Basic Research Of TCM Incompatibility Theory Based On"Eighteen Incompatible Medicament"

This work was supported by National Basic Research Program of China (973Program): Basic Research of TCM Incompatibility Theory based on "Eighteen Incompatible Medicament", and was part of toxicity-effect characterization and basic material research on the compatibility of "Haizao, Daji, Gansui, Yuanhua Reverse Gancao". This dissertation is divided into four chapters and the contents are summarized as follows:Chapter1. Literature researchThe relevant literature was summarized, and a systematic review was given about the origin, material basis, biological effect and clinical application on the incompatibility of Genkwa Flos (GF) and Glycyrrhizae Radix et Rhizoma (GRR). On this basis, the research thoughts and experimental program were designed.Chapter2. Toxicity-effect characterization and mechanism researchSection1Toxicity evaluation of GF-GRR anti-drug combinationPart1Acute toxicity evaluation of GF and GRR single drugAcute toxic reaction was observed in mice by orally given GF or GRR. The results showed that the median lethal dose (LD50) of GF water extract was52.2g·kg-1,95%confidence limit was41.7-66.7g·kg-1, the LD50was about87times the clinical equivalent dosage. The LD50of GF total extract was18.7g·kg-1,95%confidence limit was14.9-23.5g·kg-1. However, due to the limitation of drug concentration, the LD50of GRR water extract was failed to detect. Therefore, the maximum tolerating dose (MTD) for mice was explored. The results showed that the MTD of GRR water extract was96.2g·kg-1, which was about48times the clinical equivalent dosage.Part2Acute toxicity evaluation of GF-GRR anti-drug combinationAcute toxic reaction and mortality was observed in mice by orally given different combination of GF-GRR decoction and GF total extract-GRR water extract. It showed obvious symptoms of toxicity after orally given GF-GRR decoction, the time of death was more than2h after dosing, and the mortality rate was GF-GRR1:1decoction group>GF-GRR2:1decoction group>GF-GRR1:2decoction group>GF-GRR1:3decoction group> GF-GRR3:1decoction group, no mice dead in GF or GRR control group. It also showed obvious symptoms of toxicity after orally given GF total extract-GRR water extract combination, but the time of death was less than2h after dosing, and the mortality rate was GF-GRR1:2combination group>GF-GRR3:1combination group>GF-GRR1:1combination group>GF-GRR1:3combination group.Part3Chronic toxicity evaluation of GF-GRR anti-drug combination In order to confirm the toxicity target organ and toxicity dose, toxic reaction was observed in rats by orally given different combination of GF-GRR decoction for8weeks. The results showed that:(1) When the ratio of GF-GRR was1:3.3, the digestive system and the reproductive system toxicity were observed in the pharmacopoeia clinical dosage range, which showed the performance of constipation, decreased testicular and epididymis factor, prostate pathological abnormalities, reduced sperm number.(2) When the ratio of GF-GRR was1:0.21, and GRR dosage was in the pharmacopoeia clinical range, the urinary system toxicity such as increased renal factor and abnomal renal pathology were observed.(3) When the ratio of GF-GRR was1:53, and GF dosage was in the pharmacopoeia clinical range, the influences on glucose and lipid metabolism were observed such as increased blood glucose and triglycerides, elevated liver factor, and weight loss of high-dosage group.Section2Biological effect evaluation of GF-GRR anti-drug combinationPart1Study on the incompatibility mechanism based on the diuretic and diarrhea effect of GF influenced by GRRTo investigate the possible incompatibility mechanism of GF-GRR anti-drug combination by comparing the diuretic and diarrhea effect before and after compatibility of GF and GRR. Compared with control group, GF powder processed by vinegar showed significant diuretic effect in the Pharmacopoeia equivalent dose range, and GRR decoction showed no influence to the diuretic effect, but the diuretic effect of GF powder processed by vinegar can be influenced by GRR in the Pharmacopoeia equivalent dose range, suggesting that the diuretic effect of GF suppressed by GRR would be one possible mechanism of incompatibility.Part2Study on the incompatibility mechanism based on the water and salt metabolism of GF-GRR anti-drug combinationThis study is to reveal the characteristic and possible mechanism of incompatibility for GF-GRR based on the investigation of water and salt metabolism in rats. Results showed that GF has a strong diuretic effect at Pharmacopoeia dose, and the mechanism may be the reduction of the antidiuretic hormone (AVP) secretion, inhibition of renal tubular Na+reabsorption, thereby promoting the discharge of Na+and water. GRR has no significant influence on rat urine volume, but has a certain impact to AVP and electrolyte level such as increased AVP level, increased urinary K+excretion, decreased urinary Na+excretion, which suggest that GRR has a certain effect of "keep sodium and excrete potassium". GRR inhibited the diuretic effect of GF was expressed as increased AVP and aldosterone (ALD) levels, increased urinary K+excretion, decreased urinary Na+excretion and urine volume. So, the electrolytes, AVP and ALD levels change caused by the combination use of GF and GRR suggesting that the water and salt metabolism imbalance would be one possible mechanism of incompatibility.Part3Study on the biological effects in mice with malignant ascites influenced by GF-GRR anti-drug combinationIn this experiment, malignant ascites model was chosen to evaluate the biological effects (ascites, electrolytes, ALD, AVP, pathology of heart, liver and kidney) of malignant ascites in mice before and after the combination use of GF and GRR. The results showed that the amount of ascites significantly increased in mice after modeling, while plasma Na+, K+, Cl-concentration reduced, ALD and ADH levels rised, the liver and kidney pathological changed. A decreasing trend of ascites amout was observed in GF group but not in GF-GRR group. Plasma Na+, K+, Cl-concentration and ALD, AVP level did not change significantly in GF or GRR group, but plasma K+concentration and ALD, AVP level increased significantly in GF-GRR combination group. In summary, malignant ascites in mice can lead to water and electrolyte imbalance, which changed more disorder while GF and GRR used together. It would be one possible mechanism of incompatibility.Section3Mechanism research of GF-GRR anti-drug combinationPart1Mechanism research of GF-GRR anti-drug combination based on metabonomic studyA metabonomic approach was used for mechanism research of GF-GRR anti-drug combination, UPLC-Q-TOF/MS was applicated for rat urine and blood metabolic spectrum analysis, and potential biomarkers related to toxicity were screened. The results show that, the toxicity of GF-GRR combination group is more obvious than GF group. Both of the two groups affected glycerophospholipid pathway, GF group also influenced bile acid biosynthesis pathway and sphingolipid metabolic pathways, while GF-GRR combination group influenced mainly phenylalanine, tyrosine and tryptophan biosynthesis pathway, tyrosine metabolic pathway and glycerol metabolic pathways. The regulation of different metabolic pathways may be one reason of the toxicity differences.Chapter3. Basic material research of GF-GRR incompatibilitySection1Study on the chemical constituents of GFThe chemical constituents of the ethanol extract were isolated and purified by various chromatographic methods. Their structures were elucidated by physical-chemical and spectroscopic methods.forty-four compounds were isolated, including eight lipids, two ceramides, four steroids, two triterpenes, eight diterpenoids, twelve flavonoids, seven phenols and one chlorophyl:tetracosane (1), palmitic acid (2), n-nonacohol (3), n-hexacosanol (4), octacosanoic acid heptadecanol ester (5), n-octacosanol (6), monopalmitin (7), glycerylmonostearate (8),(2S,3S,4R,8E)-2-[(2’R)-2’-hydroxydocosanosylamino]-octadecane-1,3,4-triol (9),(2S,3S,4R,8E)-2-[(2’R)-2’-hydroxytricosanosylamino]-octadecane-1,3,4-triol(10), friedelin (11),δ-Amyrone (12), β-sitosterol (13),5α,8α-epidioxyergosta-6,22-dien-3β-ol (14),7α-hydroxysitosterol (15), daucosterol (16), yuanhuacine (17), yuanhuadine (18), yuanhuafine (19), yuanhuatine (20), yuanhuapine (21), genkwanine F (22), genkwanine N (23), Wikstroemia factor Mi (24), genkwanin (25), apigenin (26),5-hydroxy-7,4’-dimethoxyflavone (27), acacetin (28),3,7-dimethoxy-5,4’-dihydroxyflavone (29),3’-hydroxy-genkwanin (30), tiliroside (31), umbelliferone (32), daphnoretin (33), zhebeiresinol (34), syringaresinol (35), p-hydroxybenzoic acid (36), daphneolon (37), daphnenone (38), genkwanin-5-O-β-D-glucoside (39), luteolin-7-methylether-5-O-β-D-glucopyranoside (40), luteolin-5-O-β-D-glucoside (41), genkwanin-5-O-β-D-primeveroside (42), genkwanin-4’-O-β-D-rutinoside (43),173-ethoxyphaeophorbide a (44). Compounds3-6,9-12,14-15,24,34,37-38,40,43-44are isolated from D. genkwa for the first time, and this is the first isolation of ceramide-, tritepene-, chlorophyl-type constituents from this plant.Section2Toxic material basis research of GFThe acute toxicity of total fatty acids, total diterpenes and total flavonoids fraction were evaluated to identify the toxic material basis, it showed that total diterpenes was the toxic part. Then the toxicity of five representative diterpenoids were evaluated by animal and cell toxicity experiment. Yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine and yuanhuapine all showed significant toxicity, such as liver toxicity, gastrointestinal toxicity and cause animal death. It also showed toxicity to intestinal epithelial cells, manifested as inhibition of cell proliferation and induce cell apoptosis. The metabonomic characters of the toxicity induced by yuanhuapine was studied, and it showed that yuanhuapine induced intestinal and hepatic toxicity were correlated with disturbance of amino acids metabolism, lipids metabolism, carbohydrate metabolism and gut microflora.Section3Study on the incompatibility mechanism based on the interaction of multiple-components for GF and GRRBy investigating the interaction between components from GF and GRR and the dissolution profile of toxic components in co-decoction, the characteristics and possible mechanism of incompatibility could be revealed. UPLC-Q-TOF/MS and UPLC-TQ/MS were applied to analyze multi-components in different herb extractions prepared by different ratios of GF/GF processed by vinegar (GFV) and GRR, which reflect the interaction and characteristics of multiple components in incompatibility combinations. The results showed that the components dissolution was influenced by compatibility ratio with certain regularity. Whether GF processed by vinegar or not, with the increase of GRR in co-decoction, the dissolution of diterpenes, especially for yuanhuacine, yuanhuadine and yuanhuajine, the toxic ingredients of GF, increased significantly. From these results, we can conclude that the material basis and one possible mechanism of incompatibility between GF and GRR is the dissolution increasing of diterpenes, toxic components of GF in co-decoction process, which caused by interaction between multi-components in these two herbs.Section4Study on ADME process and drug interactions of GF and GRRPart1The pharmacokinetic study of diterpenoids in GFThe toxicity of diterpenoids was evaluated after oral administration of total diterpenoids extract from GF to rats, and the blood concentration of diterpenoids was analyzed by UPLC-TQ-MS. The diterpenoids were confirmed to be the toxic components. The pharmacokinetic profile of these diterpenoids was quite different due to their different structures. Although the contents of yuanhuafine and yuanhuapine were low in the extract, the blood concentrations were extremely high. In contrary, the contents of genkwanine F and Wikstroemia factor Mi in the extract were much higher, but they could not be detected in the blood. This result implied that yuanhuafine and yuanhuapine but not genkwanine F and Wikstroemia factor Mi were the potentail toxic components of GF in vivo.Part2The pharmacokinetic study of flavonoids in GFSD rats were administrated with GF extract orally, and plasma samples were collected at different time points, which was then analyzed by UPLC-TQ-MS. Eight flavonoids could be detected in plasma samples, including four glycosides and four aglycone: genkwanin-5-O-β-D-glucoside,3’-hydroxy-genkwanin-5-O-β-D-glucoside, luteolin-5-O-β-D-glucoside, genkwanin-5-O-β-D-primeveroside, genkwanin, apigenin,3’-hydroxy-genkwanin and3,7-dimethoxy-5,4’-dihydroxyflavone. Flavonoid glycosides reach maximum plasma concentration in about1h, and flavonoid aglycone showed bimodal phenomenon, that is, after reach maximum plasma concentration in1h, another absorption peak appeared at about7h, probably due to the glycosides were metabolized to aglycone and then re-absorbed. This study provides a certain foundation for the later perspective of incompatibility of GF and GRR.Part3ADME process of toxic component before and after combination useAs a representative toxic component, yuanhuapine was chosen to explore the differences of ADME process before and after combination use by LC-MS. It showed that after combination use tmax reached earlier, Cmax was lower and MRT was longer, which suggested that GRR inhibit absorption and elimination of yuanhuapine in vivo. Tissue distribution of yuanhuapine before and after combination use of GRR was:stomach, intestine> liver>kidney>heart>spleen>lung> brain, which may be associated with intestinal damage and liver damage. Yuanhuapine excreted mainly from the feces, GRR inhibited its excretion process, indicating that GRR can slow down the excretion rate of toxic component, which may be related with the toxicity mechanism increased by GRR.Chapter4. Dosage-toxicity-effect research of GF-GRR incompatibilityBased on Multi-dose and multi-index optimization of experimental design, the toxicity (toxicity to the gastrointestinal tract, liver and reproductive organs) and the related effects (diuretic and purgative effect, motor function to the stomach and intestinal) before and after combination use was tested to analyze the dynamic safe dosage range of compatibility, and to reveal the dosage-toxicity-effect relationship of GF-GRR anti-drug combination.Section1Dosage-toxicity-effect research of GF (water extract)-GRR anti-drug combinationGF was safe within the range of Pharmacopeia, and it showed liver and kidney damage exceed three times of Pharmacopeia high dosage. When GF used in combination with GRR, no obvious toxicity was observed in Pharmacopoeia range. When the dose of GF was3times the upper limit for pharmacopoeia and the ratio of GF:GRR was1:10liver toxicity was observed. When the dose of GF was9times the upper limit for pharmacopoeia, liver toxicity was observed as the ratio of GF:GRR was1:10, the liver, kidney and reproductive toxicity was observed as the ratio was1:3, the liver and reproductive toxicity was observed as the ratio was1:1. When the dose of GF was27times the upper limit for pharmacopoeia and the ratio was1:1, it showed liver, kidney and genital toxicity. Males showed obvious toxicity compared with females. As the dose of GF was low, the combination group showed toxicity only when GRR had a large proportion, which is consistent with the rule that with the increase of GRR in co-decoction, the dissolution of the toxic ingredients increased significantly, and the toxicity increased. In the pharmacopoeia dose range, GF had a diuretic effect, no purgative effect and gastrointestinal motility effect was observed. The diuretic effect of GF was inhibited when combined with GRR in Pharmacopoeia range. The gastrointestinal motility effect was also inhibited when the ratio was1:10,1:1and3:1, the absorption of certain toxic ingredients may increase due to the prolonged residence time, thereby the toxicity may be increased.Section2Dosage-toxicity-effect research of GF (powder)-GRR anti-drug combinationGF was safe within the range of Pharmacopeia. When GF used in combination with GRR, no obvious toxicity was observed in Pharmacopoeia range, and the toxicity reaction get more obvious with the increasing of GF and GRR beyond Pharmacopoeia range. When the dose of GF was twice the upper limit for pharmacopoeia and the ratio of GF:GRR was1:10,1:1and3:1, the liver and kidney toxicity was observed. When the dose of GF was4times the upper limit for pharmacopoeia and the ratio was1:10,1:1and3:1, it also showed liver and kidney toxicity. Males showed obvious toxicity compared with females. In the pharmacopoeia dose range, GF had a diuretic effect, and it was inhibited when combined with GRR. The gastrointestinal motility effect was also inhibited when combined with GRR in Pharmacopoeia range. The absorption of certain toxic ingredients may increase due to the prolonged residence time, thereby the toxicity may be increased.