Daphne genkwa
1、 Pharmacopoeia standard of Daphne genkwa       This product is the dried flower buds of Daphne genkwa sieb. et zucc., a Daphne family plant. .
      ; This product usually has 3-7 flowers clustered on the short flower axis, 1-2 bracts at the base, and most of them fall off as a single flower. A single flower is mallet shaped, mostly curved, 1-1.7cm long, about 1.5mm in diameter; the surface of the perianth tube is lavender or grayish green, densely pubescent, the apex is 4-lobed, and the lobes are lavender or yellowish brown. Soft. It tastes sweet and pungent.
      [identification] (1) powder: grayish brown. The pollen grains are yellow, quasi spherical, 23-45 μ m in diameter, with obvious reticular carvings on the surface, and most germination pores scattered. There are non glandular hairs on the lower surface of the perianth, which are single-cell, multi curved, 88 ～ 780 μ m long, 15 ～ 23 μ m in diameter, with thick walls and slightly warty protrusions.
      (2) . Another 1g of Daphne genkwa reference drug was prepared into the reference drug solution by the same method. Then take the reference substance of genkwanin and add methanol to make a solution containing 2mg per 1ml as the reference solution. According to the thin-layer chromatography (general rule 0502), suck 4 μ l of each of the above three solutions and dot them on the same silica gel G thin-layer plate. Use toluene ethyl acetate formic acid (8:4:0.2) as developing agent, develop, take out, dry, and inspect under UV light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control sample.
      [extract] according to the hot leaching method under the determination method of alcohol soluble extract (general rule 2201), the use of dilute ethanol as solvent shall not be less than 20%.
      [content determination]   Determine according to high-performance liquid chromatography (general rule 0512).
      ; Octadecylsilane bonded silica gel was used as filler; The mobile phase was methanol water glacial acetic acid (65:35:0.8); The detection wavelength was 338nm. The number of theoretical plates should not be less than 6000 according to the peak of genkwanin.
      Preparation of reference solution   Take a proper amount of genkwanin reference substance, weigh it accurately, add methanol to make a solution containing 90 μ g per 1ml.
      Preparation of test solution   Take about 0.5g of this product powder (passing through No. 4 screen), weigh it accurately, place it in a corked conical flask, add 25ml of methanol precisely, weigh it, heat and reflux for 1 hour, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate.
      Assay   Precisely suck 10 μ l of the reference solution and 10 μ l of the test solution respectively, inject them into the liquid chromatograph, and determine.
      Calculated as a dry product, the content of Daphne genkwanin (c16h12o5) shall not be less than 0.20%.
      Decoction pieces
      [processing] remove impurities from Wuhua.
      [character] [identification] [extract] [content determination]   Same as medicinal materials.
          . 30kg vinegar for every 100kg of Daphne genkwa.
        This product is shaped like Daphne genkwa, with a yellowish surface. It has a slight vinegar aroma.
      [nature, taste and meridian tropism]   Bitter, pungent, warm; . It belongs to the lung, spleen and kidney meridians.
        [function and indications]   ; Topical insecticidal treatment of sores. It is used for edema, fullness, hydrothorax, accumulation of phlegm and drink, cough and asthma due to Qi inversion, and unfavorable defecation; External treatment of scabies, alopecia, carbuncle, chilblain.
      [usage and dosage]   1.5 ～ 3G. Swallow vinegar Daphne genkwa powder, 0.6 ～ 0.9g once a day. .
      [note]   Forbidden for pregnant women; It should not be used with licorice.
      [storage]   Place in a ventilated and dry place to prevent mold and moth.


2、 Chemical constituents of Daphne genkwa
The flowers and flower buds contain diterpenoid orthoacid esters: the flowers contain yuanhuacin, yuanhuadin, yuanhuafin, genkwadaphnin, or 12 benzoxydaphnetoxin; Flower buds contain yuanhuatin and yuanhuapin. Flavonoids:Daphnetin（genkwanin）， 3 '- hydroxygenkwanin (luteolin-7-methyl), yuankanin,Apigenin（apigenin），Luteolin（luteolin）， Tilirosid is kaempferol-3-o - β -d- (6 ″ - p-coumaroyl) glucopyranoside.
Flower volatile oil contains a lot of fatty acids,palmitic acid（palmitic aicd）、 ; It also contains n-tetracosane, n-Pentadecane, n-dodecanal, undecanonal, benzaldehyde, a-furaldehyde, phenylethanol, 1-octene-3-ol, humulene, geraniolpropionate and nerol pentanoate.

3、 Pharmacological effects of Daphne genkwa
. At a dose of 20g / kg, the rate of urination, sodium excretion and potassium excretion were significantly increased, but 2.5 or 5g / kg was ineffective. It is also reported that intravenous injection of 50% Daphne genkwa Decoction at 0.4-1.0g / kg to anesthetized dogs can more than double the urine output and maintain it for about 20 minutes. Rats with ascites formed by intraperitoneal injection of 3% sodium chloride solution were gavaged with 10g / kg Daphne genkwa decoction or alcohol extract, which had diuretic effect.
2. antitussive and expectorant effects: the results of ammonia spray induced cough experiment showed that mice were given 1.25g/kg vinegar made Daphne genkwa and benzene made Daphne genkwa alcohol water extract, or 0.625g/kg hydroxygenkwa had antitussive effects. Phenol red excretion experiment showed that mice were given 5g / kg vinegar made Daphne genkwa and benzene made Daphne alcohol water extract or 0.625g/kg hydroxygenkwanin by gavage, which had certain expectorant effect. The expectorant mechanism may be related to the reduction of inflammation and sputum viscosity after treatment.
3. effects on the central nervous system: according to xiaoqingci and other reports, 90 Kunming white mice weighing 15-25g, both male and female, were randomly divided into 9 groups, 10 in each group. The drugs were administered by gavage according to the doses listed in Table 4. Ysd-4 pharmacological and physiological experiment multi-purpose instrument and electric stimulation box were used to measure and record, and the voltage (MV) that caused the mouse to scream at different times was observed. The results are shown in Table 4. . And the baked Daphne genkwa lies on the licorice. Compared with the single decoction of the same dose, the analgesic effect of the combined decoction was better than that of licorice and inferior to that of fried Daphne genkwa. According to weichengwu and other reports, intraperitoneal injection of 500mg / kg of Daphne genkwa ethanol extract (converted into crude drug, the same below) has obvious analgesic effect on mice in hot plate method, antimony potassium tartrate writhing method and electric shock method, and naloxone, a morphine receptor antagonist, can block its analgesic effect. In the mouse rotarod test, intraperitoneal injection of l000mg / kg ethanol extract of Daphne genkwa showed obvious sedative effect. In the anti convulsive test of strychnine or sodium benzoate caffeine, it had obvious anticonvulsant effect, and the anti convulsive effect of tudinine was stronger; .
4. effect on digestive system:.
4.1. effects on isolated intestinal segments of animals: several healthy adult rabbits weighing about 2kg, both male and female, were taken out of the jejunum after acute sacrifice, processed routinely, and stored in Tyrode's solution for future use. The experiment was divided into 20 groups. The results showed that at high concentration, the excitatory effect of baked Daphne genkwa on intestinal segments was better than that of liquorice, and at low concentration, liquorice was buried in Daphne genkwa. . The alcohol extract of raw Daphne genkwa and vinegar made Daphne genkwa can cause mild diarrhea in rabbits, vomiting in dogs, but not in mice. It is also reported that the irritating oil of 1% Daphne genkwa can make the isolated duodenum of rats show tetanic contraction, while the isolated duodenum of rabbits is first excited and then inhibited. 10% vinegar and 10% benzene Daphne (prepared after removing the oil with benzene) have similar effects.
4.2. effect on intestinal peristalsis in rats: rats were randomly divided into three groups by whole intestine propulsion method. After fasting for 24h, rats were orally gavaged with raw Daphne genkwa and vinegar baked Daphne genkwa (40g / kg), and 5% charcoal powder was given at the same time. Rats were treated 1 hour later. The control group was set up to observe the pushing length of charcoal powder in raw and vinegar baked Daphne genkwa groups. The total length of small intestine of rats (mm) was measured and the pushing rate was calculated (10 rats in each group). The results showed that raw Daphne genkwa and vinegar baked Daphne genkwa had a mild excitatory effect on the intestinal peristalsis of rats, which accelerated the intestinal peristalsis. The charcoal staining length of raw Daphne genkwa group increased by 6.4% on average compared with the control group; The average increase of vinegar baked Daphne genkwa group was 9.7% compared with the control group; It shows that the effect of vinegar baked Daphne genkwa on intestinal peristalsis is stronger than that of raw product.
5. antifertility effect: the combination of Daphne genkwa terpene and estradiol has synergistic effect on rat uterus in vitro, and its effect on cervix is weaker than that of uterine body, and it has no synergistic effect with estradiol. Injection of 100 μ g/kg Daphne genkwa terpene into rabbit cervix caused strong uterine contraction. . Intrauterine administration of drugs in pregnant monkeys can also cause abortion. And the local administration effect is strengthened, and the intravenous injection reaction is slow or not obvious. The causes of abortion can be seen from the pathological examination results of the placenta and fetus induced by Daphne genkwa terpene. The degenerative chorionic Decidua Tissue thrombosis, red blood cell destruction, and a large number of neutrophilic polymorphonuclear leukocytes under the placental chorionic plate are gathered, which is the reason for the inflammatory cell infiltration after drug injection; The pathological changes such as tissue edema, hemorrhage and cell swelling are the direct effects of drugs on local tissues; The degeneration and necrosis of decidual cells, as well as the inflammatory cell infiltration and edema of the adhesive tape, may be due to the increased secretion and release of endogenous prostaglandins, resulting in the enhanced contraction of uterine smooth muscle cells, so as to achieve the purpose of labor induction. From the perspective of cell ultrastructural function, after administration, it can cause obvious degeneration and necrosis of decidua and placenta, so that lysosomes are destroyed and a large amount of phospholipase A is released, resulting in accelerated synthesis and release of prostaglandins in the rough endoplasmic reticulum of decidua that continues to maintain secretory function. The experiment proved that the amniotic fluid of induced labor pregnant women, with the increase of prostaglandin content, accompanied with the beginning of labor, stimulated uterine smooth muscle contraction and caused abortion. This further shows that endogenous prostaglandins play an important role in the mechanism of genkwa terpene induced labor. . Another study showed that Daphne genkwa terpene (20 μ g/ml) had a certain inhibitory effect on the synthesis of mouse embryo deoxyribonucleic acid (DNA). .
6. inhibitory effect on xanthine oxidase (XO): in 1983, Noro, tacataka, etc. found that the flowers and buds of Daphne genkwa had a strong inhibitory effect on XO when looking for XO inhibitors from crude drugs and botanical drugs, and isolated four components with inhibitory effects on XO, including genkwanin, apigenin, 3-hydroxygenkwanin, and luteolin. Their IC50 of inhibitory activities were 7 × 10 (-5) m, 7.4 × 10 (-7) m, 10-5m, and 5.9 × 10 (-7) m, respectively. Apigenin and luteolin are the strongest inhibitors of XO. These flavonoids did not show strong inhibitory activity against monoamine oxidase under this test condition.
7. anti leukemia effect: Hall IH and Noro t isolated two diterpenoid compounds with strong anti-p-388 lymphocytic leukemia from the methanol extract of Daphne genkwa flower, Daphne genkwa Daphne Ning and Daphne genkwa ester a, respectively. At a low dose (0.8mg / kg) in vivo, they showed potent inhibitory activity, with T / C values of 175% and 151%, respectively. The study also showed that Daphne genkwa Daphne and genkwa ester a could inhibit the synthesis of nucleic acid and protein in P-388 cancer cells. The inhibition of the former was the phosphoribosyl aminotransferase, inosine dehydrogenase and dihydrofolate reductase in the synthesis of DNA polymerase and purine; For the latter system, the reaction of peptidyl transferase was inhibited and interfered in the extension step.
8. antibacterial effect: in vitro test, 1:50 concentration of alcohol water extract of vinegar made Daphne genkwa and benzene made Daphne genkwa have inhibitory effect on pneumococcus, Streptococcus hemolyticus and influenza bacilli. The water extract of Daphne genkwa (1:4) had different degrees of inhibitory effects on skin fungi such as Schlumberger, Microsporum audouang, and Nocardia starum in the test tube. Daphnetin has no antibacterial effect.
. The experiment without quadriceps stimulation showed that 5mg, 0.5ml genkwa terpene alcohol solution had serious local stimulation reaction.
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10.1 effect on cardiovascular system:.
10.1.1. effect on isolated hearts of guinea pigs: eight guinea pigs weighing 350-600g were taken out, and the hearts were quickly removed after sacrifice. According to Langendorff's method, the hearts were perfused with renluo's solution at 37 ℃, constant temperature and oxygen flow at a constant pressure of 70cm in height. After about 15 minutes of cardiac activity and flow stabilization, add 0.6g/100ml of Daphne genkwa leaf extract (crude drug content, the same below) into the perfusate, and observe the changes of coronary flow and heart rate. The results showed that Daphne genkwa leaves significantly increased coronary flow, but the heart rate did not change significantly.
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. After 30 minutes, one administration mouse and one control mouse with the same sex and similar weight were selected and placed in an airtight container with 10g sodium lime. The average survival time of mice in the administration group was 877.9 ± 33.0 seconds, and that of mice in the control group was 715 ± 32.3 seconds. There was a very significant difference between the two groups in t-value test (P < 0.01).
10.1.4. effect on serum phosphocreatine kinase (CPK) in cats with coronary artery ligation the cat weighed 2.0-3.8kg, both male and female, was fixed in the back position under pentobarbital abdominal anesthesia, the trachea was connected to an artificial respirator, and the left carotid artery blood pressure was recorded with a mercury manometer. Open the chest, expose the heart, thread a silk thread 12-14mm from the origin of the coronary artery under the anterior descending branch of the left coronary artery, and ligate the anterior descending branch after the blood pressure is stable. After ligation of the coronary artery, the cats in the administration group were immediately injected with 0.75g/kg Daphne genkwa leaf injection through the femoral vein, and supplemented with 0.375g/kg/h by a constant flow pump within the next 5 hours. The control group was given the same volume of normal saline. 1ml of blood was taken from the left common carotid artery every hour 5 minutes before and 5 hours after coronary artery ligation, and the serum creatine phosphokinase (CPK) activity was measured. Considering that the skeletal muscle injury caused by surgery can lead to the increase of serum CPK activity, another blank group was set up. The cats in this group only wore a silk thread under the anterior descending branch of the left coronary artery without ligation. The same volume of normal saline was given by femoral vein injection. All other surgical procedures were the same as those in the above two groups. The first blood was taken 5 minutes before threading, and the interval between subsequent blood taking was the same as those in the above two groups. According to the determination, the serum CPK activity of the three groups of cats within 5 hours was enhanced compared with that before coronary artery ligation or myocardial threading, but the degree was different, and the CPK activity of the control group was the most obvious. Since all the surgical procedures of the control group and the blank group are the same, the difference between the two groups can be seen as caused by ligation of the coronary artery. Both the control group and the administration group are animals with ligation of the coronary artery, so the difference between the latter two groups is caused by drugs.
10.1.5. acute hypotensive effect on anesthetized cats and effect on heart rate six anesthetized cats, weighing 2.0-2.0kg, were injected with genkwa leaf injection 1g / kg through femoral vein for about 1 minute. The blood pressure rapidly decreased from 116.2 ± 7mmhg to 65.8 ± 6.1mmhg, a drop of 43.5 ± 3.0%. The hypotensive effect maintained for about 2 minutes. Two cats were injected again after bilateral vagus nerve transection, and its hypotensive effect was not obvious. The hypotensive effect of Daphne genkwa leaf was slightly weakened after intravenous injection of atropine LMG / kg in the other three cats. According to this analysis, the hypotensive mechanism may not be mainly through the vagus nerve. The cat's heart rate did not change significantly within 10 minutes, and the heart rate was not abnormal.
10.2. coronary artery dilation: perfusion of 1 / 100000 Daphne genkwa B aqueous solution in isolated intestinal rat hearts has a significant dilation effect, and its potency is slightly lower than that of dimethoxymethyl furan chromogen (khellin).
10.3. fish poisoning effect: a 1 / 100000 aqueous solution of Daphne genkwa B can kill goldfish within 30 minutes, similar to the fish poisoning effect of khellin like furan chromones.