) and tumor necrosis factor (TNF-a) in the serum of different groups of mice were detected to screen out the extraction fractions of A. caespitosa with good antitussive and anti-inflammatory activity.
［Results］ Antitussive experiments on mice showed that the water layer extraction fraction, ethyl acetate extraction fraction and petroleum ether extraction fraction of Miao medicine A. caespitosa could prolong the incubation period of cough in mice and reduce the number of coughs within 2 min to varying degrees, and its different extraction fractions all had different inhibitory effects on mouse ear swelling, granuloma and footpad swelling. Compared with the blank control group, the different extraction fractions could reduce IL-6, TNF-a and other indicators to different degrees.
［Conclusions］Ethyl acetate extraction fraction of A. caespitosa has good antitussive and anti-inflammatory effects.
Key words Aspidistra caespitosa; Antitussive effect; Anti-inflammatory effect; Screening
Received: June 10, 2021 Accepted: August 15, 2021
Supported by Scientific and Technological Research Project of Traditional Chinese Medicine and Ethnic Medicine of Guizhou Administration of Traditional Chinese Medicine (QZYY2017-116); Scientific Research Project of Guizhou University of Traditional Chinese Medicine (GZYKYN［2017］28).
Yongyue GAO (1986-), male, P. R. China, associate chief pharmacist, master, devoted to research about ethnic medicine.
*Corresponding author. E-mail: 125193312@qq.com.
With the acceleration of the pace of life and the aggravation of environmental pollution, climate change and population aging, the incidence and mortality of cough and respiratory system inflammation are increasing year by year, and the harm to the human body cannot be ignored［1］. A large number of studies have proved that Chinese medicine can significantly improve various lung diseases［2］. Miao medicine Aspidistra caespitosa C. Pei is a species included in Guizhou Chinese Herbal Medicine Resources. It refers to the whole plant of Aspidistra in Liliaceae. It is widely distributed in Guizhou, Yunnan, Guangxi and other places. A. caespitosa is mainly used for cough, acute and chronic bronchitis, and chronic pharyngitis in Guizhou Miao medicine［3］, and thus has high medicinal value and development prospects. Therefore, in order to further study its mechanism of action and lay a foundation for follow-up research and new drug development, its anti-tussive and anti-inflammatory effects were investigated in this study conducted studies. Materials and Methods
Materials
Experimental animals
KM mice, SPF grade, half male and half male, weighing (20±2) g, animal quality certificate number: No: 00200406, purchased from the Experimental Animal Center of Guizhou Medical University, license number: SCXK-(Q) 2012-001; Feed: full-price pellet feed provided by the Experimental Animal Center of Guizhou Medical University; feeding conditions: clean and dry litter, temperature: 16-22 ℃, humidity: 45%-75%, ventilation 10-18 times, lighting: 8-12 h.
Control drug
Yan Guo Zhi Ke Ye (Guang Zheng Pharma Co., Ltd., National Medicine Standard: Z20013188, product batch number: HAF0416), adult dosage: 20 ml once, 3 times a day, which is equivalent to 1 ml/kg.
Experimental reagents and instruments
99% sulfuric acid, Chengdu Kelong Chemical Test Site, lot number: 20120704; anhydrous sodium sulfite, Chongqing Jiyuan Chemical Reagent Co., Ltd., lot number: 20170326; xylene, Shanghai Qiangshun Chemical Reagent Co., Ltd., lot number: 20120122; sodium chloride injection, Sichuan Kelun Pharmaceutical Co., Ltd., lot number: 20180115; carrageenan, imported from Fluka, Switzerland, lot number: CH-9826; electronic balance, Shanghai Jinghai Instrument Co., Ltd.; table centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; electric thermostatic drying oven, Tianjin Taisite Instrument Co., Ltd.; vernier caliper, Shandong Meike Tools Co., Ltd.
Methods
Experimental drug preparation
A. caespitosa was accurately identified as the dry rhizome and aboveground part of A. caespitosa in Aspidistra of Liliaceae by Professor Sun from Guizhou University of Traditional Chinese Medicine. First, 10 kg of A. caespitosa was reflux-extracted with 95% ethanol for 4, 3, and 3 h respectively. Ethanol was recovered, concentrated, and then extracted with ethyl acetate and petroleum ether, respectively, obtaining 336.25 g of ethyl acetate extraction fraction, 39.12 g of petroleum ether extraction fraction, and 135.32 g of water layer extraction fraction, which were stored in a refrigerator at 4 ℃ for later use.
Grouping and model preparation
Mice, half male and half male, weighing (20±2) g, 12 mice in each group, were divided into 11 groups according to body weight, namely blank group, and high, medium and low doses of ethyl acetate, petroleum ether and water extraction fraction groups. Cough model: The mice were quickly put into a beaker filled with SO2 at 1 h after the last administration, and a cough model was made by inducing cough with SO2. Inflammation models: Each mouse in the xylene group was coated with xylene on the front and back of the right ear 1 h after the last administration, with a dosage of 0.03 ml/mouse, and the left ear was used as a control; the mice in each group were anesthetized with ether, and a 5 mg of autoclaved cotton ball was placed under the skin of the mouse’s right armpit which was then sutured; for each mouse in the foodpad group, 20 μl of 1% carrageenan was subcutaneously injected to the foodpad of the right foot of the hind limb, and the same amount of normal saline was subcutaneously injected to the foodpad of the left foot of the hind limb; and for the inflammatory factor detection group, each mouse in various groups was subcutaneously injected with 20 μl of 1% freshly-prepared carrageenan to the foodpad of the right foot of the hind limb, and the mice were decapitated for blood 3 h later. Method of administration
The mice in each group were given intragastric administration and fasted overnight before the administration. The dose of each group of mice was 0.1 ml/10 g, that is, 10 ml/kg, once a day for 10 d consecutively. For the experimental groups, according to the average dosage of A. caespitosa for adults in Mao medicine, 20 g/day, high-, medium- and low-dose groups of 20, 10, and 5 times the adult dosage were prepared respectively for administration. The mice in the control group were given a dose of 10 ml/kg, which was 10 times the adult dose, and in the experiment, the medicine was prepared to 1.55% with distilled water and administrated to mice at 20 ml/kg. The normal group was given the same amount of normal saline.
Observation methods and indicators
Antitussive experiment
The incubation period of cough and the number of coughs within 2 min were recorded from the first cough after the mice entered in beakers filled with SO2, and the differences between different groups were compared. Cough of mice was judged based on the contraction of the abdominal muscles, chest contraction, or opening of mouth. Cough relieving rate was calculated according to Cough relieving rate (%)=Cough reaction time in the experimental group/Cough reaction time in the control group×100%.
Effects on xylene-induced ear swelling in mice
Xylene was applied on both sides of the right ear of each mouse, and the mice were decapitated 4 h later. Then, the left and right ears of each mouse were cut along the auricles, and a hole punch with a diameter of 8 mm was used to take the ear pieces from the same part of the ears. The obtained pieces were accurately weighed, and the swelling degree, swelling rate and swelling inhibition rate were calculated according to following formulas: Swelling degree=Weight of ear piece from the right ear-Weight of ear piece from the left ear, Swelling rate (%)=Weight of ear piece from the right ear-Weight of ear piece from the left ear/Weight of ear piece from the right ear×100%, and Swelling inhibition rate (%)=Average swelling rate of the normal group-average swelling rate of the experimental group/Average swelling rate of the normal group×100%.
Effects on cotton ball-induced granulomas in mice
One hour after the last administration, the mice were killed by dislocation of cervical vertebra. The cotton ball and the surrounding connective tissue were taken out together, and the fat tissue was removed. The tissues were placed in an oven at 70 ℃, continuously dried for 18 h to constant weight, and weighed accurately. The degree of granulation swelling in mice and the inhibition rate of granulation swelling were calculated according to following formulas: Degree of granulation swelling=Weight of the cotton ball-Weight of the original cotton ball; Inhibition rate of granulation swelling (%)=Average swelling rate of the normal group-Average swelling rate of the experimental group/Average swelling rate of the normal group×100%. Effects on carrageenan-induced footpad swelling in mice
After injecting 20 μl of 1% carrageenan into mouse footpads, the thickness of footpads was measured with a vernier caliper at 0.5, 1, 2, 4, and 6 h after the administration, and the swelling rate was calculated: Swelling rate (%)=(Swelling degree of normal group-Swelling degree of administration group)/Swelling degree of normal group×100%.
Effects on inflammatory factors
The collected blood of each group of mice was spontaneously coagulated at room temperature for 15-20 min and then centrifuged for 15 min at a rotation speed of 3 000 r/min. The serum was collected and stored in liquid nitrogen at minus 20 ℃ for later use. ELISA kits were used to detect the levels of IL-6, TNF-a and other indicators, and differences between different groups were compared.
Statistical methods
SPSS 2.0 statistical software was used to analyze the data, which were expressed as mean±standard deviation (x±s). Differences between groups and with the control group were compared for significance.
Results and Analysis
Antitussive effect on mice［4-5］
Compared with the normal group and the control group, the ethyl acetate, petroleum ether and water extraction fraction groups of A. caespitosa had lower cough inoculation periods and cough frequencies, but the antitussive effect of the ethyl acetate extraction fraction was significant (P<0.01). The details are shown in Table 1.
Inhibition of xylene-induced ear swelling in mice［6-7］
Different concentrations of ethyl acetate extraction fraction, petroleum ether extraction fraction and water layer extraction fraction of A. caespitosa all had obvious anti-inflammatory effects (P<0.01), and compared with the normal group and the control group, the inhibitory rates of different extraction fractions on swelling degree were all greater than 30%, indicating that different extracts of A. caespitosa all had an inhibitory effect on the acute inflammation model caused by xylene. The details are shown in Table 2.
Inhibition of cotton ball-induced granulomas in mice［8］
Compared with the normal group, different extracts of A. caespitosa had significant inhibitory effects on cotton ball-induced granulomas in mice (P<0.01). The inhibitory effects of the ethyl acetate groups with different doses were higher than that of the control group, and the petroleum ether groups also had strong inhibitory effects. The details are shown in Table 3. Inhibition of carrageenan-induced foodpad swelling in mice［8］
Compared with the normal group, the A. caespitosa groups from different extraction fractions all inhibited carrageenan-induced footpad swelling in mice, but the ethyl acetate groups had a significant inhibitory effect on carrageenan-induced inflammatory footpad swelling in mice (P<0.01). The details are shown in Table 4.
Agricultural Biotechnology2021
Effects on the contents of tumor necrosis factors alpha and interleukin 6［9］
The results showed that the TNF-α and IL-6 indexes of the groups of different A. caespitosa extraction fractions decreased compared with the normal group; and compared with the control group, the high- and medium-dose groups of the ethyl acetate extraction fraction had significant inhibitory effects on TNF-α and IL-6 (P<0.01). The details are shown in Table 5.
Conclusions and Discussion
Coughing is a protective reflex of the respiratory system, but pathological coughing can lead to increased pressure in lungs, and severe coughing can also weaken the elasticity of the alveolar wall and rupture alveoli［10］. Inflammation refers to a series of pathological reactions or pathological processes caused by the body’s own defense after biological tissues are infected by pathogens after being wounded or infected［10］. Early inflammation is mainly characterized by increased permeability, telangiectasia, and inflammation and swelling, and is often accompanied by inflammation and swelling, chemotaxis and migration of leukocytes, etc.; and late inflammation is mainly characterized by hoof tissue hyperplasia and granuloma formation. It usually manifests as redness, swelling, heat, and pain. Some autoimmune diseases and even cancers are related to long-term inflammation.
A. caespitosa belongs to the whole plant of Aspidistra in Liliaceae and is a species included in Guizhou Chinese Herbal Medicine Resources. It is mainly used for lung deficiency cough, acute and chronic bronchitis, pneumonia recovery period and chronic pharyngitis in Guizhou Miao People’s habitual medication, and has the advantages of definite curative effect, uneasy relapse, and less toxic and side effects. It can be seen that A. caespitosa has a long history of antitussive and anti-inflammatory treatment, and the research on its antitussive and anti-inflammatory effect is of great value for the further research and development of this medicinal plant［11-12］. To study the antitussive and anti-inflammatory effects of drugs, animal models of cough and inflammation must be constructed. In this study, we used the SO2 cough-inducing method to make a mouse cough model, and induced acute inflammatory ear swelling in mice with xylene, granulomas in mice with cotton balls, and footpad swelling in mice with carrageenan. The models have the characteristics of small differences and good reproducibility, and are the most commonly used for the study of antitussive and anti-inflammatory drugs. This study explored the antitussive and anti-inflammatory effects of different extracts from Miao medicine A. caespitosa in mice. The results showed that compared with the normal group, the ethyl acetate extraction fraction had significant inhibitory effects on SO2-induced mouse cough, xylene-induced mouse ear swelling, cotton ball-induced mouse granulomas, and carrageenan-induced mouse footpad swelling, and could significantly reduce the levels of TNF-α and IL-6 in the serum of inflammation model mice. In summary, we verified the antitussive and anti-inflammatory effects of Miao medicine A. caespitosa from a scientific point of view, and through a series of experiments found that its antitussive and anti-inflammatory active ingredients were mainly present in the ethyl acetate extraction fraction, providing a reliable scientific basis for the clinical application of A. caespitosa, and laying a foundation for the rational development and utilization of A. caespitosa.
References
［1］ LAHOUSSE L, SEYS LJM, JOOS GF, et al. Epidemiology and impact of chronic bronchitis in chronic obstructive pulmonary disease［J］. Eur Respir J, 2017, 50(20): 160.
［2］ KIM V, CRINER GJ. Chronic bronchitis and chronic obstructive pulmonary disease［J］. Am J Respir Crit Care Med, 2013, 187(3): 228-237.
［3］ HE SZ, XU WF. Congsheng Chinese herbal medicine resources［M］. Congsheng: Congsheng Technology Press, 2007: 734. (in Chinese)
［4］ CHEN Q. Research methods of pharmacology of traditional Chinese medicine［M］. Beijing: People’s Medical Publishing House, 1993: 6361. (in Chinese)
［5］ HE XY, LI Q, ZHOU Y, et al. Experimental study on anti-inflammatory, antitussive and expectorant effects of Viburnum burejaeticum Regel et Herd.［J］. Lishizhen Medicine and Materia Medica Research, 2015, 26(2): 306-308. (in Chinese)
［6］ MA SB, LI KB, SUI YH. Experimental study on the effects of San’ao Capsule in relieving cough, resolving phlegm and relieving asthma［J］. Journal of Guiyang College of Traditional Chinese Medicine, 2008, 30(5): 74-76. (in Chinese)
［7］ SHI YJ, SI LJ, LIU Y, et al. Effects of total flavonoids from Artemisia Scoparia on anti-inflammatory, relieving cough and antipyretic［J］. Journal of Xinjiang Medical University，2015,38(5): 574-577. (in Chinese)
［8］ LIANG SL, HUANG FH, ZHONG XH, et al. Screening of anti-inflammatory and analgesic effective fractions from Cayratia japonica［J］. Chinese Traditional and Herbal Drugs, 2016, 47(4): 634-638. (in Chinese)
［9］ WANG ZM, DONG LR, JIA P, et al. Observation on antitussive, expectorant and anti inflammation effects of Sonchus oleraceus water extract on mice［J］. World Chinese Medicine，2018,13(10): 2604-2612. (in Chinese)
［10］ MA SB, SUI YH, WANG SH, et al. Screening effective parts of antitussive and anti-inflammatory from Miao medicine Guoshangye［J］. Journal of Guiyang College of Traditional Chinese Medicine, 2016, 38(1): 1-5. (in Chinese)
［11］ LYU HZ, HUANG BY, PAN CL, et al. Research review of Aspidistra (Asparagaceae)［J］. Guangdong Agricultural Sciences, 2016, 43(9): 17-25. (in Chinese)
［12］ ZHANG JL, ZHU H, PENG ZJ, et al. Investigation of Aspidistra plants in Guizhou Province［J］. Guizhou Agricultural Sciences, 2012, 40(12): 23-26. (in Chinese)