Fritillaria cirrhosa
1、 Pharmacopoeia standard of Fritillaria cirrhosa     This product is Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et K.C.Hsia, Fritillaria przewalskii maxim., Fritillaria delavayi franch., Fritillaria taipaiensis p.y.li or Fritillaria wabuensis Fritillaria unibracteata Hsiao et K.C.Hsiavar wabuensis（S.Y.Tang et S.C.Yue）Z.D.Liu，S.Wang et S. C. Chen's Dried bulb. According to different characters, they are commonly known as "pine shellfish", "green shellfish", "furnace shellfish" and "cultivated product". Excavate in summer and autumn or after the snow melts, remove fibrous roots, coarse skin and sediment, and dry in the sun or at low temperature.
      [character] Pinctada Pinctada is conical or nearly spherical, 0.3 ～ 0.8cm high and 0.3 ～ 0.9CM in diameter. Its surface is white. The outer scale leaves have 2 petals, which vary greatly in size. The large petals hold the small petals tightly, and the unbuckled part is crescent shaped, which is known as "holding the moon in the arms"; ; The apex is blunt or slightly pointed, the bottom is flat, slightly concave, and there is a grayish brown bulb disc in the center, with occasional residual fibrous roots. It is hard and brittle, with white section and rich powder. The breath is slight, and the taste is slightly bitter.
      The green shellfish is oblate in shape, 0.4-1.4cm high and 0.4-1.6cm in diameter. The outer scale leaves are 2-petaled, similar in size, relatively close, and the top is cracked. There are 2-3 heart buds and small scale leaves and thin cylindrical residual stems inside.
      The furnace shell is long conical, 0.7 ～ 2.5cm high and 0.5 ～ 2.5cm in diameter. Its surface is white or light brownish yellow, and some have brown spots. The outer scale leaves are 2-petaled, similar in size, with the top cracked and slightly pointed, and the base slightly pointed or blunt.
      The cultivated products are oblate or short cylindrical, 0.5 ~ 2cm high and 1 ~ 2.5cm in diameter. The surface is white or light brownish yellow, slightly rough, and some have light yellow spots. .
      [identification] (1) the powder of this product is white or light yellow.
      Pine shellfish, green shellfish and cultivated products have many starch grains, which are widely oval, oblong or irregular round, and some of the edges are uneven or slightly branched, with a diameter of 5-64 μ M. the umbilicus is short seam shaped, punctate, herringbone shaped or horseshoe shaped, and the laminar lines are faintly visible. The epidermal cells are rectangular in shape, and the vertical walls are microwave-shaped curved, with occasional adventitious stomata, round or oblate. The diameter of threaded conduit is 5 ～ 26 μ M.
      The starch granules of the Fritillaria are broadly oval, shell shaped, kidney shaped or oval, with a diameter of about 60 μ m, and have herringbone shaped, star shaped or dot shaped tickles, with obvious laminar lines. The diameter of striated duct and reticulated duct can reach 64 μ M.
      (2) Take 10g of this product powder, add 10ml of concentrated ammonia test solution, tightly plug, soak for 1 hour, add 40ml of dichloromethane, sonicate for 1 hour, filter, evaporate the filtrate, add 0.5ml of methanol to dissolve the residue, and use it as the test solution. In addition, take the reference substance of peimine B and add methanol to make a solution containing 1mg per 1ml as the reference solution. . In the chromatogram of the test sample, spots with the same color appear at the corresponding position of the chromatogram of the control sample.
      (3) Polymerase chain reaction restriction enzyme length polymorphism method.
      Template DNA extraction take 0.1g of this product, wash it with 1ml of 75% ethanol and 1ml of sterilized ultrapure water in sequence, suck up the water on the surface, and grind it into very fine powder in a mortar. Take 20mg, put it into a 1.5ml centrifuge tube, and extract DNA with a new broad-spectrum plant genomic DNA rapid extraction kit[add 400 μ l buffer AP1 and 4 μ l RNase solution (10mg/ml), vortex and shake, heat it in a 65 ℃ water bath for 10 minutes, add 130 μ l buffer ap2, mix well, cool it in an ice bath for 5 minutes, and centrifuge (at 14000 RPM) for 10 minutes; ; Add 500 μ l of rinsing solution, centrifuge (at 12000 RPM) for 30 seconds, and discard the filtered solution; Centrifuge again (at 13000 revolutions per minute) for 2 minutes, take out the adsorption column, put it into another centrifuge tube, add 50 μ l elution buffer, place it at room temperature for 3-5 minutes, centrifuge (at 12000 revolutions per minute) for 1 minute, add the eluent into the adsorption column again, place it at room temperature for 2 minutes, centrifuge (at 12000 revolutions per minute) for 1 minute], take the eluent, take it as the test solution, and put it in the refrigerator at 4 ℃ for standby. Another 0.1g of Fritillaria thunbergii reference material was prepared into the template DNA solution of the reference material by the same method.
      PCR-RFLP reaction identification primers: 5'cgtaacaaggttt-ccgtaggtgaa3'and 5'gctacgttcttcatcgat3'. . The centrifuge tube was placed in a PCR instrument, and the PCR reaction parameters were as follows: pre denaturation at 95 ° C for 4 minutes, 30 cycles (95 ° C for 30 seconds, 55-58 ° C for 30 seconds, 72 ° C for 30 seconds), and extension at 72 ° C for 5 minutes. Take the PCR reaction solution, place it in a 500 μ l centrifuge tube, and perform the enzymatic digestion reaction. The total volume of the reaction is 20 μ L. the reaction system includes 10 × 2 μ l of enzymatic digestion buffer, 6 μ l of PCR reaction solution, 0.5 μ l of SMA I (10u/ μ L), 11.5 μ l of sterile ultrapure water. The enzymatic digestion reaction is carried out in a 30 ℃ water bath for 2 hours. In addition, take sterile ultrapure water and operate the PCR-RFLP reaction in the same way as above as a blank control.
      Agarose gel electrophoresis (general rule 0541) was used for electrophoresis detection. The gel concentration was 1.5%, and GelRed, a nucleic acid gel stain, was added to the gel; The loading amount of enzymatic digestion reaction solution of the test sample and the control medicinal material was 8 μ L, and the loading amount of DNA molecular weight marker was l μ L (0.5 μ g/ μ L). After electrophoresis, take the gel slice and inspect it on the gel imager or UV transmission instrument. In the gel electrophoresis pattern of the test sample, there should be two DNA bands at 100 ~ 250bp in the position corresponding to the gel electrophoresis pattern of the control medicinal material, and there is no band in the blank control.
      [inspection] the moisture content shall not exceed 15.0% (the second method of general rule 0832).
      The total ash content shall not exceed 5.0% (general rule 2302).
      [extract] according to the hot leaching method under the alcohol soluble extract determination method (general rule 2201), using dilute ethanol as solvent, it shall not be less than 9.0%.
      [content determination] preparation of reference solution take an appropriate amount of West Fritillaria base reference, weigh accurately, add chloroform to make a solution containing 0.2mg per 1ml.
      Preparation of standard curve accurately measure 0.1ml, 0.2ml, 0.4ml, 0.6ml, 1.0ml of the reference solution, put it into a 25ml plugged test tube, add chloroform to 10.0ml respectively, add 5ml of water precisely, and then add 0.05% bromocresol green buffer accurately (take 0.05g of bromocresol green, dissolve it with 6ml of 0.2mol/l sodium hydroxide solution, add LG of potassium dihydrogen phosphate, add water to dissolve it and dilute it to 100ml, get it), close the plug, shake vigorously for 1 minute, transfer it to a separatory funnel, and leave it for 30 minutes. Take the trichloromethane solution, filter it with dry filter paper, take the continuous filtrate, take the corresponding reagent as blank, measure the absorbance at the wavelength of 415nm according to the UV visible spectrophotometry (general rule 0401), and draw the standard curve with the absorbance as the ordinate and the concentration as the abscissa.
      Determination method take about 2G of this product powder (passing through No. 3 screen), accurately weigh it, put it into a corked conical flask, add 3ml of concentrated ammonia test solution, soak it for 1 hour, add 40ml of trichloromethane methanol (4:1) mixed solution, heat it in an 80 ℃ water bath and reflux it for 2 hours, cool it, filter it, put the filtrate into a 50ml volumetric flask, wash the drug residue with an appropriate amount of trichloromethane methanol (4:1) mixed solution for 2-3 times, incorporate the washing solution into the same volumetric flask, add the trichloromethane methanol (4:1) mixed solution to the scale, and shake it well. Accurately measure 2-5ml, put it into a 25ml plugged test tube, evaporate it on a water bath, precisely add 10ml of chloroform to dissolve it, according to the method under the preparation of the standard curve, measure the absorbance according to the law from "5ml of precise water", read the weight (mg) of imperialine in the test solution from the standard curve, and calculate.
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      [nature, taste and meridian tropism] bitter, sweet and slightly cold. Return to lung and heart meridian.
      [functions and indications] clearing heat and moistening lungs, resolving phlegm and relieving cough, dispersing nodules and eliminating carbuncle. It is used for lung heat dry cough, dry cough with less phlegm, yin deficiency and cough, blood in sputum, scrofula, breast carbuncle and lung carbuncle.
      [usage and dosage] 3-10g; powder, 1-2g at a time.
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      [storage] store in a ventilated and dry place to prevent moths.


2、 Chemical constituents of Fritillaria cirrhosa
1. the bulb of Fritillaria thunbergii contains alkaloids: songbeisine and songbeinine. It also contains sucrose, stearicacid,palmitic acid（palmiticacid），β - sitosterol（β-sitosterol）。
,Imperialine（sipeimine）。
3. alkaloids in Fritillaria Fritillaria Bulb:Fritillaria angustifolia（delavine），Fritillaria angustifolia base（delavinone）， Chuanbeinone, delafrine, delafrinone, (22r, 25s) - 5-solanid-5-en-3 β, 5 α, 6 β - triol, PEIMISINE,ImperialineImperialine refers to imperialine, chuanbeine and Fritimine.

3、 Pharmacological effects of Fritillaria cirrhosa
1. effect on respiratory system:
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  1.2 expectorant effect: the phenol red excretion method in mice was used, and the dose of 3.9g/kg (Ig) was used as the time-lapse curve. 0.25% phenol red (0.2ml/10g) was immediately IPED at 30, 60 and 120 minutes after administration, and the absorbance at 558nm was measured by spectrophotometry. . The results showed that the expectorant effects of the two kinds of Fritillaria were enhanced with increasing dose. Other mice were treated with 30g/kg Fritillaria thunbergii decoction, and the pain threshold was reduced 60 minutes after treatment compared with that before treatment.

2. anti ulcer effect
  2.1 effect on ligation of pylorus ulcer in rats: rats were anesthetized and opened 48 hours after fasting, ligated gastric pylorus, and sutured the abdominal wall with 3 and 1.5mg/100g of total alkaloids (total alkaloids for short), and the control group was treated with equal volume of normal saline. Nine hours after administration, the rats were decapitated and sacrificed, and the gastric juice was collected. At the same time, the amount of gastric juice, free acid, total acid and pepsin activity were measured, and the ulcer formation was observed with the naked eye. Results the two doses had significant inhibitory effect on pepsin, P < 0.01 compared with the control group, and the ulcer index of the 1.5mg/100g group was also significantly lower than that of the control group, P < 0.01.

  2.2 effect on stress ulcer: observe the ulcer index of stress ulcer rats with the above doses. Results the 1.5mg/100g group had obvious inhibitory effect, P < 0.05 compared with the normal saline control group.

  . Results 3.0mg/100g total alkaloids had inhibitory effect on indomethacin induced gastric ulcer, compared with the control group (P < 0.01).

3. other functions. The intravenous injection of Fritillaria thunbergii can produce a lasting drop in blood pressure in anesthetized cats, accompanied by short-term respiratory depression, can enhance the contraction of isolated uterus of guinea pigs, inhibit isolated rabbit intestine, and have no effect on pupil expansion. The minimum lethal dose to mice is 40mg/ kg. There is convulsion before death, and it also causes blood glucose elevation in rabbits.

Imperialine has hypotensive effect on anesthetized dogs, which is mainly due to peripheral vasodilation and has no obvious effect on ECG. .
The intravenous injection of frittimine (4mg/kg) produced a persistent drop in blood pressure accompanied by transient respiratory depression in anesthetized cats; 7.5-16mg/kg intravenously can increase blood glucose in rabbits; 1: 50000 concentration can make the isolated guinea pig uterus contract, and 1:100000 concentration can inhibit the movement of isolated rabbit intestine; The minimum lethal dose to mice is 40mg/kg.
The extract of Fritillaria ussuriensis has an inhibitory effect on the central nervous system of experimental animals.