Study On Toxicity,substance Basis And Target Organs Of Momordica Cochinchinensis Seed

Objective:In this study,the acute toxicity,long-term toxicity,general pharmacology experiment and toxic substance basis were studied in order to clarify the substance basis and target organ of the toxic effect of Momordica cochinchinensis seed,provide scientific basis for the safe and effective clinical medication of Momordica cochinchinensis seed,and promote the further development and utilization of Momordica cochinchinensis seed.Methods:1.Acute toxicity study of Momordica cochinchinensis:1.1 Acute toxicity test of the Momordica cochinchinensis seed:according to the results of the pre experiment,the powder,water extract and 95%ethanol extract of the seed of Momordica cochinchinensis were divided into 5 groups according to the interval of 1:0.7 between 0%lethal dose and 100%lethal dose,respectively.The mice were given 0.4ml/10g by gavage to determine the acute toxicity LD₅₀.The mice were observed continuously for 7 days,and the death was recorded to calculate the LD₅₀and Compare the toxicity.1.2 Acute toxicity test of the Momordica cochinchinensis seed shell:As the pre experi-ment could not detect the 100%lethal dose of water extract and 95%ethanol extract from the seed shell of Momordica cochinchinensis,the maximum dose experiment was conducted.The mice were observed continuously for 14 days.On the 15th day,the main organs of mice were dissected,observed and weighed,and the organ index was calculated.2.Study on the long-term toxicity of the Momordica cochinchinensis seed:20 times,50 times,and 100 times the clinical dose of Momordica cochinchinensis seed to set low（0.34g/kg）,medium（0.86g/kg）,high（1.71g/kg）and blank control 4 dose groups.The rats were given 1m L/100g by intragastric administration for 3 months,and half of the rats in each group were examined for organ index,blood biochemical,blood routine and pathological section after drug withdrawal.The remaining 1/2 rats were continued to observe for 2 weeks after withdrawal of the drug,and the same check were continued.3.General pharmacological experiment of Momordica chinensis seed:3.1 Effects on the respiratory system and cardiovascular system of rats:40 SD rats were selected and randomly divided into high,medium,low and blank control 4 dose groups of Momordica chinensis extract.After anesthesia,the rats in each group were intubated into the carotid artery,and each measuring instrument was connected to the BL-420F biological function experiment system to measure the respiratory rate,heart rate and blood pressure before intragastric administration.After duodenal administration,repeat the pre-dose measurement at 30 min,60 min,90 min,and 120 min.3.2 Effect on spontaneous activities of mice:KM mice were randomly divided into a blank control group,a diazepam group,water extract and 95%ethanol extract of Momordica chinensis seed high,medium and low dose groups.0.2m L/10g was administered once by gavage.After 60 minutes,the mice of each group were placed in a multifunctional mouse activity recorder to measure the number of spontaneous activities within 2 minutes.3.3 Effect on the hypnotic effect of the subthreshold dose of sodium pentobarbital in mice:the grouping and administration method of mice are the same as"3.2".After 60 minutes of administration,intraperitoneal injection of sodium pentobarbital 30 mg/kg.The number of mice falling asleep in each group within 30 minutes and the rate of falling asleep were recorded,and the X²test method was used for comparison between groups.3.4 Effect on the sleep time of mice caused by the threshold dose of sodium pentobarbital:The grouping and administration method of mice are the same as"3.2",60 minutes after the administration,the mice are injected with 47 mg/kg sodium pentobarbital into the intraperitoneal cavity.Observe and record the duration of sleep in mice.3.5 Effect on the coordinated movement of mice:Except that the positive drug group is changed to chlorpromazine,the grouping and administration method of mice are the same as"3.2".The mice will be transferred 30 min,60 min,and 90min after administration.the rotation speed is 10 r/min,and the time of the mouse in the rod within 1 min and the number of drops in each group are recorded.4.The basic research on the toxic substances of Momordica cochinchinensis seed:using the acute toxicity experiments of mice and zebrafish juveniles as the guidance of toxicity screening,combining the separation and analysis methods of macroporous resin,semi-preparative liquid,high performance liquid chromatography to separate the Momordica cochinchinensis Toxic compounds in seeds.The compound structure is identified by ¹H-NMR,¹³C-NMR,high resolution mass spectrometry and other spectral methods and literature data.5.Study on target organs of toxic compound toxicity of Momordica cochinchinensis seed:5.1 Study on zebrafish embryo development and cardiotoxicity:select 6hpf zebrafish embryos and set toxic compounds LC₁₀,1/2 LC₁₀,1/4LC₁₀,solvent control group And the blank control group,30embryos in each group,cultured to 72hpf after administration,were divided into 24 hpf,48 hpf,72hpf to take pictures to observe and record the number of spontaneous twitches,mortality,hatching rate,deformity rate,heart rate of the embryos,using Image J measurement analyzes the eye size,body length,SV-BA distance and pericardial area of zebrafish juveniles.5.2 Study on liver and nephrotoxicity of zebrafish juveniles:Select Gz15Tg/+（AB）liver fluorescent zebrafish with liver fluorescence and normal development for 4days.The setting method of each dose group is the same as that of"5.1",and each group has 60 juveniles Fish.72 hours after administration,were photographed with a fluorescence microscope,and the fluorescence area and intensity of zebrafish liver were calculated using Image-J software.After the end,clean the fish,and measure the liver and kidney function indexes such as AST,ALT,Cr,Bu N,according to the instructions.Results:1.Acute toxicity study of Momordica cochinchinensis:1.1 Acute toxicity test of the Momordica cochinchinensis seed:After a large dose of the extracts of Momordica cochinchinensis seed,the mice experienced toxic reactions such as lethargy,decreased voluntary activity,convulsions of the limbs,decreased body temperature,and slowed breathing.The LD₅₀of the Momordica chinensis seed fine powder was 10.54g/kg,the LD₅₀of the water extract of Momordica chinensis seed was 31.26g/kg,and the LD₅₀of the 95%ethanol extract of the Momordica chinensis seed was 123.09g/kg.1.2 Acute toxicity test of the Momordica cochinchinensis seed shell:The maximum dose of water extract from seed shell of Momordica chinensis was 1376g/kg,and the maximum dose of 95%ethanol extract from seed shell Momordica chinensis was 1872g/kg.There was no obvious toxic reaction and no animal death.2.Study on the long-term toxicity of Momordica chinensis seed:Long-term oral administration of Momordica chinensis seed will reduce the appetite and weight growth rate of experimental rats.The medium and high-dose group Compared with the blank group,the weight was significantly different（P<0.05）.After 12 weeks of administration,the number of WBC and LYM#in the female and male high-dose groups increased（P<0.05 or P<0.01）.MCV,MCH and the ratio of reticulocyte count decreased（P<0.05）.Compared with the blank group,the albumin（ALB）,triacylglycerol（TG）and total cholesterol（TCHO）contents of the male high-dose group rats were significantly reduced（P<0.01）.Compared with the blank group,the heart index of the high-dose group was significantly decreased,and the lung index was significantly increased（P<0.05）.The lung tissues of rats with medium and high doses showed mild to moderate inflammation.After the recovery period,the lung inflammation of the rats was reduced,and no abnormal lesions were seen in other organs.3.General pharmacological experiment of Momordica chinensis seed:Within the dose range of this experiment,the water and alcohol extract of Momordica chinensis seed had no significant effect on the respiratory frequency and heart rate of rats,But it has a hypotensive effect on rats,and the difference is significant compared with the blank group（P<0.05）.After intragastric administration,the number of voluntary activities in the Momordica chinensis seed extract and the high-dose group of mice was significantly reduced（P<0.05）.Momordica chinensis seed extract can enhance the hypnotic effect of pentobarbital sodium subthreshold dose on mice,and increase the sleep time of mice at the threshold dose of pentobarbital sodium,which is significantly different from the blank group（P<0.05）.There were significant differences in the staying time and the number of rods dropped on the fatigue rotating rod instrument after the administration of the extract of Momordica chinensis seeds to the mice in the high-dose group compared with the blank group（P<0.05）.4.Basic research on the toxic substances of Momordica chinensis seed:7toxic compounds have been isolated.At present,2 triterpene saponins have been resolved,and they are compound 3:quillaic acid 3-O-β-D-galactopyran osyl（1→2）-[α-L-rhamnopyranosyl（1→3）]-β-D-glucuronopyranosideand compound 5:gypsogenin3-O-β-D-galactopyranosyl（1→2）-[α-L-rhamnopy ranosyl（1→3）]-β-D-glucuronopyranoside.5.Study on the target organs of toxic compound toxicity of Momordica chinensis seed:5.1 Toxicity to zebrafish embryo development:The number of autonomous twitches of embryos at high doses of toxic compound 3 and compound 5 of Momordica chinensis seed was significantly reduced at 24 hpf（P<0.05）.After 72hpf,it can be observed that the hatched juveniles have deformities such as spinal curvature and pericardial edema.The body length and eyes of the zebrafish juveniles in the high-dose group are shortened,and there is a significant difference compared with the blank group（P<0.05）.5.2Cardiotoxicity to zebrafish:Momordica chinensis toxic compound 3 and compound 5 can cause pericardial edema,slow heart rate,delayed heart development of zebrafish embryos,and induce apoptosis of zebrafish embryonic cardiomyocytes.The higher the dose Cardiomyocyte apoptosis is more obvious.5.3 Liver and kidney toxicity to zebrafish:The two toxic compounds of Momordica chinensis deform the liver of fluorescent zebrafish in the high-dose group,reduce the fluorescent area,and significantly increase the content of AST and ALT in the zebrafish.Compared with the blank group,there is a difference Significant（P<0.05 or P<0.01）.The content of creatinine（BUN）and urea nitrogen（Cr）was not significantly different from that of the blank group（P>0.05）.Conclusion:1.Acute toxicity experiments show that the main toxicity of Momordica chinensis is the seeds of Momordica chinensis,and the shells of Momordica chinensis are non-toxic.The toxicity of Momordica chinensis seed is:Momordica chinensis seed fine powder>water extract>95%ethanol extract.2.The long-term high-dose toxic target organ of Momordica chinensis seed powder is the lung,which may also affect the hematopoietic system,heart and liver to a certain extent.The toxic effect on the body is reversible to a certain extent after stopping the drug.However,it is safe to use within the clinical dosage range prescribed by the Pharmacopoeia and will not produce obvious toxic effects.3.General pharmacological studies have shown that,in the range of safe dose prescribed in clinical practice,the seeds of Momordica chinensis will not have obvious effects on the nervous system,respiratory system and cardiovascular system of experimental animals,but large dose administration may cause the decrease of blood pressure and inhibit the nervous system.4.The toxic effect of Momordica sinensis is based on the triterpene saponins quillaic acid 3-O-β-D-galactopyranosyl（1→2）-[α-L-rhamnopyranos-yl（1→3）]-β-D-glucuronopyranoside and gypsogenin3-O-β-D-galactopyranos-yl（1→2）-[α-L-rhamnopyranosyl（1→3）]-β-D-glucuronopyranoside.5.The two triterpene saponins have teratogenic and delayed developm-ental toxicity to zebrafish embryonic development.The target organs of toxicity to zebrafish juveniles are the heart and liver.The preliminary judgment that the mechanism of toxicity may be the induction of zebrafish cardiomyocyte apoptosis.