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Abstract [Objectives] This study was conducted to investigate the inhibitory effect of pratia extract on H22 tumorbearing mice and the effects on immune organs.
[Methods] With the application of H22 liver tumorbearing mice as an animal model， the animals were divided into such three Pratia extract groups as the high， medium and low dose groups （400， 200 and 100 mg/kg） and cyclophosphamide CTX group （20 mg/kg）. 15 d after the administration， the animals were killed by cervical dislocation， and the tumors， thymuses and spleens were taken and weighed， followed by the calculation of the tumor inhibitory rate and the thymus and spleen index， and the serum tumor necrosis factorα （TNFα） and interleukin2 （IL2） levels were determined by ELISA assay.
[Results] The inhibitory rates were 54.1， 32.6 and 8.2%， respectively， and there were significant differences from the model group （P<0.05）; and the spleen index of the tumorbearing mice was reduced， while the thymus index was improved. The serological results showed that the drugadministrated groups significantly improved the IL2 levels in the tumorbearing mice， but had no effects on TNFα.
[Conclusions] Pratia extract has an antitumor effect on H22 tumorbearing mice， and show certain doseeffect relationship， and its mechanism may be related to enhancing the immune function in tumorbearing mice by regulating IL2.
Key words Pratia; H22; Tumorbearing mice; TNFα; IL2
Pratia refers to the dry whole plant of Pratia begonafolia （Wall） Lindl. or P. nummularia （Lam.） A. Br. et Aschers， also known as Xiaotongchui， Dikouzi， Tongchuicao， Dishiliu， etc. It is a herbaceous plant in Campanulaceae， which can be eaten as a wild vegetable[1]. Pratia has the effects of dispelling wind and eliminatingdampness， promoting blood circulation and relieving internal heat or fever， and can be used to treat rheumatic pain， bruises， acute mastitis and innominate inflammation of unknown origin. Its chemical composition studies have shown that it mainly contains active ingredients such as triterpene esters[2-3]， flavonoids and polyacetylene[4].
In this study， an invivo antitumor experiment was carried out on mice subcutaneously transplanted with H22 cells， so as to explore the antitumor effect of pratia and its possible action mechanism. This study provides a theoretical basis for the further development and utilization of pratia.
Materials and Methods Experimental materials
Experimental plants
The pratia was collected from Guangxi Province， and identified as the dry whole plant of Pratia begonafolia or P. nummularia in Campanulaceae by Professor Li from the College of Traditional Chinese Medicinal Materials， Jilin Agricultural University. 100 g of pratia powder was extracted with 65% methanol as the extracting solvent at 40 ℃ for 3 times， 30 min each time， and the extracts were merged for later use.
Experimental instruments
Electronic analytical balance; full wavelength scanning microplate reader; rotary evaporator.
Experimental reagents
Positive drug： cyclophosphamide， purchased from Jilin Cancer Hospital; sodium carboxymethyl cellulose （food grade）; normal saline; tissue fixation liquid， Beijing Chemical Works; tissue homogenizer， Jintan Hengfeng Instrument Manufacturing Co.， Ltd.; ALT and AST kits， Nanjing Jiancheng Bioengineering Institute; TNFα kit， CUSABIO Biotech Co.， Ltd.; IL12 （IL12） kit， CUSABIO Biotech Co.， Ltd.; tumor necrosis factor （TNFα） kit， CUSABIO Biotech Co.， Ltd.; mouse maintenance feed， provided by the Experimental Animal Center of Jilin University; tweezers; scissors; syringe.
Experimental animals
Clean grade male ICR mice， with a weight of （20±2） g.
Experimental methods
Grouping of animals
C57 mice were inoculated with H22 tumor cells via armpits， and then subdivided into six groups， which were as follows：
Control group： normal saline （CON）， positive drug group： cyclophosphamide group （CTX， intraperitoneal injection）， and three test groups， i.e.， the lowdose extract group 50 mg/kg （PBEL）， the mediumdose extract group 100 mg/kg （PBEM） and the highdose extract group 200 mg/kg （PBEH）.
Preparation of drugs
Positive drug： cyclophosphamide 20 mg/kg， which was dissolved in normal saline， for intraperitoneal injection; blank group （negative control group）： normal saline for injection， administrated by intragastric administration; test drug， for intragastric （oral） administration.
Preparation of mice bearing ascites sarcoma
Three normal ICR mice were taken and raised for 1 week for experiment. The frozen H22 cells were taken from a liquid nitrogen tank and resuscitated at 37 ℃. The cell preservation solution was discarded， suspended in normal saline and injected into the peritoneal cavities of three ICR mice， which were weighed 3 d later. The abdomens of the mice bulged 5-7 d after inoculation， which meant the inoculation was successful. Preparation of tumor mice
Ascites carcinoma cells in the ICR mice inoculated for 5-7 d were prepared with normal saline to a cell suspension （1×106 cells/ml）， which was then inoculated subcutaneously into mice via the right armpits at 0.1 ml per mouse. On the second day， the tumor mice were randomly divided into different administration groups， 12 in each group， and the administration test was started.
The control group was given only normal saline. For the administration groups， the extract was prepared with sodium carboxymethylcellulose aqueous solution to different concentrations， which were administered intragastrically according to the doses as above at 0.2 ml/10 g. The positive drug， cyclophosphamide， was prepared with normal saline and administered intraperitoneally.
14 d （2 weeks） after administration， the mice were subjected to eyeball removal to collect blood and killed. The blood was stood for 2 h and centrifuged， and the serum was taken for the determination of various biochemical indicators. The levels of TNFα （tumor necrosis factor） and IL2 （interleukin2） were determined by enzymelinked immuno sorbent assay （ELISA） according to the procedure given in the kit.
The tumors， thymuses and spleens were carefully removed and weighed separately， for the calculation of each index.
Data processing
The tumor inhibitory rate in C57 mice was calculated according to Inhibitory rate = （Average tuber weight in the control group-Average tuber weight in the drugadministrated group）/Average tuber weight in the control group×100%.
The thymus index is the number of milligrams of thymus per gram of body thymus. The spleen index is the number of milligrams of spleen per gram of body weight. All data were subjected to mean and variance processing. The parameter values were expressed as mean ± standard deviation （Mean±SD）. The intergroup comparison adopted the T test with P<0.05as a significant level. The significance of difference between the drugadministrated groups was investigated with the significant level *P<0.05.
Results
Effects on the body weights of C57 mice
After the start of the experiment， the body weight variations of the mice were calculated every other day. It can be seen from the statistical results that there were no significant differences between the drugadministered groups and the blank control group （P>0.05）. The body weights of the blank control group， the cyclophosphamideadministered group and the highdose drug group showed an obvious decreasing trend， but there were no significant differences between these groups （P>0.05）. In general， after successful modeling of the mouse tumor model， each group has no significant effect on body weight. Inhibitory effect on the H22 tumor born by C57 mice
The statistical results are shown in Table 1. From the statistical results， it can be seen that the positive group （CTX） was significantlydifferent from the negative control group （P<0.01）. 14 d after administration， the tumor proliferation was significantly inhibited. The PBEL group showed no obvious inhibitory effect on tumor growth， and the highdose PBEH and mediumdose PBEM groups inhibited the tumor proliferation significantly， both showing a significant difference （P<0.01）.
Effects on immune organ indexes in mice
The thymus and spleen are important immune organs， and their organ indexes can reflect the strength of the bodys immune function to some extent. The main function of the thymus is to produce T lymphocytes and secrete thymosin， which are mainly involved in cellular immunity; and the spleen is rich in lymphocytes and macrophages， but the proportion of B lymphocytes is relatively large， so it is more closely related to humoral immunity.
The spleen is an important immune organ， the function of which is affected in tumor patients， and can be partially improved after drug administration. In this experiment， 14 d after administration， the spleens were taken to observe whether they had been affected after drug administration. The results showed that 14 d of administration， the liver index of the positive cyclophosphamide group increased， and the highdose pratia group in the drugadministeredgroups can significantly reduce the liver indexes of the tumor mice. Compared with the chemical drug， the traditional Chinese medicine pratia had a protective effect on the liver.
Meanwhile， the mouse thymus index was also observed in this experiment. 14 d after administration， whether the drugadministrated groups affected the thymus index was observed. The results showed that 14 d after administration， the thymus index of the positive drug group decreased significantly， but the highdose pratia extract group can significantly increase the mouse thymus index and improve the immunity of the tumor mice. The mediumdose and lowdose groups had no effect on the thymus index.
Effects on IL12 and TNFα levels in mice
Interleukin 12 （IL12） is considered to be a key regulator in the process of cellular immune response， and has obvious antitumor activity， which has become a hot spot in tumor immunotherapy and comprehensive treatment. A large number of animal experiments have shown that IL12 not only inhibits tumor growth， but also inhibits tumor metastasis and prolongs the survival time of tumorbearing animals. For TNFα （tumor necrosis factorα）， an elevated expression of this substance can effectively kill cancer cells. The effects of the active extract of pratia on the expression of IL12 and TNFα in H22 tumorbearing mice were investigated. The results showed that the high dose can effectively increase the content of IL12， reaching the significant level （P≤0.05）， but had no effect on TNFα. The mediumdose and lowdose groups had no statistically significant effects on the two indexes （Fig. 1）.
Discussion
Pratia extract can counteract the tumor proliferation of H22 tumorbearing mice， and improve the body immunity and enhance the antioxidant effect， indicating that the active extract has a significant inhibitory effect on tumor growth. Therefore， the pratia extract is expected to be developed into a new generation of safe and effective antitumor drug or healthcare product.
Agricultural Biotechnology2019
References
[1] Jiangsu New Medical College. Chinese medicine dictionary[M]. 1992. （in Chinese）
[2] LIU XK， QIU MH， LI ZR. Two new triterpene ester from Pratia begonafolia[J]. Natural Product Research and Development， 1999， 10（3）： 20-23. （in Chinese）
[3] KANJI ISHIMARUA， MAIKO OSABEA， LI YANA. Plyacetylene glycosides from Pratia nummularia cultures[J]. Phytochemistry， 2003， （62）： 643-646.
[4] ISHIMARU KANJI， MATSUURA ETSUKO. Flavonoids and a polyacetylene in Pratia nummulariachemistry， bioactivity analysis and biotechnology[J]. Foods & Food Ingredients Journal of Japan. 2000， 186： 33-44.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] With the application of H22 liver tumorbearing mice as an animal model， the animals were divided into such three Pratia extract groups as the high， medium and low dose groups （400， 200 and 100 mg/kg） and cyclophosphamide CTX group （20 mg/kg）. 15 d after the administration， the animals were killed by cervical dislocation， and the tumors， thymuses and spleens were taken and weighed， followed by the calculation of the tumor inhibitory rate and the thymus and spleen index， and the serum tumor necrosis factorα （TNFα） and interleukin2 （IL2） levels were determined by ELISA assay.
[Results] The inhibitory rates were 54.1， 32.6 and 8.2%， respectively， and there were significant differences from the model group （P<0.05）; and the spleen index of the tumorbearing mice was reduced， while the thymus index was improved. The serological results showed that the drugadministrated groups significantly improved the IL2 levels in the tumorbearing mice， but had no effects on TNFα.
[Conclusions] Pratia extract has an antitumor effect on H22 tumorbearing mice， and show certain doseeffect relationship， and its mechanism may be related to enhancing the immune function in tumorbearing mice by regulating IL2.
Key words Pratia; H22; Tumorbearing mice; TNFα; IL2
Pratia refers to the dry whole plant of Pratia begonafolia （Wall） Lindl. or P. nummularia （Lam.） A. Br. et Aschers， also known as Xiaotongchui， Dikouzi， Tongchuicao， Dishiliu， etc. It is a herbaceous plant in Campanulaceae， which can be eaten as a wild vegetable[1]. Pratia has the effects of dispelling wind and eliminatingdampness， promoting blood circulation and relieving internal heat or fever， and can be used to treat rheumatic pain， bruises， acute mastitis and innominate inflammation of unknown origin. Its chemical composition studies have shown that it mainly contains active ingredients such as triterpene esters[2-3]， flavonoids and polyacetylene[4].
In this study， an invivo antitumor experiment was carried out on mice subcutaneously transplanted with H22 cells， so as to explore the antitumor effect of pratia and its possible action mechanism. This study provides a theoretical basis for the further development and utilization of pratia.
Materials and Methods Experimental materials
Experimental plants
The pratia was collected from Guangxi Province， and identified as the dry whole plant of Pratia begonafolia or P. nummularia in Campanulaceae by Professor Li from the College of Traditional Chinese Medicinal Materials， Jilin Agricultural University. 100 g of pratia powder was extracted with 65% methanol as the extracting solvent at 40 ℃ for 3 times， 30 min each time， and the extracts were merged for later use.
Experimental instruments
Electronic analytical balance; full wavelength scanning microplate reader; rotary evaporator.
Experimental reagents
Positive drug： cyclophosphamide， purchased from Jilin Cancer Hospital; sodium carboxymethyl cellulose （food grade）; normal saline; tissue fixation liquid， Beijing Chemical Works; tissue homogenizer， Jintan Hengfeng Instrument Manufacturing Co.， Ltd.; ALT and AST kits， Nanjing Jiancheng Bioengineering Institute; TNFα kit， CUSABIO Biotech Co.， Ltd.; IL12 （IL12） kit， CUSABIO Biotech Co.， Ltd.; tumor necrosis factor （TNFα） kit， CUSABIO Biotech Co.， Ltd.; mouse maintenance feed， provided by the Experimental Animal Center of Jilin University; tweezers; scissors; syringe.
Experimental animals
Clean grade male ICR mice， with a weight of （20±2） g.
Experimental methods
Grouping of animals
C57 mice were inoculated with H22 tumor cells via armpits， and then subdivided into six groups， which were as follows：
Control group： normal saline （CON）， positive drug group： cyclophosphamide group （CTX， intraperitoneal injection）， and three test groups， i.e.， the lowdose extract group 50 mg/kg （PBEL）， the mediumdose extract group 100 mg/kg （PBEM） and the highdose extract group 200 mg/kg （PBEH）.
Preparation of drugs
Positive drug： cyclophosphamide 20 mg/kg， which was dissolved in normal saline， for intraperitoneal injection; blank group （negative control group）： normal saline for injection， administrated by intragastric administration; test drug， for intragastric （oral） administration.
Preparation of mice bearing ascites sarcoma
Three normal ICR mice were taken and raised for 1 week for experiment. The frozen H22 cells were taken from a liquid nitrogen tank and resuscitated at 37 ℃. The cell preservation solution was discarded， suspended in normal saline and injected into the peritoneal cavities of three ICR mice， which were weighed 3 d later. The abdomens of the mice bulged 5-7 d after inoculation， which meant the inoculation was successful. Preparation of tumor mice
Ascites carcinoma cells in the ICR mice inoculated for 5-7 d were prepared with normal saline to a cell suspension （1×106 cells/ml）， which was then inoculated subcutaneously into mice via the right armpits at 0.1 ml per mouse. On the second day， the tumor mice were randomly divided into different administration groups， 12 in each group， and the administration test was started.
The control group was given only normal saline. For the administration groups， the extract was prepared with sodium carboxymethylcellulose aqueous solution to different concentrations， which were administered intragastrically according to the doses as above at 0.2 ml/10 g. The positive drug， cyclophosphamide， was prepared with normal saline and administered intraperitoneally.
14 d （2 weeks） after administration， the mice were subjected to eyeball removal to collect blood and killed. The blood was stood for 2 h and centrifuged， and the serum was taken for the determination of various biochemical indicators. The levels of TNFα （tumor necrosis factor） and IL2 （interleukin2） were determined by enzymelinked immuno sorbent assay （ELISA） according to the procedure given in the kit.
The tumors， thymuses and spleens were carefully removed and weighed separately， for the calculation of each index.
Data processing
The tumor inhibitory rate in C57 mice was calculated according to Inhibitory rate = （Average tuber weight in the control group-Average tuber weight in the drugadministrated group）/Average tuber weight in the control group×100%.
The thymus index is the number of milligrams of thymus per gram of body thymus. The spleen index is the number of milligrams of spleen per gram of body weight. All data were subjected to mean and variance processing. The parameter values were expressed as mean ± standard deviation （Mean±SD）. The intergroup comparison adopted the T test with P<0.05as a significant level. The significance of difference between the drugadministrated groups was investigated with the significant level *P<0.05.
Results
Effects on the body weights of C57 mice
After the start of the experiment， the body weight variations of the mice were calculated every other day. It can be seen from the statistical results that there were no significant differences between the drugadministered groups and the blank control group （P>0.05）. The body weights of the blank control group， the cyclophosphamideadministered group and the highdose drug group showed an obvious decreasing trend， but there were no significant differences between these groups （P>0.05）. In general， after successful modeling of the mouse tumor model， each group has no significant effect on body weight. Inhibitory effect on the H22 tumor born by C57 mice
The statistical results are shown in Table 1. From the statistical results， it can be seen that the positive group （CTX） was significantlydifferent from the negative control group （P<0.01）. 14 d after administration， the tumor proliferation was significantly inhibited. The PBEL group showed no obvious inhibitory effect on tumor growth， and the highdose PBEH and mediumdose PBEM groups inhibited the tumor proliferation significantly， both showing a significant difference （P<0.01）.
Effects on immune organ indexes in mice
The thymus and spleen are important immune organs， and their organ indexes can reflect the strength of the bodys immune function to some extent. The main function of the thymus is to produce T lymphocytes and secrete thymosin， which are mainly involved in cellular immunity; and the spleen is rich in lymphocytes and macrophages， but the proportion of B lymphocytes is relatively large， so it is more closely related to humoral immunity.
The spleen is an important immune organ， the function of which is affected in tumor patients， and can be partially improved after drug administration. In this experiment， 14 d after administration， the spleens were taken to observe whether they had been affected after drug administration. The results showed that 14 d of administration， the liver index of the positive cyclophosphamide group increased， and the highdose pratia group in the drugadministeredgroups can significantly reduce the liver indexes of the tumor mice. Compared with the chemical drug， the traditional Chinese medicine pratia had a protective effect on the liver.
Meanwhile， the mouse thymus index was also observed in this experiment. 14 d after administration， whether the drugadministrated groups affected the thymus index was observed. The results showed that 14 d after administration， the thymus index of the positive drug group decreased significantly， but the highdose pratia extract group can significantly increase the mouse thymus index and improve the immunity of the tumor mice. The mediumdose and lowdose groups had no effect on the thymus index.
Effects on IL12 and TNFα levels in mice
Interleukin 12 （IL12） is considered to be a key regulator in the process of cellular immune response， and has obvious antitumor activity， which has become a hot spot in tumor immunotherapy and comprehensive treatment. A large number of animal experiments have shown that IL12 not only inhibits tumor growth， but also inhibits tumor metastasis and prolongs the survival time of tumorbearing animals. For TNFα （tumor necrosis factorα）， an elevated expression of this substance can effectively kill cancer cells. The effects of the active extract of pratia on the expression of IL12 and TNFα in H22 tumorbearing mice were investigated. The results showed that the high dose can effectively increase the content of IL12， reaching the significant level （P≤0.05）， but had no effect on TNFα. The mediumdose and lowdose groups had no statistically significant effects on the two indexes （Fig. 1）.
Discussion
Pratia extract can counteract the tumor proliferation of H22 tumorbearing mice， and improve the body immunity and enhance the antioxidant effect， indicating that the active extract has a significant inhibitory effect on tumor growth. Therefore， the pratia extract is expected to be developed into a new generation of safe and effective antitumor drug or healthcare product.
Agricultural Biotechnology2019
References
[1] Jiangsu New Medical College. Chinese medicine dictionary[M]. 1992. （in Chinese）
[2] LIU XK， QIU MH， LI ZR. Two new triterpene ester from Pratia begonafolia[J]. Natural Product Research and Development， 1999， 10（3）： 20-23. （in Chinese）
[3] KANJI ISHIMARUA， MAIKO OSABEA， LI YANA. Plyacetylene glycosides from Pratia nummularia cultures[J]. Phytochemistry， 2003， （62）： 643-646.
[4] ISHIMARU KANJI， MATSUURA ETSUKO. Flavonoids and a polyacetylene in Pratia nummulariachemistry， bioactivity analysis and biotechnology[J]. Foods & Food Ingredients Journal of Japan. 2000， 186： 33-44.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU