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Abstract [Objectives] This study was conducted to investigate the analgesic effects and acute toxicities of Bidens alba （L.） DC.
[Methods] The alcohol extract of B. alba （L.） DC was extracted and separated with petroleum ether and chloroform successively. The acute toxicities of the two extracts on mice were measured， and then the analgesic effects were measured with writhing pain model induced by acetic acid.
[Results] No mice died when the crude dosages of B. alba （L.） DC from petroleum ether extract and chloroform extract were 5 016 and 5 100 mg/kg， respectively. When the petroleum ether extract was 60.0 mg/kg， the percentage of twisted mice induced by acetic acid was 40%， the analgesic rate was 77.5%， and the time of the first writhing was （294.0±165.8） s; when the chloroform extract was 20.0 mg/kg， the percentage of twisted animals was 55.6%， the analgesic rate was 51.5%， and the time of the first writhing was （273.8 ±153.4） s; and when the chloroform extract was 4.0 mg/kg， the percentage of twisted animals was 40%， and the analgesic rate was 62.1%， and the time of the first writhing was （370.6±231.3） s.
[Conclusions] The petroleum ether extracts and chloroform extracts of B. alba （L.） DC have good analgesic effects and no acute toxicities.
Key words Bidens alba （L.） DC; Mouse; Analgesic effect; Acute toxicity; Petroleum ether; Chloroform
Bidens alba （L.） DC is an annual or perennial herb belonging to Bidens in Compositae， native to tropical America and is now widely distributed in high heat and high humidity areas around the world[1]. According to records of Chinese Materia Medica （1999 version）， there are eight species of Bidens plants in China. Most of them are mainly used for treating colds， throat swelling and pain， rheumatism， jaundice， and carbuncle. However， B. alba （L.） DC was not included.
The research on B. alba （L.） DC mainly focused on its ecological effects[2-6]， and there were few reports on its pharmacological activity. B. alba （L.） DC has anti inflammatory[7-8]， anti tumor[9]， anti ulcer effects， and its pollen has allergenic effect[10]. However， no studies on its acute toxicities and analgesic activity have been reported so far.
Materials and methods
Materials
Instruments
XS205DU 1/100000 electronic analytical balance （METTLER TOLEDO）; BS124S electronic balance （Sartorius）; UV visible spectrophotometer （MAPADA， 3100PC）.
Raw materials
The whole herb was collected from Huadu District， Guangzhou City， and identified by deputy researcher Yi Qifei from South China Botanical Garden of Chinese Academy of Sciences as B. alba （L.） DC. Reagents
Glacial acetic acid， petroleum ether， chloroform， dimethyl sulfoxide （DMSO）， and Tween 80 were all analytically reagents. Aspirin enteric coated tablets was produced by Hunan Xinhui Pharmaceutical Co.， Ltd. with batch number 160102.
Methods
Preparation of drugs
B. alba （L.） DC was harvested during flower and fruit stage， and then was dried and crushed. 170. 6 g of crude powder was combined with 1 L of 95% ethanol， then refluxed for 1 h， and repeated for 3 times. The extract was combined and the ethanol was recovered to a proper volume by decompression. After the chlorophyll was removed by centrifugation， the solution was concentrated until it had no alcoholic taste. Petroleum ether and chloroform were used for extraction， and then the extractant was removed by rotary evaporation. The petroleum ether extract and chloroform extract were dissolved in DMSO at 204 and 67 mg/ml， and stored in a refrigerator at 4 ℃.
Acute toxicity experiments
The acute toxicity test （LD50 assay） was carried out in accordance with the "Guiding Principles of Veterinary Drug Research Technology Compilation 2006-2011". The 204 mg/ml petroleum ether extract was suspended with 10% Tween 80 to 6 mg/ml. Four Kunming mice， half male and half female， with a weight of （20±2） g， were selected. According to the weight of mice， 0.2 ml/20 g， the petroleum ether extract was gavaged at 60 mg/kg. The dosage of 60 mg/kg petroleum ether extract was equivalent to that of 5 016 mg/kg of crude drug. If without death， the experiment was stopped and 10 mice were enlarged to repeat the above experiment.
The 67 mg/ml chloroform extract was suspended with 10% Tween 80 to 2 mg/ml. The acute toxicity test was carried out by the same method. The dosage of 20 mg/kg of chloroform extract was equivalent to 5 100 mg/kg of crude drug.
Analgesic experiments
60 Kunming mice， half male and half female， with a weight of （20±2） g， were randomly divided into N group （negative control group， saline group）， P group （positive group， Aspirin group， 20 mg/kg）， PEH group （petroleum ether high dose group）， PEL group （petroleum ether low dose group）， CH group （chloroform high dose group）， CL group （chloroform low dose group）， 10 mice in each group. The petroleum ether extract was diluted to 6 and 1.2 mg/ml with 10% Tween 80; the chloroform extract was diluted to 0.4 and 2 mg/ml ; and aspirin was diluted to 2.0 mg/ml.
Mice in each group were given 0.2 ml of drug solution by gavage once a day for 3 d. 60 min after the last administration， 10 mg/kg of 0.6% acetic acid was injected intraperitoneally. The starting time and times of twisting were recorded within 15 min. The twist counting criteria of mice were as follows： ① the abdomen of mice contracted inward and depressed， and the body distorted obviously; ② the abdomen contracted and adhered to the wall， and the body curled obviously; ③ hind abdomen attached to the bottom of the cage， the hind feet stretched out and crawled forward; and ④ unobvious curling of the body is excluded due to obstacles encountered in the course of moving forward.
Statistical analysis
The data were analyzed by one way ANOVA and Duncan multiple comparisons with SPSS21.0， and P<0.05 as the significant difference standard.
Results and Analysis
Acute toxicity of B. alba （L.） DC
It was found that no mice died when the crude dosages of B. alba （L.） DC from petroleum ether extract and chloroform extract were 5 016 and 5 100 mg/kg， respectively， indicating that the petroleum ether extract and chloroform extract of B. alba （L.） DC had no acute toxicities.
The effects of B. alba （L.） DC analgesic rate
The analgesic effects of B. alba （L.） DC on mice is shown in Table 1. Compared with the N group， the analgesic rates （ARs） of all the drug groups of petroleum ether extract were more than 50%， and the analgesic rate of the 60.0 mg/kg of petroleum ether extract was 77.5%， similar to that of the P group.
Analgesic rate （%）=（Writhing times of N group-Writhing times of drug group）/Writhing times of N group×100%（1）
The effects of B. alba （L.） DC on the rate of twisted mice
Compared with the negative control group， the rates of twisted mice （RTM） in petroleum ether high dose group， chloroform high dose group and chloroform low dose group， were 40%， 55.6% and 40%， respectively. It can be concluded that the petroleum ether extract and chloroform extract at the concentrations of 60.0 mg/kg and 4.0 or 20.0 mg/kg could significantly reduce the percentage of twisted mice.
The effects of B. alba （L.） DC on the writhing times
Compared with the negative control group， the writhing times （WT） in the 20 mg/kg aspirin group， petroleum ether high dose group and petroleum ether low dose group， were 4.6±13.5 （P<0.05） ， 5.1±10.1 （P<0.05） and 10.3±14.8， respectively; and the writhing times in the chloroform high dose group and chloroform low dose group were 11.0±18.7 and 8.6±16.7， respectively. The twisting times of the 60.0 mg/kg petroleum ether group were similar to those of the 20 mg/kg aspirin group. The effects of B. alba （L.） DC on the occurrence time of first writhing
The occurrence time of first writhing （OTFW） is from the beginning to the first writhing. The longer the occurrence time of first writhing occurs， the more obvious the analgesic effect is. From Table 1， it can be found that when the petroleum ether extract of B. alba （L.） DC was 60.0 mg/kg， and the chloroform extract was 4.0 or 20.0 mg/kg， the occurrence time of first writhing was significantly prolonged， compared with the N group， indicating that petroleum ether extract and chloroform extract at this concentration had better analgesic effects. When the petroleum ether concentration of B. alba （L.） DC was 60.0 mg/kg， and when the chloroform concentration was 4.0 or 20.0 mg/kg， the occurrence time of first writhing was similar or longer than that of the positive drug group. The results showed that the analgesic effects of petroleum ether extract and chloroform extract was equivalent to or slightly better than that of aspirin at 20 mg/kg.
Discussion
Analgesics include narcotic ones represented by morphine and non steroidal antipyretic ones represented by aspirin （a non steroidal anti inflammatory drug， NSAID）. Narcotic analgesics generally act on the central nervous system， but they are addictive， while non steroidal antipyretic analgesics generally act on the peripheral nervous system. Non steroidal analgesics can inhibit the synthesis of inflammatory factors such as PG， so they have good analgesic and anti inflammatory effects on chronic dull pain. However， NSAIDs have side effects such as gastric ulcer. The development of new drugs with good anti inflammatory effect and analgesic function will bring good news to mankind.
It is reported that B. odorata Cav[11]， B. pilosa[12]， B. tripartita L.[13]， and B. parviflora willd[14] all have certain analgesic effects. However， the analgesic effects of B. alba （L.） DC are not comparable with that of other plants because of their different pain models， reference drugs and preparation techniques.
In conclusion， the petroleum ether and chloroform extracts of B. alba （L.） DC have good analgesic effects and no acute toxicities. The results provide a theoretical basis for the development of the invasive plant B. alba （L.） DC as a new analgesic drug.
Agricultural Biotechnology2019
References
[1] YUE MF， FENG L， CUI Y， et al. Prediction of the potential distribution and suitability analysis of the invasive weed， Bidens alba （ L.） DC[J]. Journal of Biosafety， 2016（3）： 222-228. [2] ZHONG JD， ZHOU XX， LI XL， et al. Effects of different seeding depths on seedling emergence and growth of Bidens alba， Praxelis clematidea and Ageratum conyzoides[J]. Chinese Journal of Tropical Agriculture， 2017（1）：1-6.
[3] ZHONG JD， ZHOU HB， LIU KD， et al. Comparative study on seed biological characteristics of three invasive compositae species[J]. Journal of Weed Science， 2016（2）： 7-11.
[4] ZOU CP， CHEN JF， SUN YB， et al. Effects on cadmiums accumulation and migration characteristics from Bidens alba L.[J]. South China Agriculture， 2015（7）： 23-26.
[5] WEI CQ， PAN YM， TANG SC， et al. Seed germination of Bidens alba L. and B. biternata L. under different light and temperature regimes[J]. Journal of Weed Science， 2013（4）： 1-4.
[6] LIU MC， WEI CQ， TANG SC， et al. Bionomics of two invasive weeds， and， and their native congeners grown under different nutrient levels[J]. Journal of Biosafety， 2012（1）： 32-40.
[7] YUAN MG， GAO QY， XU ZH， et al. Comparative study on anti inflammatory activities of two extracts from Bidens alba（L.） DC[J]. Heilongjiang Animal Science and Veterinary Medicine， 2018（13）： 167-169.
[8] GAO QY， QIN YW， YUAN MG. Studies On the Anti Inflammatory Activity of Three Extraction Processes of Bidens Alba（L.） DC[J]. Medicinal Plant， 2018（6）： 68-71.
[9] ONG PL， WENG BC， LU FJ， et al. The anticancer effect of protein extract from Bidens alba in human colorectal carcinoma SW480 cells via the reactive oxidative species and glutathione depletion dependent apoptosis[J]. Food Chem Toxicol， 2008， 46（5）： 1535-1547.
[10] LI Y. Identification and purification of allergens in Bidens alba （L.） DC. pollen and verification through a model of mouse asthma and rhinitis[D]. Jinzhou： Liaoning Medical University， 2015.
[11] ZAPATA MORALES JR， ALONSO CASTRO AJ， DOMINGUEZ F， et al. The antinociceptive effects of a standardized ethanol extract of the Bidens odorata Cav （Asteraceae） leaves are mediated by ATP sensitive K+ channels[J]. J Ethnopharmacol， 2017， 207： 30-33.
[12] FOTSO AF， LONGO F， DJOMENI PD， et al. Analgesic and antiinflammatory activities of the ethyl acetate fraction of Bidens pilosa （Asteraceae）[J]. Inflammopharmacology， 2014， 22（2）： 105-114.
[13] POZHARITSKAYA ON， SHIKOV AN， MAKAROVA MN， et al. Anti inflammatory activity of a HPLC fingerprinted aqueous infusion of aerial part of Bidens tripartita L[J]. Phytomedicine， 2010， 17（6）： 463-468.
[14] LI SY， LI SZ， WANG XP. Effects of bidens injection on central nervous system[J]. Henan Traditional Chinese Medicine， 1984（3）： 44-45.
[Methods] The alcohol extract of B. alba （L.） DC was extracted and separated with petroleum ether and chloroform successively. The acute toxicities of the two extracts on mice were measured， and then the analgesic effects were measured with writhing pain model induced by acetic acid.
[Results] No mice died when the crude dosages of B. alba （L.） DC from petroleum ether extract and chloroform extract were 5 016 and 5 100 mg/kg， respectively. When the petroleum ether extract was 60.0 mg/kg， the percentage of twisted mice induced by acetic acid was 40%， the analgesic rate was 77.5%， and the time of the first writhing was （294.0±165.8） s; when the chloroform extract was 20.0 mg/kg， the percentage of twisted animals was 55.6%， the analgesic rate was 51.5%， and the time of the first writhing was （273.8 ±153.4） s; and when the chloroform extract was 4.0 mg/kg， the percentage of twisted animals was 40%， and the analgesic rate was 62.1%， and the time of the first writhing was （370.6±231.3） s.
[Conclusions] The petroleum ether extracts and chloroform extracts of B. alba （L.） DC have good analgesic effects and no acute toxicities.
Key words Bidens alba （L.） DC; Mouse; Analgesic effect; Acute toxicity; Petroleum ether; Chloroform
Bidens alba （L.） DC is an annual or perennial herb belonging to Bidens in Compositae， native to tropical America and is now widely distributed in high heat and high humidity areas around the world[1]. According to records of Chinese Materia Medica （1999 version）， there are eight species of Bidens plants in China. Most of them are mainly used for treating colds， throat swelling and pain， rheumatism， jaundice， and carbuncle. However， B. alba （L.） DC was not included.
The research on B. alba （L.） DC mainly focused on its ecological effects[2-6]， and there were few reports on its pharmacological activity. B. alba （L.） DC has anti inflammatory[7-8]， anti tumor[9]， anti ulcer effects， and its pollen has allergenic effect[10]. However， no studies on its acute toxicities and analgesic activity have been reported so far.
Materials and methods
Materials
Instruments
XS205DU 1/100000 electronic analytical balance （METTLER TOLEDO）; BS124S electronic balance （Sartorius）; UV visible spectrophotometer （MAPADA， 3100PC）.
Raw materials
The whole herb was collected from Huadu District， Guangzhou City， and identified by deputy researcher Yi Qifei from South China Botanical Garden of Chinese Academy of Sciences as B. alba （L.） DC. Reagents
Glacial acetic acid， petroleum ether， chloroform， dimethyl sulfoxide （DMSO）， and Tween 80 were all analytically reagents. Aspirin enteric coated tablets was produced by Hunan Xinhui Pharmaceutical Co.， Ltd. with batch number 160102.
Methods
Preparation of drugs
B. alba （L.） DC was harvested during flower and fruit stage， and then was dried and crushed. 170. 6 g of crude powder was combined with 1 L of 95% ethanol， then refluxed for 1 h， and repeated for 3 times. The extract was combined and the ethanol was recovered to a proper volume by decompression. After the chlorophyll was removed by centrifugation， the solution was concentrated until it had no alcoholic taste. Petroleum ether and chloroform were used for extraction， and then the extractant was removed by rotary evaporation. The petroleum ether extract and chloroform extract were dissolved in DMSO at 204 and 67 mg/ml， and stored in a refrigerator at 4 ℃.
Acute toxicity experiments
The acute toxicity test （LD50 assay） was carried out in accordance with the "Guiding Principles of Veterinary Drug Research Technology Compilation 2006-2011". The 204 mg/ml petroleum ether extract was suspended with 10% Tween 80 to 6 mg/ml. Four Kunming mice， half male and half female， with a weight of （20±2） g， were selected. According to the weight of mice， 0.2 ml/20 g， the petroleum ether extract was gavaged at 60 mg/kg. The dosage of 60 mg/kg petroleum ether extract was equivalent to that of 5 016 mg/kg of crude drug. If without death， the experiment was stopped and 10 mice were enlarged to repeat the above experiment.
The 67 mg/ml chloroform extract was suspended with 10% Tween 80 to 2 mg/ml. The acute toxicity test was carried out by the same method. The dosage of 20 mg/kg of chloroform extract was equivalent to 5 100 mg/kg of crude drug.
Analgesic experiments
60 Kunming mice， half male and half female， with a weight of （20±2） g， were randomly divided into N group （negative control group， saline group）， P group （positive group， Aspirin group， 20 mg/kg）， PEH group （petroleum ether high dose group）， PEL group （petroleum ether low dose group）， CH group （chloroform high dose group）， CL group （chloroform low dose group）， 10 mice in each group. The petroleum ether extract was diluted to 6 and 1.2 mg/ml with 10% Tween 80; the chloroform extract was diluted to 0.4 and 2 mg/ml ; and aspirin was diluted to 2.0 mg/ml.
Mice in each group were given 0.2 ml of drug solution by gavage once a day for 3 d. 60 min after the last administration， 10 mg/kg of 0.6% acetic acid was injected intraperitoneally. The starting time and times of twisting were recorded within 15 min. The twist counting criteria of mice were as follows： ① the abdomen of mice contracted inward and depressed， and the body distorted obviously; ② the abdomen contracted and adhered to the wall， and the body curled obviously; ③ hind abdomen attached to the bottom of the cage， the hind feet stretched out and crawled forward; and ④ unobvious curling of the body is excluded due to obstacles encountered in the course of moving forward.
Statistical analysis
The data were analyzed by one way ANOVA and Duncan multiple comparisons with SPSS21.0， and P<0.05 as the significant difference standard.
Results and Analysis
Acute toxicity of B. alba （L.） DC
It was found that no mice died when the crude dosages of B. alba （L.） DC from petroleum ether extract and chloroform extract were 5 016 and 5 100 mg/kg， respectively， indicating that the petroleum ether extract and chloroform extract of B. alba （L.） DC had no acute toxicities.
The effects of B. alba （L.） DC analgesic rate
The analgesic effects of B. alba （L.） DC on mice is shown in Table 1. Compared with the N group， the analgesic rates （ARs） of all the drug groups of petroleum ether extract were more than 50%， and the analgesic rate of the 60.0 mg/kg of petroleum ether extract was 77.5%， similar to that of the P group.
Analgesic rate （%）=（Writhing times of N group-Writhing times of drug group）/Writhing times of N group×100%（1）
The effects of B. alba （L.） DC on the rate of twisted mice
Compared with the negative control group， the rates of twisted mice （RTM） in petroleum ether high dose group， chloroform high dose group and chloroform low dose group， were 40%， 55.6% and 40%， respectively. It can be concluded that the petroleum ether extract and chloroform extract at the concentrations of 60.0 mg/kg and 4.0 or 20.0 mg/kg could significantly reduce the percentage of twisted mice.
The effects of B. alba （L.） DC on the writhing times
Compared with the negative control group， the writhing times （WT） in the 20 mg/kg aspirin group， petroleum ether high dose group and petroleum ether low dose group， were 4.6±13.5 （P<0.05） ， 5.1±10.1 （P<0.05） and 10.3±14.8， respectively; and the writhing times in the chloroform high dose group and chloroform low dose group were 11.0±18.7 and 8.6±16.7， respectively. The twisting times of the 60.0 mg/kg petroleum ether group were similar to those of the 20 mg/kg aspirin group. The effects of B. alba （L.） DC on the occurrence time of first writhing
The occurrence time of first writhing （OTFW） is from the beginning to the first writhing. The longer the occurrence time of first writhing occurs， the more obvious the analgesic effect is. From Table 1， it can be found that when the petroleum ether extract of B. alba （L.） DC was 60.0 mg/kg， and the chloroform extract was 4.0 or 20.0 mg/kg， the occurrence time of first writhing was significantly prolonged， compared with the N group， indicating that petroleum ether extract and chloroform extract at this concentration had better analgesic effects. When the petroleum ether concentration of B. alba （L.） DC was 60.0 mg/kg， and when the chloroform concentration was 4.0 or 20.0 mg/kg， the occurrence time of first writhing was similar or longer than that of the positive drug group. The results showed that the analgesic effects of petroleum ether extract and chloroform extract was equivalent to or slightly better than that of aspirin at 20 mg/kg.
Discussion
Analgesics include narcotic ones represented by morphine and non steroidal antipyretic ones represented by aspirin （a non steroidal anti inflammatory drug， NSAID）. Narcotic analgesics generally act on the central nervous system， but they are addictive， while non steroidal antipyretic analgesics generally act on the peripheral nervous system. Non steroidal analgesics can inhibit the synthesis of inflammatory factors such as PG， so they have good analgesic and anti inflammatory effects on chronic dull pain. However， NSAIDs have side effects such as gastric ulcer. The development of new drugs with good anti inflammatory effect and analgesic function will bring good news to mankind.
It is reported that B. odorata Cav[11]， B. pilosa[12]， B. tripartita L.[13]， and B. parviflora willd[14] all have certain analgesic effects. However， the analgesic effects of B. alba （L.） DC are not comparable with that of other plants because of their different pain models， reference drugs and preparation techniques.
In conclusion， the petroleum ether and chloroform extracts of B. alba （L.） DC have good analgesic effects and no acute toxicities. The results provide a theoretical basis for the development of the invasive plant B. alba （L.） DC as a new analgesic drug.
Agricultural Biotechnology2019
References
[1] YUE MF， FENG L， CUI Y， et al. Prediction of the potential distribution and suitability analysis of the invasive weed， Bidens alba （ L.） DC[J]. Journal of Biosafety， 2016（3）： 222-228. [2] ZHONG JD， ZHOU XX， LI XL， et al. Effects of different seeding depths on seedling emergence and growth of Bidens alba， Praxelis clematidea and Ageratum conyzoides[J]. Chinese Journal of Tropical Agriculture， 2017（1）：1-6.
[3] ZHONG JD， ZHOU HB， LIU KD， et al. Comparative study on seed biological characteristics of three invasive compositae species[J]. Journal of Weed Science， 2016（2）： 7-11.
[4] ZOU CP， CHEN JF， SUN YB， et al. Effects on cadmiums accumulation and migration characteristics from Bidens alba L.[J]. South China Agriculture， 2015（7）： 23-26.
[5] WEI CQ， PAN YM， TANG SC， et al. Seed germination of Bidens alba L. and B. biternata L. under different light and temperature regimes[J]. Journal of Weed Science， 2013（4）： 1-4.
[6] LIU MC， WEI CQ， TANG SC， et al. Bionomics of two invasive weeds， and， and their native congeners grown under different nutrient levels[J]. Journal of Biosafety， 2012（1）： 32-40.
[7] YUAN MG， GAO QY， XU ZH， et al. Comparative study on anti inflammatory activities of two extracts from Bidens alba（L.） DC[J]. Heilongjiang Animal Science and Veterinary Medicine， 2018（13）： 167-169.
[8] GAO QY， QIN YW， YUAN MG. Studies On the Anti Inflammatory Activity of Three Extraction Processes of Bidens Alba（L.） DC[J]. Medicinal Plant， 2018（6）： 68-71.
[9] ONG PL， WENG BC， LU FJ， et al. The anticancer effect of protein extract from Bidens alba in human colorectal carcinoma SW480 cells via the reactive oxidative species and glutathione depletion dependent apoptosis[J]. Food Chem Toxicol， 2008， 46（5）： 1535-1547.
[10] LI Y. Identification and purification of allergens in Bidens alba （L.） DC. pollen and verification through a model of mouse asthma and rhinitis[D]. Jinzhou： Liaoning Medical University， 2015.
[11] ZAPATA MORALES JR， ALONSO CASTRO AJ， DOMINGUEZ F， et al. The antinociceptive effects of a standardized ethanol extract of the Bidens odorata Cav （Asteraceae） leaves are mediated by ATP sensitive K+ channels[J]. J Ethnopharmacol， 2017， 207： 30-33.
[12] FOTSO AF， LONGO F， DJOMENI PD， et al. Analgesic and antiinflammatory activities of the ethyl acetate fraction of Bidens pilosa （Asteraceae）[J]. Inflammopharmacology， 2014， 22（2）： 105-114.
[13] POZHARITSKAYA ON， SHIKOV AN， MAKAROVA MN， et al. Anti inflammatory activity of a HPLC fingerprinted aqueous infusion of aerial part of Bidens tripartita L[J]. Phytomedicine， 2010， 17（6）： 463-468.
[14] LI SY， LI SZ， WANG XP. Effects of bidens injection on central nervous system[J]. Henan Traditional Chinese Medicine， 1984（3）： 44-45.