Semen ziphi Spinosae
1、 Pharmacopoeia standard of semen ziphi Spinosae       . The mature fruits were harvested at the end of autumn and the beginning of winter, the pulp and core shell were removed, and the seeds were collected and dried in the sun.
      [properties] the product is oblate or oblate, 5-9mm long, 5-7mm wide, and about 3mm thick. The surface is purplish red or purplish brown, smooth and shiny, and some have cracks. Some of the two sides are round protrusions; Some surfaces are relatively flat, with a raised longitudinal line in the middle; . One end is concave, and a linear hilum can be seen; At the other end, there is a small protruding commissure. The seed coat is brittle, the endosperm is white, the cotyledons are 2, light yellow, and rich in oil. The air is weak and the taste is light.
      [identification] (1) the powder of this product is brownish red. The seed coat grid cells are brownish red, polygonal in appearance, about 15 μ m in diameter, thick in wall, lignified, and small in lumen; The lateral view shows a long strip, the outer wall is thickened, the upper and middle parts of the side wall are very thick, and the lower part is gradually thin; The bottom view is polygonal or round polygonal. The epidermal cells in the seed coat are brownish yellow, rectangular or square like on the surface, with bead like thickening and lignification of the pericarp wall. .
      (2) Take 1g of this product powder, add 30ml of methanol, heat and reflux 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 wild Jujuboside A reference substance and wild Jujuboside B reference substance, add methanol to make a mixed solution containing 1mg per 1ml as the reference solution. Test according to thin-layer chromatography (general rule 0502), suck 5 μ l of each of the above two solutions, dot them on the same silica gel G thin-layer plate, use water saturated n-butanol as the developing agent, develop, take out, dry, spray 1% vanillin sulfuric acid solution, and inspect immediately. 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) Take 1g of this product powder, add 30ml of petroleum ether (60 ～ 90 ℃), heat and reflux for 2 hours, filter, discard the petroleum ether liquid, volatilize the drug residue, add 30ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate, add 2ml of methanol to dissolve the residue, and use it as the test solution. Another 1g of semen Ziziphi Spinosae reference medicinal material was prepared into the reference medicinal material solution by the same method. . According to the test of thin layer chromatography (general rule 0502), absorb 2 μ l of each of the above three solutions, respectively dot on the same silica gel G thin layer plate, use water saturated n-butanol as the developing agent, develop, take out, dry, spray 1% vanillin sulfuric acid solution, and inspect under the ultraviolet light (365nm). In the chromatogram of the test sample, the same blue fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the control sample.
      [inspection] impurities (core and shell, etc.) shall not exceed 5% (general rule 2301).
      The moisture content shall not exceed 9.0% (the second method of general rule 0832).
      The total ash content shall not exceed 7.0% (general rule 2302).
      Aflatoxin was determined according to the aflatoxin assay (general rule 2351).
      Take about 5g of this product powder (passing through No. 2 screen), accurately weigh it, add 3G of sodium chloride, measure and calculate according to the preparation method of the test article under the aflatoxin determination method.
      The content of aflatoxin B1 should not exceed 5 μ g per 1000g, and the total content of aflatoxin G2, aflatoxin G1, aflatoxin B2 and aflatoxin B1 should not exceed 10 μ G.
      [content determination] Jujuboside A was determined by high performance liquid chromatography (general rule 0512).
      Chromatographic conditions and system suitability test octadecylsilane bonded silica gel was used as filler; Acetonitrile was used as mobile phase A and water as mobile phase B; gradient elution was performed according to the following table; Evaporative light scattering detector detection. The number of theoretical plates shall not be less than 2000 according to the peak of Jujuboside a.
      Time (min) mobile phase a (%)         Mobile phase B (%)
    0～15                 20→40           80→60
    15～28                40               60
    28～30                40→70           60→30
    30～32                70→100          30→0
      Preparation of reference solution take an appropriate amount of Jujuboside A reference substance, accurately weigh it, add methanol to make a solution containing 0.1mg per 1ml.
      Preparation of test solution take about 1g of powder (passing through No. 4 screen), weigh accurately, place in Soxhlet extractor, add an appropriate amount of petroleum ether (60 ～ 90 ℃), heat and reflux for 4 hours, discard the petroleum ether liquid, volatilize the solvent from the drug residue, transfer to a conical flask, add 20ml of 70% ethanol, heat and reflux for 2 hours, filter, wash the filter residue with 5ml of 70% ethanol, combine the wash solution and filtrate, recover the solvent to dryness, dissolve the residue with methanol, transfer to a 5ml volumetric flask, add methanol to the scale, shake well, filter, and take the continued filtrate.
      The determination method is to precisely suck 5 μ L and 20 μ l of the reference solution and 10 μ l of the test solution, inject them into the liquid chromatograph, determine, and calculate with the external standard two-point method logarithmic equation.
      The content of Jujuboside A (c58h94o26) in this product shall not be less than 0.030% according to the dry product.
      Spinozin was determined by HPLC (general rule 0512).
      Chromatographic conditions and system suitability test octadecylsilane bonded silica gel was used as filler; Use acetonitrile as mobile phase A and water as mobile phase B to perform gradient elution according to the following table; The detection wavelength is 335nm. The number of theoretical plates should not be less than 2000 according to the peak of spinozin.
      Time (min) mobile phase a (%)         Mobile phase B (%)
    0～10                 12→19           88→81
    10～16                19→20           81→80
    16～22                20→100          80→0
    22～30                100              0
      Preparation of reference solution take appropriate amount of spironolactone reference, weigh accurately, add methanol to make a solution containing 0.2mg per 1ml.
      Preparation of the test solution take the continued filtrate under Jujuboside A [content determination] as the test solution.
      The determination method is to precisely suck 10 μ l of the reference solution and 10 μ l of the test solution, inject them into the liquid chromatograph, and determine.
      .
      Decoction pieces
      [processing] the residual core and shell of semen ziphi Spinosae were removed. Mash when needed.
      [character], [identification], [inspection] (moisture and total ash) [content determination] is the same as that of medicinal materials.
      Stir fry the sour jujube kernels. Take the pure sour jujube kernels and stir fry them according to the clear frying method (general rule 0213) until they bulge and become slightly darker. Mash when needed.
      . The surface is slightly bulging, with slight focal spots. Slightly burnt aroma, light taste.
      [inspection] the moisture content is the same as that of medicinal materials, and shall not exceed 7.0%.
      The total ash content is the same as that of medicinal materials, and shall not exceed 4.0%.
      [identification] [content determination] is the same as that of medicinal materials.
      [nature, taste and meridian tropism] sweet, sour and flat. Return to liver, gallbladder and heart meridian.
      [functions and indications] it can nourish the heart and liver, calm the heart and mind, arrest sweat, and generate fluid. It is used for restlessness, palpitations and dreams, body weakness and sweating, body injury and thirst.
      [usage and dosage] 10 ～ 15g.
      [storage] store in a cool and dry place to prevent moths.


2、 Chemical constituents of semen ziphi Spinosae
Semen ziphi Spinosae contains alkaloids: sanjoinine a, B, D, e, F, G1, G2, IA, IB, K, of which base a is frangu foline, and base e isNuciferine（nuciferine）， Base IA is nornu ciferine, base IB is norisocorydine, and base K is dextralHengzhou AconitineCoclaurine, n-methylamilo-bine, zizyphusine, caaver ine, 5-hydroxy-6-methoxynoraporphine, amphibine D. It also contains sanjoinenine. It also contains triterpenoids:Betulinic acid（betulinic acid），Betulin（betulin），Theanoic acid（ceanothic acid），Maltic acid（alphitolic acid），Jujuboside A（jujuboside）、Zizyphus jujuba saponinB, andCarotene（daucosterol）。 It also contains flavonoids:Spinosol（spinosin）， Namely, 2-o- β - d-glucopyranosyl-sylswertisin, zivulgarin, 6 '- sinapoylspinosin, 6' - feruloylspinosin, 6 '- p-coumaroylspinosin,Angelica flavin（swertisin）， Vicenin Ⅱ, apigenin-6-c - [(6-o-p-hydroxybenzoyl) - β - D-glucopyranosyl (1 → 2)] - β - d-glucopyranoside, etc. Also containsL-threonine（L-threonine），L-valine （L-valine），L-Methionine （L-methionine），l-leucine （L-leucine），L-isoleucine（L-isoleucine），L-lysine（L-lysine），L-phenylalanine. Also containsFerulic acid（ferulic acid）， Vitamin C, phytosterols, cyclic adenosine 3 ', 5' -monophosphate, etc.

3、 Pharmacological effects of semen ziphi Spinosae
1. sedative and hypnotic effects: the water-soluble extract of semen ziphi Spinosae, equivalent to 7.5G crude drug per ml, dilute the original solution to 1:1, l：0.5， 1: 0.25 3 concentrations, pH＝9， .
1.1. effect on general activities of mice: 1.1.1. direct observation test: take mice weighing 18.0 ± 1.0g, half each, observe their normal activities before administration, and then give different concentrations of water-soluble extract of Zizyphus jujuba seed 1.0ml/20g Ig.
1.1.2. optoelectronic sedation test: use two light tubes of cross beam in series to form a optoelectronic sedator, and use an integrator to record the number of spontaneous activities of mice within 10 minutes. The activity of mice in the drug concentration of 1:1 and 1:0.7 groups was significantly reduced.
1.1.3. displacement method test: refer to komlos' cage shaking drainage device and use the displacement in unit time to express the activity of mice. The mice were divided into a high-dose group (1:0.5), a low-dose group (1:0.25) and a control group. After one Ig administration, the water displacement in 15 and 30 minutes was observed after 30 minutes. The water displacement in the 1:0.5 drug group was significantly lower than that in the control group (p< 0.01).
1.1.4. cage shaking test: mice weighing 18 ± 1.0g were randomly divided into two groups, with 10 mice in each group. The drug group was treated with 1:1 Suanzaoren aqueous solution Ig 1.0ml/20g body weight, and then put into the cage to observe the activity within 10 minutes after 30 minutes. Results the spontaneous activity of the drug group was significantly reduced. The petroleum ether extract, n-butanol extract, ethyl acetate extract of semen ziphi Spinosae (each 1ml of the above extract is equivalent to 10g of the original crude drug) water decoction (the concentration is 1g of the original crude drug for each 1ml), pinosin is prepared into 1mg of the original crude drug for each 1ml. The shaking cage method was used for self-control test. The results are shown in Table 4. Except that the petroleum ether extract was ineffective, it showed a significant reduction in spontaneous activity.
. Forty mice were randomly divided into four groups according to body weight and sex, and given different doses of semen ziphi Spinosae water extract (each 1ml is equivalent to 7.5G crude drug, and diluted to 1:1, 1:0.5, and 1:0.7) and normal saline as control. The experiment was carried out with cage rolling method. The cage rolling speed was 6.5wk/ min, and 10 mice were put into the cage each time. After ig Administration for 30 minutes, the mice were put into the cage, and the number of animals was observed for 60 minutes. The results are shown in Table 5. The three concentrations fell by more than 60%, which was significantly different from the saline group control.
1.3. effect on animal sleep: 1.3.1. effect of water-soluble extract of semen ziphi Spinosae and sodium pentobarbital: 20 mice of 18.0 ± 1.0g were randomly divided into two groups according to weight and sex. ; The other group was given equal volume normal saline as control. 30 minutes after ig administration, each mouse was IP 0.2% sodium pentobarbital 0.1ml/10g, and the righting reflex test was conducted every 5 minutes until the righting reflex disappeared. The number of disappeared within 30 minutes was recorded. The water-soluble extract of semen ziphi Spinosae can strengthen the hypnotic effect of sodium pentobarbital. The water-soluble extract of semen ziphi Spinosae could also significantly prolong the sleep time of pentobarbital sodium on mice (p< 0.05), but it was less than that of chlorpromazine (p< 0.01).
1.3.2. effect of fried jujube kernel decoction on sleep in rats: Eighteen Wistar rats weighing 160-240g were randomly divided into experimental group and control group. They were placed in plexiglass cylinder and raised alone, with unrestricted activities, free feeding and drinking, maintaining natural light, and cortical electrodes were installed under sodium pentobarbital anesthesia for EEG recording. Silver Wire electrodes were inserted into the bilateral temporal muscles and neck muscles to record the EMG for 6 hours. Each rat was recorded for 3 days before Ig and 3 days after ig. Each 1ml of fried jujube seed decoction is equivalent to 0.5g of the original drug. The experimental group was treated with Ig of fried jujube seed decoction, 1ml each time, twice a day, starting from the night of recording sleep, for a total of 3 days. The control group was treated with the same amount of normal saline. The sleep data were analyzed by visual inspection, and every 30 seconds was a segmented time. The sleep wake cycle is divided into: 1) the wake period is characterized by low amplitude fast wave EEG and obvious myoelectric activity guided by frontal parietal lobe; 2) Slow wave sleep (SWS) is characterized by fusiform waves (10-15hz) and high amplitude slow waves (Q waves). 0.5-5hz), and the EMG activity was significantly reduced in this period. ; The second stage of slow wave sleep (SWS2) occurs when the Q wave exceeds 50%, which is equivalent to the deep sleep stage; 3) Paradoxical sleep (PS) is characterized by the EEG of low amplitude fast waves (6-9h2), with no significant muscle activity except occasional muscle jerks; 4) The total sleep phase (TS) included SWS1, SWS2 and PS. all experimental data were expressed as mean ± standard error (x ± SD) and statistically processed by t-test. Results: the duration of TS and SWS2 in the experimental group increased significantly after ig. Compared with that before Ig, the duration of TS increased by 51.0 minutes (26.0%) and the duration of SWS2 increased by 41.4 minutes (116.3%) within 6 hours, with significant differences (p< 0.001). There was no significant change in SWS1 and PS duration. There was no significant change in the control group before and after ig normal saline. The attack frequency and duration of TS and SWS2 in the experimental group changed significantly after ig. Compared with pre Ig, the frequency of TS episodes decreased by 22.7 times (-36.3%) on average within 6 h, and the duration of each episode increased by 3.5 minutes (+ 95.6%); ; 001), while the duration of each episode of SWS2 was prolonged by 0.3 hours (+ 31.6%), which was statistically significant (p< 0.05). In the control group, there was no significant change in seizure frequency and duration before and after ig. In the experiment, it was also found that the EEG wave patterns of most rats after ig had obvious changes, mainly in the slow wave sleep stage, the high amplitude slow waves increased, the amplitude increased, and the frequency slowed down, especially in the rats with less sleep before Ig. There was no significant change in the control group before and after ig.
1.3.3. effect of the effective components of Zizyphus jujuba seed on polysomnography in rats: the Zizyphus jujuba seed was degreased with petroleum ether and extracted with water, then ammonium sulfate was added to a certain saturation, precipitated, dissolved with water, and the insoluble matter was filtered out. After adjusting the pH of the clear solution to 3.0, it was chromatographed with cation exchange column, washed with water to neutral, then eluted with NH4OH and collected in parts to obtain two parts, 1.90g (SZ1) and 1.86g (SZ2), respectively. SZ1 and SZ2 were mixed with normal saline (NS) to form 4g/l. The rats were randomly divided into three groups, with 11 rats in each group, IP SZ1, SZ2 and NS respectively. Polysomnography was performed at the same time every day (2:00-6:00 p.m.) to avoid the influence of circadian rhythm on the experiment. Each rat was continuously recorded for 4H before and on the day of administration, and the sleep of rats was according to the usdin method. The results were expressed as mean ± standard deviation (x ± SD) and statistically processed by t-test. Results: the total sleep time (TST) and slow wave sleep (SWS) were significantly increased in SZ1 and SZ2 groups after administration, while the arousal (W) was significantly reduced. Compared with 1H before administration, TST increased by 21.7 ± 19.4 minutes (± 17.2%, P< ; SWS increased by 20.0 ± 18.6 minutes (+ 17.0%, p< 0.01) and 28.2 ± 19.0 minutes (+ 21.9%, p< 0.001), respectively, and SWS2 increased by 8.4 ± 12.1 minutes (+ 48.8%, p< 0.001) respectively; 005) and 25.3 ± 19.4 min (+ 138.2%, p< 0.01); W decreased by 21.7 ± 19.4 minutes (-19.0%, p< 0.01) and 27.7 ± 18.5 minutes (-27.3%, p< 0.001), respectively, while there was no significant change in slow wave sleep light sleep (SWS1) and fast wave sleep (PS), and there was no significant change in each phase of NS group before and after administration. From the perspective of time effect relationship of active ingredients, the time effect relationship of SZ1 and SZ2 was analyzed by comparing the hourly TST and SWS2 at the same time on the day of administration and the previous LD. According to the peak time of TST and SWS2 effect after administration, at h4-5, the TST and SWS of SZ1 increased by 45.3% (p< 0.01) and 186.7% (p< At 3-4 hours, TST and SWS2 increased by 23.6% (p< 0.05) and 191.4% (p< 0.01), respectively, when the effect of SZ2 reached its peak.
1.4. action against central stimulants: 1.4.1. action against pentylenetetrazol: mice weighing 18 ± 1.0g were randomly divided into three groups. . Normal saline was used as control. The results showed that the water-soluble extract of semen ziphi Spinosae had an antagonistic effect on the convulsion induced by pentylenetetrazol.
1.4.2. anti caffeine effect: 20 mice weighing 18.0 ± 1.0g, half each, were randomly divided into two groups according to body weight and sex. One group was given 1.0m1/20g of 1:1 water-soluble extract of semen ziphi Spinosae (each 1ml is equivalent to 7.5G of crude drug, diluted to 1:1), which was given twice, with an interval of 30 minutes. After the last dose of 30 minutes, 50mg/kg of sodium benzoate caffeine was given. The control group was treated with normal saline, and sodium benzoate caffeine 50mg/kg was given 30 minutes after the last administration. The activity curves of the two groups of mice within 30 minutes were recorded by shaking cage tracing method. Results the water-soluble extract of semen ziphi Spinosae could reduce the tracing curve.
1.4.3. the anti convulsion effect of strychnine in mice showed that 0.3ml/10g of Zizyphus jujuba seed in mice had no obvious antagonism.
2. effect on cardiovascular system: 2.1. effect of semen ziphi Spinosae on experimental arrhythmia: 2.1.1. effect on arrhythmia induced by barium chloride in rats: 39 rats, weighing 110-180g, of either sex, were divided into two groups, 20 in the semen ziphi Spinosae group and 19 in the control group. After IP 3% sodium pentobarbital 1ml/kg anesthesia, IP 10% chloral hydrate 2ml/kg for 5 minutes, the ECG of leads I, II and III were observed and recorded with rm-46 physiological recorder. Then inject 1ml/kg of sublingual iv0.4% barium chloride solution within 5 seconds; After the occurrence of arrhythmia, the administration group received the above-mentioned aqueous solution of Zizyphus spinosa 2ml/kg sublingually, and the control group was injected with the same amount of normal saline, and the changes of ECG were observed for 10 minutes. After barium chloride injection, 39 white rats immediately appeared arrhythmia waveform dominated by biphasic ventricular tachycardia. In the semen ziphi Spinosae group, the sinus rhythm turned to normal within 20 seconds on average (within 10 seconds in 14 rats), and no arrhythmia waveform was observed in 10 minutes. In the control group, there was no obvious change in ECG after the administration of normal saline, and the duration of arrhythmia was 1-10 minutes.
2.1.2. effect on aconitine induced arrhythmia in rats: 27 rats, weighing 110-220g, 14 in the semen ziphi Spinosae group and 3 in the control group. . The changes of electrocardiogram were observed and recorded with rm-46 physiological recorder. With 0.002% aconitine 1ml/kg sublingual IV, when arrhythmia occurred, the administration group IV Suanzaoren solution 2ml/kg, the control group IV the same amount of normal saline, observed and compared the changes of ECG in 15 minutes between the two groups. After aconitine injection, arrhythmic waveforms such as ventricular premature dyads and ventricular tachycardia appeared within seconds to 4 minutes. Eight of the 14 white rats in the semen ziphi Spinosae group had improved ECG waveforms (i.e., they could recover sinus rhythm for several minutes, then they developed arrhythmias, and recovered in a short time, and repeated for several times); However, in the normal saline group, all 13 rats sustained arrhythmia waveform within 15 minutes. The results of the two groups were calculated by x2 test and p< was measured; 0.01, the difference is very significant.
2.2. effect of semen Ziziphi Spinosae on myocardial ischemia induced by Pituitrin in rats: 2.2.1.ip administration group: 18 rats weighing 160-220g were selected and divided into two groups with 9 rats in each group. After anesthesia, one group was pre IP with 4ml/kg of semen ziphi Spinosae solution (1ml is equivalent to 1g of crude drug), and the other group was treated with normal saline. After 10 minutes, 0.5u/kg of hypoglossal IV pituitrin injection was used to record the changes of lead II of ECG at 1, 2, 15, 30 seconds and 1, 2, 5, 10 minutes after injection with xdh-3 electrocardiograph.
2.2.2.iv administration group: take 10 rats, weighing 160-290g, with 5 rats in each group. After anesthesia, one group received 1.5ml/kg of semen ziphi Spinosae solution under the tongue in advance, and the other group received normal saline. . Myocardial ischemia is defined as the occurrence of one ECG change in the following first and second phases. Indications for myocardial ischemia: the first phase T wave is more than 50% higher than the original level, or the S-T segment moves down significantly or there is pre phase contraction. In the second phase, the S-T segment was significantly downward or the T wave was inverted and flat, and the heart rate was significantly slowed down. The difference of myocardial ischemia between IP and IV Zizyphus jujuba seed group and normal saline group was measured by X-test direct calculation probability method, and the results were very significant (p< 0.01).
2.3. effects on the heart: semen ziphi Spinosae can slow down the heart rate of isolated and in vivo frog hearts, strengthen the cardiac contractility, and have a cardiac strengthening effect.
3. effect on microcirculation: semen ziphi Spinosae can significantly expand the diameter of microvessels.
4. effects on the anti hypoxia ability of animals: 120 adult mice were randomly divided into hypoxia control group and semen ziphi Spinosae group according to body weight, with 60 mice in each group. One day before decompression and hypoxia, each mouse was Ig with 0.4ml of Suanzaoren Decoction (containing 0.1g of crude drug) and 0.4ml of Ig distilled water in the hypoxia control group. The Ig was repeated once 2 hours before decompression and hypoxia, and the dose was the same as above. After the animal enters the decompression device, it is first rapidly (1000m/ min) decompressed to a simulated height of 3000m, and then slowly (200m/ min) to 10000m. After stopping for 1 hour, it returns to the plain height, and the survival rate of the animal is calculated. Results 27 rats survived in the control group and 48 rats survived in the medication group, P< 0.01. , P< 0.05 and 0.01, respectively. The oxygen consumption of brain tissue was significantly reduced after treatment.
. The semen ziphi Spinosae and polysaccharide were orally administered 0.1g/kg per day for 16 days, which could enhance the humoral and cellular immune functions of mice, and had a certain protective effect on radiation-induced injury mice.