| Objective:(1)To establish a qualitative and quantitative analysis method for Gymnadenia conopsea,and provide a basis for improving its quality standard.(2)The long-term toxicity experiment of Gymnadenia conopsea was carried out to evaluate its safety and provide experimental basis for clinical application.Methods:(1)To study the qualitative identification of Gymnadenia conopsea by character identification,microscopic identification and thin layer identification.(2)A quantitative analysis of multi-components by single marker method(QAMS)was established for simultaneous determination of dactylorhin A,p-hydroxybenzyl alcohol,loroglossin and militarine in Gymnadenia conopsea.The analysis was performed on a Agilent Zorbax SB-C18column(250mm×4.6mm,5μm),with a mobile phase consisting of methanol-aqueous solution for gradient elution.The volume flow rate,column temperature and sample injection volume were set at 1.0 mL/min,25°C,and 10 μL,respectively.The relative correction factors of dactylorhin A and p-hydroxybenzyl alcohol,loroglossin and militarine were calculated and the durability was also investigated.The contents of these four compounds in ten batches of Gymnadenia conopsea from different batches were determined by external standard method(ESM)and quantitative analysis of multi-components with a single-marker method(QAMS),respectively.(3)HPLC method was used to establish the common pattern of fingerprint of 10 batches of Gymnadenia conopsea,and the common peaks were identified qualitatively based on the retention time of the reference substance and spectrogram comparison.(4)According to the general principles of the Chinese Pharmacopoeia(2020 edition,Part IV),the moisture,total ash content,acid-insoluble ash content and alcohol-soluble extract of Gymnadenia conopsea were determined.(5)A total of 120 SD rats were randomly divided into blank control group,low dose of Gymnadenia conopsea group,medium dose of Gymnadenia conopsea group and high dose of Gymnadenia conopsea group,with 30 rats in each group.The rats were fed with ordinary feed and medicated feed every day for 3 months,the blank group was fed with ordinary feed and the same volume of distilled water.The general status of rats in each group was continuously observed,their weight was measured weekly,and 10 rats in each group were killed at 45 days,90 days,and 15 days after drug withdrawal.Blood routine indexes,blood biochemical indexes and major organ indexes were measured,and HE staining was used to observe the lesions of various organs and comprehensively evaluate the long-term toxicity of Gymnadenia conopsea.Results:(1)By observing appearance,color,shape and smell,the characters and characteristics of Gymnadenia conopsea were determined.The microscopic characteristics of calcium oxalate needle crystal,ladder tube,calcium oxalate square crystal and starch granules were determined by microscopic identification method.In the TLC identification of Gymnadenia conopsea,the chromatogram of test solution and reference solution such as p-hydroxybenzyl alcohol and β-sitosterol showed the same color spots,the color of the spots was clear and the separation was good.(2)The content ranges of p-hydroxybenzyl alcohol,loroglossin,dactylorhin A and militarine were 0.07% ～ 0.51%,0.18% ～ 12.80%,0.40% ～4.75% and 0.09% ～ 1.33%,respectively.Using dactylorhin A as reference,the relative correction factors of p-hydroxybenzol,loroglossin and militarine were 0.2146,0.8154 and0.5478,respectively.RSDs of the relative correction factor were less than 2% at different column temperatures,flow rates,instruments and columns,indicating that the changes of column temperature,flow rates,instruments and columns had little effect on the relative correction factors.RSDs of relative retention time and retention time difference were less than5% when different columns were used,indicating that the chromatographic peaks could be located by relative retention time and retention time difference.Relative error was used to evaluate QAMS method and ESM method,and the results showed that RE was less than 5%,indicating that there was little difference between QAMS method and ESM method.The contents of P-hydroxy benzyl alcohol,loroglossin,dactylorhin A and militarine in different batches of Gymnadenia conopsea were significantly different,and the contents of p-hydroxy benzyl alcohol and dactylorhin A were the highest in Gymnadenia conopsea grown in Alute of Chifeng City.The content of loroglossin in Gymnadenia conopsea growing in Kezuohouqi of Tongliao City was the highest,and the content of militarine in Keyouzhongqi of Xingan was the highest.(3)There were 12 common peaks in fingerprints of 10 batches of Gymnadenia conopsea,and the similarity was above 0.9.P-hydroxybenzyl carbinol,loroglossin,dactylorhin A and militarine were identified.(4)The moisture,total ash content,acid-insoluble ash content and alcohol-soluble extract of Gymnadenia conopsea were8.13%～11.98%,1.31%～5.73%,0.17%～1.96%,14.98%～25.44%,respectively.(5)During the long-term toxicity experiment,no death was observed in the animals in the administration groups,and the general condition,body weight and food intake were normal.At the middle stage of administration,the mean platelet volume of the medium and high dose groups of Gymnadenia conopsea was significantly increased compared with the blank control group,and recovered to normal at the recovery stage.At the end of administration,the creatinine and total protein were significantly decreased in the high-dose and medium-dose groups.After convalescence,cholesterol in medium dose group decreased significantly.There were no significant differences in organ weight and organ index among all groups,and no pathological changes were observed in organ histopathology.Conclusion:(1)The microscopic identification method established in this paper is simple,reliable and stable.The established thin layer identification method has clear spots and strong specificity.The above two methods can be used for qualitative identification of Gymnadenia conopsea.(2)The QAMS method based on dactylorhin A was simple,practical and reliable,and could be used for the content determination and quality evaluation of Gymnadenia conopsea.(3)No obvious toxic reaction was observed in rats after continuous intragastric administration of Gymnadenia conopsea for 3 months,indicating that Gymnadenia conopsea was safe. |
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