Preliminary Study On The Effective And Toxic Components In Radix Tripterygii Based On Spectrum-effect Relationship Analysis

ObjectiveWith commonly used effective and poisonous Radix Tripterygii as the research object, to explore the material foundation for anti-inflammatory effect and liver damage of tripterygium wilfordii, and to provide reference to enhance the quality control methods of Radix Tripterygii. MethodsBy sampling common varieties Tripterygium wilfordii Hook. F. and local alternative Tripterygium hypoglaucum(Levl.) Hutch, a total of 23 batches of samples were included.LC-Q/TOF-MS technology was introduced to characterize the chemical composition of Tripterygium wilfordii Hook. F. samples of different regions. The liquid phase methods: chromatographic column: Agilent ZORBAX Eclipse Plus C18 600 Bar(1.8 mu m, 2.1 * 100 mm). Mobile phase: A: CH3CN(HCOOH) 0.1%, B: H2O(HCOOH) 0.1%; Flow rate: 0.3 m L, min- 1; Sample quantity: 4 μL; Column temperature: 30 ℃; The sample temperature: 4 ℃; Detection wavelength: 190-420 nm; Mass spectrometry conditions: ion source: ESI; Capillary voltage: 4000 V; Mass/nuclear than range: 80-1000 m/z; degasser temperature: 225 ℃; desolvation gas flow: 11 L/min; Atomizer pressure: 45 psig. Sheath temperature: 350 ℃; 12 L/min; sheath gas flow rate: Nozzle: voltage 500 V. Through the high resolution mass spectrometry combined with literatures, including 10 kinds of main components were preliminary identified; Fingerprint similarity calculation software-USES, adopted by the pharmacopoeia to study the similarity of all herbs, was further carried out to study the similarities between the original medicinal materials. By subjecting each sample to SPSS software for system cluster analysis, the study preliminarily identified 10 kinds of main components of highest abundance.For tripterygium wilfordii induced liver toxicity and anti-inflammatory effects, with normal liver cell(L02 cell line) as the carrier and acetaminophen as positive medicine, cell inhibition rate as testing index, to optimize the liver cell poison value, and to evaluate the toxicity potency of different tripterygium wilfordii Hook. F. and its alternatives. With macrophages(BMDM) as the carrier, nitrogen pyridine as positive drugs, and interleukin 6(IL-6), interleukin 10(IL-10) and nitric oxide synthase(i NOS) as indicators, in vitro anti-inflammatory effect of tripterygium wilfordii Hook. F. was established and in vitro anti-inflammatory effect of tripterygium wilfordii was evaluated.Based on spectrum-effect correlation analysis, by combining chemical detection and biological detection technology, with the adoption of simple correlation analysis and multivariate statistics, ingredients responsible for tripterygium wilfordii induced liver toxicity and anti-inflammatory effects were analyzed, which could provide reference basis for its quality control. Results(1) Collection and preparation of sample: Taking 23 batches of Radix Tripterygii collected from 2 kinds of origin(national main habitats) as the study subject, the result of extraction rate shows that extraction rate varies greatly in different habitat.(2) Chemical characterization and fingerprint analysis: According to the chemical fingerprint, the chemical component varies greatly among 23 batches of Radix Tripterygii, and chemical component similarities across samples from different habitats are greater than the differences. Furtherly analyzed by high-resolution Mass Spectroscopy and relevant literature, 10 kinds of main component was preliminarily identified from the extract ion flow profile: tripterygium wilfordii lactone alcohol(EIC + M/Z: 361.1641), tripterygium wilfordii alkaloid e(EIC + M/Z: 780.2709), tripterygium wilfordii soda ash(EIC + M/Z: 874.2764), tripterygium wilfordii lactone(EIC + M/Z: 313.1798), tripterygium wilfordii alkaloid(EIC + M/Z: 884.2972), tripterygium wilfordii jin alkali(EIC + M/Z: 858.2815), tripterygium wilfordii time base(EIC + M/Z: 868.3022), Demethylzeylasteral(EIC + M/Z: 481.2585), tripterygium wilfordii red element(EIC + M/Z: 451.2848), tripterygium wilfordii ester methyl(EIC + M/Z: 455.3520). Hierarchical cluster analysis was carried out based on aforesaid 10 kinds of main components by SPSS, and the results showed that 8 batches of Tripterygium wilfordii Hook. f. materials were classified into the same cluster, which suggested that those components had a fine aggregation degree in Radix Tripterygii(T. wilfordii). In addition, 5 batches of samples from Tripterygium hypoglaucum(Levl.) Hutch materials couldn’t classified into the same cluster between each other, suggesting that the component content differentiated greatly in kunmingshan haitang(T. hypoglaucum) materials.(3) Biological and poisonous value: Hepatotoxicity value of 18 samples of Radix Tripterygii was 17.78 ~ 4131.4 U/g(far more than 200 times), and hepatotoxicity value of 5 samples of kunmingshan haitang was 209.42 ~ 7422.2 U/g(far more than 30 times). Hepatotoxicity varies significantly in different habitat. Willow nitrogen cycling arsenic organism(positive comparison) could significantly reduce the concentration of IL-6 and i NOS in the BMDM supernatant fluid, and significantly increased the concentration of IL-10, which showed good anti-inflammatory efficacy. The IL-6 inhibition capacity of 18 samples was stronger than the positive drug group(P < 0.05), the IL-10 releasing effect of 7 samples was stronger than that of positive medicine(P < 0.05), and the i NOS inhibition capacity of the 16 samples was significantly stronger than the positive medicine(P < 0.05);(4) Spectrum-effect correlation analysis: through simple correlation analysis of toxicity potency and ten main ingredients, the results showed that, with correlation coefficient R > 0.80 as index, it can be determined primarily that tripterine, triptolide, Demethylzeylasteral may be the main toxic compounds of tripterygium wilfordii, of which ripterine showed highest correlation; Through principal component regression analysis of ten main component, in fitting regression model, R2 = 0.745, and there was a significant statistical significance, as P = 0.0049 < 0.01. The regression model showed that variable tripterine has a larger parameter values, illustrating the great contribution of tripterine to toxicity of tripterygium wilfordii. Simple correlation and multiple regression analysis indicated that tripterine and triptolide has greater contribution to the toxicity of tripterygium wilfordii. Through analysis of the correlation of inflammatory factor IL-10 with 10 constituents, it was found that 10 main ingredients and IL-10 had no significant correlation; Through principal component regression analysis of 10 main components and inflammatory factor of IL- 10, it was found that in the fitting regression model, R2 = 0.4601, and there was a significant statistical significance, with P = 0.0.0154 < 0.05. The regression model showed that triptolide, wilforgine has a larger parameter values, which indicted that triptolide wilforgine have greater contribution to the anti-inflammatory effect of tripterygium wilfordii.Further we made a comprehensive analysis of the efficacy and toxicity of 23 batches of Tripterygium wilfordii samples, and screened "low-poison high-effectiveness" samples, including 5 batches of Tripterygium wilfordii medicinal materials, all of which the origin were Tripterygium wilfordii Hook. F., and their origin are mainly distributed in southwest region. Also we screened the “high-poison low-effectiveness " samples, which were originated from anhui bozhou. ConclusionThe method built for in vitro bio-potency detection of tripterygium wilfordii Hook. F. can effectively evaluate difference between toxicity of tripterygium wilfordii Hook. F. induced liver toxicity and anti-inflammatory effect; Spectrum- effect correlation analysis study found that triptolide may be the toxic and anti-inflammatory component, which is consistent with the traditional literature. In addition, this paper found that tripterine mainly contributed to hepatotoxicity, whereas wilforgine is highly correlated to anti-inflammatory effect of tripterygium wilfordii Hook. F., special attention should be paid to the three components in the quality control of tripterygium wilfordii Hook. F.; tripterygium wilfordii Hook. F. of different varieties, origins, showed big difference, tripterygium wilfordii Hook. F. of southwest origin showed relatively high efficacy and low toxicity, indicating that it can be used as the important resources in the subsequent development of high-efficiency but low toxicity medicinal materials.