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AbstractSafety of a compound Lonicera rupicola Hook.f.et & Thomson injection was evaluated by local irritation experiments including conjunctiva, skin and muscle irritant experiments, and the effect on weight gain was studied. The results showed that the compound L. rupicola Hook.f.et & Thomson injection had no irritation to rabbit eyes and weak irritation to rabbit ears, and had relatively strong irritation to the leg muscles of the mice. Its metabolism in mice had no abnormal toxicity.
Key wordsLonicera rupicola Hook.f.et & Thomson; Injection; Safety evaluation
Received: October 20, 2018Accepted: December 7, 2018
Supported by Project of Science and Technology Department of Sichuan Province, China (2016KZ0007); Innovative Scientific Research Project for Postgraduates of Southwest Minzu University (CX2018SZ44).
Haoyuan FENG (1994-), female, P. R. China, master candidate, devoted to research about veterinary pharmacology and toxicology.
*Corresponding author. Email: xnmdylsys@163.com.
Lonicera rupicola Hook.f.et & Thomson belongs to Lonicera, Caprifoliaceae and has great reserved resources. It is mainly distributed in Gansu, southern Ningxia, southeastern Qinghai, western Sichuan, northwest Yunnan and the vast areas from eastern to southwestern Tibet, and is mostly found in alpine shrub meadows, on the edge of runoff beaches, in the river beaches and grassland at forest edge, or in hillside shrubs at an altitude of 2 100-4 900 m［1-4］. The chemical constituents of Lonicera palnts are mainly flavonoids, volatile oils, chlorogenic acids and glycosides, of which the main components are flavonoids. The mother nucleus of flavonoids have strong chelation, and some flavonoid drugs combine with metal elements to form metal complexes, which may increase their efficacy and even produce new pharmacological activity［5-8］. In recent years, the adverse reactions caused by TCM injections account for more than 80% of total number of adverse reactions caused by TCM(TCM). According to relevant statistical analysis, the adverse reactions caused by TCM injections happen generally, show different clinical symptoms and have differences between various batches［9］. Most of the adverse reactions caused by TCM injections in clinical reports are various forms of allergic reactions, which can occur in any system and organ［10］. The adverse reactions caused by TCM are mainly related to the administration route, certain components, process and unstable control of TCM in quality, so safety evaluation of TCM preparations is necessary. In this study, the safety evaluation of a compound L. rupicola Hook.f.et & Thomson injection was evaluated to judge whether the reagent can be used in clinical practice or the issues should be paid attention to during use, and the compound L. rupicola Hook.f.et & Thomson injection was studied to understand the method of safety evaluation for TCM preparations. Experimental Materials
Experimental animals
Experimental animals included one SPF rabbit(1 kg in weight) and 20 SPF mice(18-22 g in weight), and they were male or female. The animals were provided by Chengdu Dashuo Experimental Animal Co., Ltd.
Drugs and reagents
In October 2016, L. rupicola Hook.f.et & Thomson was collected from Danqing Yaowang Shenshan in Sertar County, Garze Tibetan Autonomous Prefecture, Sichuan Province, and was identified by the associate professor Chaoxi Chen of Southwest Minzu University. The compound L. rupicola Hook.f.et & Thomson injection were mainly composed of copper sulfate (CuSO4?5H2O), zinc sulfate (ZnSO4?7H2O), manganese sulfate (MnSO4?H2O), sodium selenite (Na2SeO3?5H2O), ferric citrate (C6H5FeO7?5H2O), cobalt sulfate (CoSO4?7H2O), C10H12N2O8CuNa2 (397.7), C10H16N2Na2O8Zn (435.6), C10H18MnN2Na2O11 (443.174 3), sodium selenite (172.94), Se yeast Selplex, and total flavonoids of L. rupicola Hook.f.et & Thomson.
Experimental Methods
Conjunctiva of the rabbit
The tested drug was adjusted to nearly isotonic solution with sodium chloride solution. Two drops of the compound L. rupicola Hook.f.et & Thomson injection were instilled into the conjunctival sac of the left eye of the rabbit, and the same amount of normal saline was dropped into the right eye as a control. The responses of the eyes were recorded after 0 min, 30 min, 1, 2, 4, 8, 16 and 24 h respectively. Whether the conjunctiva of the left eye suffered from congestion and edema, whether the eye secretion increased, and whether the cornea was turbid were observed compared with the right eye［11-12］.
Subcutaneous injection of rabbit ears
The tested drug was adjusted to nearly isotonic solution with sodium chloride solution. 0.5 ml of the compound L. rupicola Hook.f.et & Thomson injection was injected into the inside of the left ear, while the same amount of normal saline was injected into the inside of the right ear as a control. The responses of the ears were recorded at the injection sites after 0 min, 30 min, 1, 2, 4, 8, 16 and 24 h respectively. Whether the left ear suffered from congestion, edema, denaturation or necrosis was observed compared with the right ear.
Injection experiment of leg muscles of the mice
The mice were forbidden to eat food and drink water for 12 h before the test. At first, each of the mice was numbered 1-20. Before the compound L. rupicola Hook.f.et & Thomson injection was injected into the mice, each of the mice was weighed. The mice were randomly divided into groups A, B, C and D, and there were five mice in each group. Among them, group D was as the control group, while groups A, B and C were the experimental groups. Afterwards, 0.1 ml of the compound L. rupicola Hook.f.et & Thomson injection was injected into the quadriceps femoris muscles of the left and right buttock of each mouse in the experimental groups, while the same amount of normal saline was injected into the quadriceps femoris muscles of the left and right buttock of each mouse in the control group. Dosing interval was one week, and if no mice died during one week, each of the mice was weighed after one week. All mice in group A were put to death and dissected. Their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. The remaining mice in groups B and C continued to be administered in the same manner and in the same dose, while the mice in group D was injected with the same amount of normal saline. After a week, the mice in groups B, C and D were weighed. Afterwards, all mice in group B were put to death, and their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. The remaining mice in groups C and D continued to be administered in the same manner and in the same dose, while the mice in group D was injected with the same amount of normal saline. After a week, the mice in groups C and D were weighed. Afterwards, all mice in groups C and D were put to death, and their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. In the above experimental processes, the mice ate food and drank water except that they were forbidden to eat food and drink water for 12 h before being weighed. The taken injected muscles and viscera of the mice were made into paraffin sections and dyed. Finally, the morphological differences of tissues of corresponding parts of the mice in groups A, B, C and D were observed. Results and Analysis
Experimental results of conjunctiva of the rabbit and subcutaneous injection of rabbit ears
According to Table 1, in compassion with the control group (the right eye), the cornea of the left eye was not turbid, while the iris and conjunctiva did not suffer from congestion, edema and hemorrhage. Moreover, there was no increase in secretion around the eye. Seen from Table 2, the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection and the right ear injected with normal saline had a slight congestion and fever in a short period, and then the symptoms gradually disappeared. The congestion and fever of the right ear injected with normal saline disappeared, while the congestion and fever of the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection did not disappear completely 8 h after the injection. The congestion and fever of the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection completely disappeared 24 h after the injection.
Table 1Experimental results of conjunctiva of the rabbit
Dosing time∥hChange of appearance (the left side was the experimental group, and the right side was the control group)
1
8
24
Table 2Experimental results of subcutaneous injection of rabbit ears
Dosing time∥hChange of appearance (the left side was the experimental group, and the right side was the control group)
1
8
24
Experimental results of injection of leg muscles of the mice
After the mice in the experimental groups were injected with the compound L. rupicola Hook.f.et & Thomson injection, no mice died but limped in a short period, so it is concluded that this drug was very irritating to the muscles or nerves of the mice. As shown in Fig. 1, after being injected for three times, the injected leg muscles of the mice were wax yellow and hard in texture and had more serious necrosis. One week after the mice were injected with the drug, the mice in group A were put to death by cervical dislocation and dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group A. Two weeks after the mice were injected with the drug, the mice in group B were put to death by cervical dislocation and dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group B. Three weeks after the mice were injected with the drug, the mice in group C were put to death by cervical dislocation and then dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group C. During the treatment, the mice had normal drinking water, feeding, motion, peeing, activity, and mental status. According to the standard, the compound L. rupicola Hook.f.et & Thomson injection had no abnormal toxicity. Analysis of weight gain of the mice
According to Table 3-Table 5, the average weight gain of the mice in the experimental groups was lower than that of the mice in the control group in the first, second and third week after they were injected with the compound L. rupicola Hook.f.et & Thomson injection.
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Table 3Weight gain of the mice in the first weekg
Group
Number of mouse
12345Average weight gain
A5.615.346.294.274.945.29
B8.275.016.795.76.386.43
C5.996.845.895.815.596.02
D6.768.327.835.424.676.60
Table 4Weight gain of the mice in the second weekg
Group
Number of mouse
12345Average weight gain
A------
B7.794.944.904.585.015.44
C5.516.446.054.945.015.59
D7.068.758.316.426.587.42
"-” mean the mice in group A had already been executed.
Table 5Weight gain of the mice in the third weekg
Group
Number of mouse
12345Average weight gain
A------
B------
C9.6010.929.578.208.759.21
D9.5713.0010.975.848.689.61
"-" mean the mice in groups A and B had already been executed.
The difference of weight gain of the mice in the experimental groups (A, B and C) and the control group (D) was analyzed by using biostatistical method. According to Table 6-Table 8, the difference of weight gain of the mice in the experimental groups and the control group was not significant one or three weeks after the treatment (FF crit (0.05)).
Changes in the muscles and organs of the mice
By observing the tissue sections of the injected muscles, heart, liver, spleen, lung and kidney of mice in each group, it was found that there was no abnormal change in the heart, liver, spleen, lung and kidney tissues of the mice, and the injected muscles showed waxy necrosis. As shown in Fig. 2, the muscle fibers in the injection site were swollen, broken, and the transverse stripes disappeared.
Table 6Difference of weight gain of the mice in the experimental groups (A, B and C) and the control group (D) after one week
Source of differenceSSdfMSFPvalueF crit
Between groups1.7613 066 711.761 3071.427 6510.247 6554.413 873
In a group22.206 773 3181.233 71
Total23.968 0819
Table 7Difference of weight gain of the mice in the experimental groups (B and C) and the control group (D) after two weeks Source of differenceSSdfMSFPvalueF crit
Between groups12.122 163 3112.122 1611.995 450.004 1984.667 193
In a group13.137 33131.010 564
Total25.259 493 314
Table 8Difference of weight gain of the mice in the experimental group (C) and the control group (D) after three weeks
Source of differenceSSdfMSFPvalueF crit
Between groups0.104 0410.104 040.025 4820.877 1285.317 655
In a group32.662 5684.082 82
Total32.766 69
Fig. 1Changes of the injected muscles of the mice after the treatment for three times
Fig. 2Tissue section of the injected muscles of a mouse
Conclusions and Discussion
According the experimental results of conjunctiva of the rabbit, it was found that there are no abnormalities in the cornea, iris and conjunctiva of the rabbit eye in different periods. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no irritation to the mucous membranes of the rabbit eye. Seen from the experimental results of subcutaneous injection of rabbit ears, both the left ear and the right ear had a slight congestion and fever in a short period, but the duration of the symptoms was not long. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no irritation to the rabbit ears. The leg muscles of the mice showed waxy necrosis after the injection according to the experimental results of injection of leg muscles of the mice. Seen from the tissue sections of the injected muscles, the muscle fibers in the injection site were swollen, broken, and the transverse stripes disappeared. It can be seen that the injection of the compound L. rupicola Hook.f.et & Thomson injection was very irritating to the muscles and could lead to the necrosis of the injected muscles. There was no obvious change in the heart, liver, spleen, lung and kidney tissues of the mice, so the compound L. rupicola Hook.f.et & Thomson injection was metabolized in the mice and had no effect on their viscera. The average weight gain of the mice in the experimental groups was lower than that of the mice in the control group in the first, second and third week after they were injected with the compound L. rupicola Hook.f.et & Thomson injection. The difference of weight gain of the mice in the experimental groups and the control group was not significant one or three weeks after the treatment but was significant two weeks after the treatment. To sum up, the average weight gain of the mice in the experimental groups was slightly lower than that of the mice in the control group, but the weight gain was not significant on the whole. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no great effect on the growth of the mice, and the small difference of weight gain of the mice in the experimental groups and the control group was related to the slight decrease in the appetite of the mice caused by the necrosis of the injected muscles. References
［1］ Chinese Pharmacopoeia Commission. Pharmacopoeia of the Peoples Republic of China (2015) (the first supplementary edition)［M］. Beijing: China Medical Science and Technology Press, 2015. (in Chinese).
［2］ Compilation Committee of the Flora of China, Chinese Academy of Sciences. The flora of China (volume 72)［M］. Beijing: Science Press,1985: 462. (in Chinese).
［3］ GOU ZP, WAN DG. Survey of resources of medicinal plants of Lonicera L. in Sichuan Province［J］. West China Journal of Pharmaceutical Sciences, 2005, 20(6): 480-482. (in Chinese).
［4］ YE HG, ZENG FY, YE YS, et al. Chinese medicinal plants (volume 4)［M］. Beijing: Chemical Industry Press, 2014. (in Chinese).
［5］ DU JM, ZHANG YM, WANG Y. Advances in the studies on plants of Lonicera［J］. Northern Horticulture, 2005(4): 11-13. (in Chinese).
［6］ XING JB, LI P. Overview and prospect of research on chemical constituents of Lonicera plants［J］. Jorunal of Chinese Medicinal Materials, 1999, 22(7): 366-370. (in Chinese).
［7］ SHE SJ, ZHU Y. Advances in studies on chemical constituents of Lonicera［J］.Strait Pharmaceutical Journal, 2008, 20(6): 1-7. (in Chinese).
［8］ SHAO ZH, WANG GX. Super delocalization of flavonoids［J］. Journal of Molecular Science, 1985,5(2):177. (in Chinese).
［9］ DU WM, WANG YM. Surveillance of adverse reaction of traditional medicine［J］. Drug Evaluation, 2004, 1(3): 183. (in Chinese).
Key wordsLonicera rupicola Hook.f.et & Thomson; Injection; Safety evaluation
Received: October 20, 2018Accepted: December 7, 2018
Supported by Project of Science and Technology Department of Sichuan Province, China (2016KZ0007); Innovative Scientific Research Project for Postgraduates of Southwest Minzu University (CX2018SZ44).
Haoyuan FENG (1994-), female, P. R. China, master candidate, devoted to research about veterinary pharmacology and toxicology.
*Corresponding author. Email: xnmdylsys@163.com.
Lonicera rupicola Hook.f.et & Thomson belongs to Lonicera, Caprifoliaceae and has great reserved resources. It is mainly distributed in Gansu, southern Ningxia, southeastern Qinghai, western Sichuan, northwest Yunnan and the vast areas from eastern to southwestern Tibet, and is mostly found in alpine shrub meadows, on the edge of runoff beaches, in the river beaches and grassland at forest edge, or in hillside shrubs at an altitude of 2 100-4 900 m［1-4］. The chemical constituents of Lonicera palnts are mainly flavonoids, volatile oils, chlorogenic acids and glycosides, of which the main components are flavonoids. The mother nucleus of flavonoids have strong chelation, and some flavonoid drugs combine with metal elements to form metal complexes, which may increase their efficacy and even produce new pharmacological activity［5-8］. In recent years, the adverse reactions caused by TCM injections account for more than 80% of total number of adverse reactions caused by TCM(TCM). According to relevant statistical analysis, the adverse reactions caused by TCM injections happen generally, show different clinical symptoms and have differences between various batches［9］. Most of the adverse reactions caused by TCM injections in clinical reports are various forms of allergic reactions, which can occur in any system and organ［10］. The adverse reactions caused by TCM are mainly related to the administration route, certain components, process and unstable control of TCM in quality, so safety evaluation of TCM preparations is necessary. In this study, the safety evaluation of a compound L. rupicola Hook.f.et & Thomson injection was evaluated to judge whether the reagent can be used in clinical practice or the issues should be paid attention to during use, and the compound L. rupicola Hook.f.et & Thomson injection was studied to understand the method of safety evaluation for TCM preparations. Experimental Materials
Experimental animals
Experimental animals included one SPF rabbit(1 kg in weight) and 20 SPF mice(18-22 g in weight), and they were male or female. The animals were provided by Chengdu Dashuo Experimental Animal Co., Ltd.
Drugs and reagents
In October 2016, L. rupicola Hook.f.et & Thomson was collected from Danqing Yaowang Shenshan in Sertar County, Garze Tibetan Autonomous Prefecture, Sichuan Province, and was identified by the associate professor Chaoxi Chen of Southwest Minzu University. The compound L. rupicola Hook.f.et & Thomson injection were mainly composed of copper sulfate (CuSO4?5H2O), zinc sulfate (ZnSO4?7H2O), manganese sulfate (MnSO4?H2O), sodium selenite (Na2SeO3?5H2O), ferric citrate (C6H5FeO7?5H2O), cobalt sulfate (CoSO4?7H2O), C10H12N2O8CuNa2 (397.7), C10H16N2Na2O8Zn (435.6), C10H18MnN2Na2O11 (443.174 3), sodium selenite (172.94), Se yeast Selplex, and total flavonoids of L. rupicola Hook.f.et & Thomson.
Experimental Methods
Conjunctiva of the rabbit
The tested drug was adjusted to nearly isotonic solution with sodium chloride solution. Two drops of the compound L. rupicola Hook.f.et & Thomson injection were instilled into the conjunctival sac of the left eye of the rabbit, and the same amount of normal saline was dropped into the right eye as a control. The responses of the eyes were recorded after 0 min, 30 min, 1, 2, 4, 8, 16 and 24 h respectively. Whether the conjunctiva of the left eye suffered from congestion and edema, whether the eye secretion increased, and whether the cornea was turbid were observed compared with the right eye［11-12］.
Subcutaneous injection of rabbit ears
The tested drug was adjusted to nearly isotonic solution with sodium chloride solution. 0.5 ml of the compound L. rupicola Hook.f.et & Thomson injection was injected into the inside of the left ear, while the same amount of normal saline was injected into the inside of the right ear as a control. The responses of the ears were recorded at the injection sites after 0 min, 30 min, 1, 2, 4, 8, 16 and 24 h respectively. Whether the left ear suffered from congestion, edema, denaturation or necrosis was observed compared with the right ear.
Injection experiment of leg muscles of the mice
The mice were forbidden to eat food and drink water for 12 h before the test. At first, each of the mice was numbered 1-20. Before the compound L. rupicola Hook.f.et & Thomson injection was injected into the mice, each of the mice was weighed. The mice were randomly divided into groups A, B, C and D, and there were five mice in each group. Among them, group D was as the control group, while groups A, B and C were the experimental groups. Afterwards, 0.1 ml of the compound L. rupicola Hook.f.et & Thomson injection was injected into the quadriceps femoris muscles of the left and right buttock of each mouse in the experimental groups, while the same amount of normal saline was injected into the quadriceps femoris muscles of the left and right buttock of each mouse in the control group. Dosing interval was one week, and if no mice died during one week, each of the mice was weighed after one week. All mice in group A were put to death and dissected. Their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. The remaining mice in groups B and C continued to be administered in the same manner and in the same dose, while the mice in group D was injected with the same amount of normal saline. After a week, the mice in groups B, C and D were weighed. Afterwards, all mice in group B were put to death, and their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. The remaining mice in groups C and D continued to be administered in the same manner and in the same dose, while the mice in group D was injected with the same amount of normal saline. After a week, the mice in groups C and D were weighed. Afterwards, all mice in groups C and D were put to death, and their parenchymatous organs and the injected muscles were taken and fixed in formalin solution. In the above experimental processes, the mice ate food and drank water except that they were forbidden to eat food and drink water for 12 h before being weighed. The taken injected muscles and viscera of the mice were made into paraffin sections and dyed. Finally, the morphological differences of tissues of corresponding parts of the mice in groups A, B, C and D were observed. Results and Analysis
Experimental results of conjunctiva of the rabbit and subcutaneous injection of rabbit ears
According to Table 1, in compassion with the control group (the right eye), the cornea of the left eye was not turbid, while the iris and conjunctiva did not suffer from congestion, edema and hemorrhage. Moreover, there was no increase in secretion around the eye. Seen from Table 2, the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection and the right ear injected with normal saline had a slight congestion and fever in a short period, and then the symptoms gradually disappeared. The congestion and fever of the right ear injected with normal saline disappeared, while the congestion and fever of the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection did not disappear completely 8 h after the injection. The congestion and fever of the left ear injected with the compound L. rupicola Hook.f.et & Thomson injection completely disappeared 24 h after the injection.
Table 1Experimental results of conjunctiva of the rabbit
Dosing time∥hChange of appearance (the left side was the experimental group, and the right side was the control group)
1
8
24
Table 2Experimental results of subcutaneous injection of rabbit ears
Dosing time∥hChange of appearance (the left side was the experimental group, and the right side was the control group)
1
8
24
Experimental results of injection of leg muscles of the mice
After the mice in the experimental groups were injected with the compound L. rupicola Hook.f.et & Thomson injection, no mice died but limped in a short period, so it is concluded that this drug was very irritating to the muscles or nerves of the mice. As shown in Fig. 1, after being injected for three times, the injected leg muscles of the mice were wax yellow and hard in texture and had more serious necrosis. One week after the mice were injected with the drug, the mice in group A were put to death by cervical dislocation and dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group A. Two weeks after the mice were injected with the drug, the mice in group B were put to death by cervical dislocation and dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group B. Three weeks after the mice were injected with the drug, the mice in group C were put to death by cervical dislocation and then dissected. It was found that there were no abnormalities in the heart, liver, spleen, lung and kidney of the mice in group C. During the treatment, the mice had normal drinking water, feeding, motion, peeing, activity, and mental status. According to the standard, the compound L. rupicola Hook.f.et & Thomson injection had no abnormal toxicity. Analysis of weight gain of the mice
According to Table 3-Table 5, the average weight gain of the mice in the experimental groups was lower than that of the mice in the control group in the first, second and third week after they were injected with the compound L. rupicola Hook.f.et & Thomson injection.
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Table 3Weight gain of the mice in the first weekg
Group
Number of mouse
12345Average weight gain
A5.615.346.294.274.945.29
B8.275.016.795.76.386.43
C5.996.845.895.815.596.02
D6.768.327.835.424.676.60
Table 4Weight gain of the mice in the second weekg
Group
Number of mouse
12345Average weight gain
A------
B7.794.944.904.585.015.44
C5.516.446.054.945.015.59
D7.068.758.316.426.587.42
"-” mean the mice in group A had already been executed.
Table 5Weight gain of the mice in the third weekg
Group
Number of mouse
12345Average weight gain
A------
B------
C9.6010.929.578.208.759.21
D9.5713.0010.975.848.689.61
"-" mean the mice in groups A and B had already been executed.
The difference of weight gain of the mice in the experimental groups (A, B and C) and the control group (D) was analyzed by using biostatistical method. According to Table 6-Table 8, the difference of weight gain of the mice in the experimental groups and the control group was not significant one or three weeks after the treatment (F
Changes in the muscles and organs of the mice
By observing the tissue sections of the injected muscles, heart, liver, spleen, lung and kidney of mice in each group, it was found that there was no abnormal change in the heart, liver, spleen, lung and kidney tissues of the mice, and the injected muscles showed waxy necrosis. As shown in Fig. 2, the muscle fibers in the injection site were swollen, broken, and the transverse stripes disappeared.
Table 6Difference of weight gain of the mice in the experimental groups (A, B and C) and the control group (D) after one week
Source of differenceSSdfMSFPvalueF crit
Between groups1.7613 066 711.761 3071.427 6510.247 6554.413 873
In a group22.206 773 3181.233 71
Total23.968 0819
Table 7Difference of weight gain of the mice in the experimental groups (B and C) and the control group (D) after two weeks Source of differenceSSdfMSFPvalueF crit
Between groups12.122 163 3112.122 1611.995 450.004 1984.667 193
In a group13.137 33131.010 564
Total25.259 493 314
Table 8Difference of weight gain of the mice in the experimental group (C) and the control group (D) after three weeks
Source of differenceSSdfMSFPvalueF crit
Between groups0.104 0410.104 040.025 4820.877 1285.317 655
In a group32.662 5684.082 82
Total32.766 69
Fig. 1Changes of the injected muscles of the mice after the treatment for three times
Fig. 2Tissue section of the injected muscles of a mouse
Conclusions and Discussion
According the experimental results of conjunctiva of the rabbit, it was found that there are no abnormalities in the cornea, iris and conjunctiva of the rabbit eye in different periods. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no irritation to the mucous membranes of the rabbit eye. Seen from the experimental results of subcutaneous injection of rabbit ears, both the left ear and the right ear had a slight congestion and fever in a short period, but the duration of the symptoms was not long. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no irritation to the rabbit ears. The leg muscles of the mice showed waxy necrosis after the injection according to the experimental results of injection of leg muscles of the mice. Seen from the tissue sections of the injected muscles, the muscle fibers in the injection site were swollen, broken, and the transverse stripes disappeared. It can be seen that the injection of the compound L. rupicola Hook.f.et & Thomson injection was very irritating to the muscles and could lead to the necrosis of the injected muscles. There was no obvious change in the heart, liver, spleen, lung and kidney tissues of the mice, so the compound L. rupicola Hook.f.et & Thomson injection was metabolized in the mice and had no effect on their viscera. The average weight gain of the mice in the experimental groups was lower than that of the mice in the control group in the first, second and third week after they were injected with the compound L. rupicola Hook.f.et & Thomson injection. The difference of weight gain of the mice in the experimental groups and the control group was not significant one or three weeks after the treatment but was significant two weeks after the treatment. To sum up, the average weight gain of the mice in the experimental groups was slightly lower than that of the mice in the control group, but the weight gain was not significant on the whole. It can be seen that the compound L. rupicola Hook.f.et & Thomson injection had no great effect on the growth of the mice, and the small difference of weight gain of the mice in the experimental groups and the control group was related to the slight decrease in the appetite of the mice caused by the necrosis of the injected muscles. References
［1］ Chinese Pharmacopoeia Commission. Pharmacopoeia of the Peoples Republic of China (2015) (the first supplementary edition)［M］. Beijing: China Medical Science and Technology Press, 2015. (in Chinese).
［2］ Compilation Committee of the Flora of China, Chinese Academy of Sciences. The flora of China (volume 72)［M］. Beijing: Science Press,1985: 462. (in Chinese).
［3］ GOU ZP, WAN DG. Survey of resources of medicinal plants of Lonicera L. in Sichuan Province［J］. West China Journal of Pharmaceutical Sciences, 2005, 20(6): 480-482. (in Chinese).
［4］ YE HG, ZENG FY, YE YS, et al. Chinese medicinal plants (volume 4)［M］. Beijing: Chemical Industry Press, 2014. (in Chinese).
［5］ DU JM, ZHANG YM, WANG Y. Advances in the studies on plants of Lonicera［J］. Northern Horticulture, 2005(4): 11-13. (in Chinese).
［6］ XING JB, LI P. Overview and prospect of research on chemical constituents of Lonicera plants［J］. Jorunal of Chinese Medicinal Materials, 1999, 22(7): 366-370. (in Chinese).
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