WO2019114676A1 - New medical use of persimmon leaf extract and of preparation of persimmon leaf extract - Google Patents

New medical use of persimmon leaf extract and of preparation of persimmon leaf extract Download PDF

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WO2019114676A1
WO2019114676A1 PCT/CN2018/120186 CN2018120186W WO2019114676A1 WO 2019114676 A1 WO2019114676 A1 WO 2019114676A1 CN 2018120186 W CN2018120186 W CN 2018120186W WO 2019114676 A1 WO2019114676 A1 WO 2019114676A1
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persimmon leaf
leaf extract
supernatant
ethyl acetate
depression
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PCT/CN2018/120186
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French (fr)
Chinese (zh)
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郭海彪
李淑如
李楚源
王德勤
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广州白云山和记黄埔中药有限公司
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Priority to JP2020552084A priority Critical patent/JP7090731B2/en
Priority to AU2018386145A priority patent/AU2018386145B2/en
Publication of WO2019114676A1 publication Critical patent/WO2019114676A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/44—Ebenaceae (Ebony family), e.g. persimmon

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a new medical use of a persimmon leaf extract and a preparation thereof.
  • Depression also known as depressive disorder, is a chronic, recurrent episode of emotional and mental illness characterized by significant and persistent mood or depression, often accompanied by anxiety, slow thinking, delusions or hallucinations. , attention and memory loss and sleep disorders. So far, the cause of depression is still unclear. It is generally believed that many factors in the biological, psychological and social environment are involved in the pathogenesis of depression.
  • the World Health Organization predicts that depression will become the second largest clinical chronic disease after hypertension in 2020 and is considered a "cold" in psychiatry. According to the survey, only 5% of people with depression in China have been treated. Most patients are not diagnosed due to lack of awareness of depression, which leads to a worsening of the condition. As the pace of life accelerates and the pressure of work in life increases, depression has become one of the most common diseases that seriously endanger human health.
  • DSM-5 The fifth edition of the American Diagnostic and Statistical Manual of Mental Disorders (DSM-5) classifies depression including destructive mood disorders, typical depressive disorders (including single and recurrent episodes), persistent depressive disorder (bad mood), and premenstrual mood.
  • Typical depressive disorder is a classic subtype of depressive disorder, a depression commonly recognized in modern medicine, characterized by episodes that last for at least 2 weeks, including significant changes in mood, cognition, and autonomic nervous system, but not by other physical problems (such as Caused by malignant tumors, cerebrovascular diseases, etc.).
  • vascular depression proposed in 1977 comes from the study of senile depression, an age-related depression syndrome closely related to cerebrovascular disease or vascular risk factors. Patients with vascular depression have less typical loss, self-sin, and milder depressive symptoms, but more likely to be accompanied by attention disorder, apathy, exercise retardation, and cognitive impairment based on executive impairment. Therefore, vascular depression is different from the above-mentioned typical depressive disorder. As the vascular injury is reduced, the depressive symptoms of vascular depression will also be relieved.
  • Drugs are currently the most important treatment for depression.
  • Currently commonly used antidepressants include selective serotonin (5-HT) reuptake inhibitors (SSRIs), 5-HT/norepinephrine (NE) double reuptake inhibitors (SNRIs), and tricyclics.
  • Chemicals such as (TCAs) and monoamine oxidase inhibitors (MAOIs). Because the cause of depression has not been studied clearly, the efficacy of these chemicals varies greatly for different types of depression, and there are delays in onset (3 to 8 weeks), more adverse reactions, and lower efficiency (50%). -70%), high recurrence rate (35% to 60%), low dependence and high price, etc.
  • the main side effects of chemical drugs include gastrointestinal symptoms (nausea, diarrhea, stomach bleeding, indigestion), liver toxicity, weight gain and metabolic abnormalities, cardiovascular problems (heart rate, QT interval prolongation, hypertension, orthostatic hypotension) ), urinary system symptoms (urinary retention, urinary incontinence), sexual dysfunction, hyponatremia, osteoporosis and fracture risk, bleeding, central nervous system disorders (lower epileptic threshold, extrapyramidal side effects, cognitive impairment), Sweat, sleep disorders, emotional symptoms (emotional dullness, mood shift, abnormal reaction), ocular symptoms (glaucoma, cataract), hyperprolactinemia. Therefore, in search of safer, more effective, less toxic, and economical antidepressants, more and more researchers are turning their attention to resource-rich natural plants.
  • Persimmon leaf is a fresh or dried leaf of Diospyros kaki Thunb. It was first used in the Ming Dynasty, "Yinnan Materia Medica”, “Frosted Leaf Applying Acne”, and “Materia Medica Renewed” It is recorded for "cough, vomiting blood, and quenching thirst.” In-depth study on the chemical constituents of persimmon leaves found that persimmon leaves contain various active ingredients and nutrients such as flavonoids, organic acids, coumarins, triterpenoids, and have a good preventive effect on cardiovascular and cerebrovascular diseases.
  • the persimmon leaf extract contained in the Chinese Pharmacopoeia is extracted from the leaves of persimmon, extracted with water, alcohol, and ethyl acetate.
  • the function is mainly to promote blood circulation and remove blood stasis. Illness.
  • Naoxinqing Tablet is an oral tablet made from persimmon leaf extract and used for cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral atherosclerosis. Previous studies have shown that flavonoids may be the material basis for the efficacy of persimmon leaf extracts.
  • persimmon leaf extract may have some degree of auxiliary improvement on vascular depression or senile depression (in fact, there is no clear evidence). However, it has not been found so far that persimmon leaf extract can be used to treat typical depressive disorders unrelated to other physical problems.
  • persimmon leaf extract in the preparation of a medicament for preventing and/or treating depression.
  • the depression refers to a typical depressive disorder, especially a depression caused by mental and affective disorders.
  • the persimmon leaf extract is the sole active ingredient of the drug for preventing and/or treating depression.
  • the medicament for preventing and/or treating depression includes or does not include a pharmaceutically acceptable excipient.
  • the drug for preventing and/or treating depression is an oral preparation or a non-oral preparation.
  • the oral preparation is one or more selected from the group consisting of a powder, a general oral tablet, a dispersible tablet, a capsule, a soft capsule, a pill, a pill, a pellet, a granule, an orally disintegrating tablet, and an oral fast dissolving film. .
  • the non-oral preparation is selected from one or more of an injection, a lyophilized powder, and a bulk infusion.
  • the drug for preventing and/or treating depression is an oral preparation.
  • the drug for preventing and/or treating depression is Naoxinqing Tablet or Naoxinqing Capsule.
  • the above-mentioned persimmon leaf extract can be prepared by the method conventional in the art, with or without the addition of a pharmaceutically acceptable excipient, for the prevention and/or treatment of depression.
  • the above persimmon leaf extract is prepared by the following method:
  • the above persimmon leaf extract is prepared by the following method:
  • the pharmaceutical administration object for preventing and/or treating depression according to the present invention is a mammal, preferably a human.
  • the persimmon leaf extract is administered in an amount of 2 to 10 mg/kg of body weight per day.
  • the present invention also provides a method of preventing and/or treating depression, the method comprising the step of administering a persimmon leaf extract to a patient in need thereof;
  • the persimmon leaf extract is prepared by the following method:
  • the persimmon leaf extract is prepared by the following method:
  • the depression refers to a typical depressive disorder, especially a depression caused by mental and affective disorders.
  • the patient in need is a mammal in need thereof, preferably a human in need thereof.
  • the method for preventing and/or treating depression includes the step of administering a persimmon leaf extract of 2 to 10 mg/kg body weight/day to a person in need thereof.
  • the above pharmaceutically acceptable excipients include, but are not limited to:
  • Diluent for example selected from the group consisting of starch, dextrin, pregelatinized starch, lactose, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, magnesium oxide, magnesium carbonate, aluminum hydroxide gel, ⁇ -cyclodextrin One or more of mannitol, sorbitol, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and sodium carboxymethylcellulose.
  • Binder For example, one or more selected from the group consisting of distilled water, ethanol, starch syrup, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and ethyl cellulose, hypromellose.
  • Lubricant for example, one or more selected from the group consisting of magnesium lauryl sulfate, polyethylene glycol, micronized silica gel, magnesium stearate, and talc.
  • Disintegrator for example selected from starch (corn, potato), microcrystalline cellulose, alginic acid, sodium alginate, ion exchange resin, effervescent-alkali system, hydroxypropyl starch, sodium carboxymethyl starch, cross-linking One or more of sodium carboxymethylcellulose, crospovidone, carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, partially alpha-starch, and microcrystalline cellulose .
  • Film forming materials one or more of gelatin, shellac, gum arabic, agar, starch, dextrin, PVA05-88, PVA17-88, and ethylene-vinyl acetate copolymer (EVA).
  • Flavoring agent for example, one or more selected from the group consisting of sodium saccharin, cyclamate, aspartame, stevioside, and flavors.
  • Solvent for injection for example, selected from water for injection, sesame oil, tea oil, peanut oil, corn oil, olive oil, cottonseed oil, soybean oil, castor oil and peach oil, ethyl oleate, benzyl benzoate, propylene glycol, polyethylene glycol 400, one or more of dimethylacetamide (DMA), ethanol, and glycerin.
  • DMA dimethylacetamide
  • the present invention finds that the persimmon leaf extract has a good antidepressant effect by a cell in vitro model and an animal model study. Through mechanism research, it is considered that the persimmon leaf extract of the present invention plays a role in preventing and/or treating depression by the following aspects:
  • Anti-neuroinflammatory effect can inhibit the release of neuroinflammatory factors such as TNF- ⁇ , IL-1 ⁇ , IL-6 in the brain.
  • the persimmon leaf extract contains flavonoids such as quercetin, kaempferol and hyperoside, and these ingredients have been reported to have certain antidepressant effects, such as 1 Bayi Sword, Wang Deqin, et al.Determination of hyperoside in Naoxinqing Tablet Extract by HPLC[J].Journal of Guangxi Traditional Chinese Medical College,2009(02):62-63;2 Bei Weijian, Luo Jie, et al.
  • the present invention provides a new medical use for persimmon leaf extract. Based on the present invention, a new safe treatment option for clinical treatment of typical depressive disorder will be provided.
  • Figure 1 shows the cell viability measured by MTT assay after the incubation of different concentrations of persimmon leaf extract (3.125-400 ⁇ g/mL) in Example 1 with normal cultured PC12 cells for 24 hours.
  • Figure 2 is a graph showing the cell viability measured by MTT assay (Fig. 2A) and the LDH assay by LDH after incubation with different concentrations of corticosterone (Cort, 0-800 ⁇ M) in normalized cells for 24 h. Activity (Fig. 2B), in the figure, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. Cort (0 ⁇ M) group (normal control group).
  • Figure 3 shows the protective effect of different concentrations of persimmon leaf extract (6.25, 12.5, 25, 50, 100 ⁇ g/mL) on corticosterone (Cort, 200 ⁇ M) damage PC12 cells in Example 1, wherein Figure 3A shows Effect on cell viability, Figure 3B shows the effect on LDH activity; in the figure: * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. Cort group (corticosterone group), # # P ⁇ 0.01vs.Veh group (normal control group).
  • Figure 4 is a graph showing that different concentrations of persimmon leaf extract (6.25, 12.5, 25, 50, 100 ⁇ g/mL) in Example 1 secreted inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ ) in corticosterone (Cort, 200 ⁇ M)-injured PC12 cells. And IL-6); among them, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. Cort group (corticosterone group), ## P ⁇ 0.01 vs. Control group (normal control group) ).
  • Fig. 5 shows the results of the open box experiment in Example 2, in which Fig. 5A shows the level scores of the mice in each group, and Fig. 5B shows the vertical scores of the mice in each group.
  • Figure 6 shows the results of the tail suspension experiment in Example 2, in which * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. Veh (solvent control group).
  • Fig. 7 shows the results of a forced swimming test of mice in Example 2, in which * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. Veh (solvent control group).
  • Figure 8 is a graph showing the results of the effect of persimmon leaf extract of Example 2 on serum corticosterone levels in acutely stressed mice, in which * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 Vs. Veh2 (solvent control group); ## P ⁇ 0.01 vs. Veh1 (normal control group).
  • FIG. 9 is a graph showing the results of measuring the effect of persimmon leaf extract of Example 2 on brain monoamine neurotransmitter levels in acutely stressed mice.
  • FIG. 9A shows the measurement results of serotonin (5-HT)
  • FIG. 9B shows the measurement results of dopamine (DA)
  • FIG. 9C shows the measurement results of norepinephrine (NE).
  • Figure 10 shows the results of the sugar water preference experiment in Example 3.
  • 1 is the normal control group
  • 2 is the model group
  • 3 is the persimmon leaf extract
  • 4 is the persimmon leaf flavonoid composition
  • 5 is quercetin
  • 6 is kaempferol
  • 7 is hyperoside
  • 8 is Rutin; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. model control group; ### P ⁇ 0.01 vs. normal control group; $ P ⁇ 0.05, $$ P ⁇ 0.01 vs. Persimmon leaf extract.
  • Figure 11 shows the results of the open box experiment in Example 3; wherein Figure 11A shows the mouse horizontal motion score and Figure 11B shows the mouse vertical motion score.
  • 1 is the normal control group
  • 2 is the model group
  • 3 is the persimmon leaf extract
  • 4 is the persimmon leaf flavonoid composition
  • 5 is quercetin
  • 6 is kaempferol
  • 7 is hyperoside
  • 8 is Rutin; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. model control group; ### P ⁇ 0.01 vs. normal control group; $ P ⁇ 0.05, $$ P ⁇ 0.01 vs. Persimmon leaf extract.
  • Figure 12 shows the results of the tail suspension experiment in Example 3.
  • 1 is the normal control group
  • 2 is the model group
  • 3 is the persimmon leaf extract
  • 4 is the persimmon leaf flavonoid composition
  • 5 is quercetin
  • 6 is kaempferol
  • 7 is hyperoside
  • 8 is Rutin; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. model control group; ### P ⁇ 0.01 vs. normal control group; $ P ⁇ 0.05, $$ P ⁇ 0.01 vs. Persimmon leaf extract.
  • Figure 13 is a graph showing the results of a forced swimming test in a mouse in Example 3.
  • 1 is a normal control group
  • 2 is a model group
  • 3 is a persimmon leaf extract
  • 4 is a persimmon leaf flavonoid composition
  • 5 is a mink skin.
  • 6, 6 is kaempferol
  • 7 is hyperoside
  • 8 is rutin; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs. model control group; ### P ⁇ 0.01vs Normal control group; $ P ⁇ 0.05, $$ P ⁇ 0.01 vs. persimmon leaf extract.
  • Figure 14 is a graph showing the results of measurement of serum corticosterone and adrenocorticotropic hormone levels in CUMS mice in Example 3; wherein the results of serum corticosterone levels shown on the left side of the figure are shown on the right side of the figure. The results of the determination of adrenocortical hormone levels.
  • Persimmon Leaf Extract (NXQ): Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd., batch number H16P006, preparation method is:
  • DMEM high sugar medium Australian fetal bovine serum, horse serum: Gibco;
  • PC12 cells rat source, Chinese Academy of Sciences cell bank;
  • Microplate reader Bio-rad
  • the persimmon leaf extract was dissolved in 100% DMSO to prepare a 400 mg/mL stock solution, which was diluted to 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 ⁇ g/mL, and the concentration of DMSO was ensured. Less than 0.1% is suitable for cell experiments.
  • the cells were cultured to logarithmic growth phase, and the cells were resuspended in DMEM medium containing 5% fetal bovine serum and 10% horse serum (containing penicillin sodium 200 kU/L, streptomycin 100 mg/L, pH 7.4), and the cell density was adjusted. 1 ⁇ 10 5 /mL.
  • the cells were seeded in a 96-well plate, 100 ⁇ L of cell liquid was added to each well, and the cells were incubated at 37 ° C in a CO 2 incubator for 24 hours. The experiment was started after the cells were full. After deprivation of the cells for 1 h, different concentrations of corticosterone (0, 25, 50, 100, 200, 400 ⁇ M) were added. After 24 h, the lesion concentration of corticosterone was observed by MTT assay and LDH assay, and the optimal model concentration of corticosterone was selected.
  • the cells were pretreated with different concentrations of persimmon leaf extract (3.125-400 ⁇ g/mL) for 1 h. Except for the control wells, the other wells were incubated with 200 ⁇ M corticosterone for 24 h. The supernatant of the cells was collected for detecting the change of lactate dehydrogenase (LDH) activity, the cell viability of the 96-well plate was measured by MTT assay, and the absorbance of each well was detected by a microplate reader at 570 nm. The average absorbance of the control group was 100%, and the changes of viability and LDH were calculated by the ratio of the absorbance value of each treatment group to the control group.
  • LDH lactate dehydrogenase
  • PC12 cells were co-cultured with different concentrations of persimmon leaf extract and corticosterone for 4 h, ELISA The concentration of inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the supernatant was determined to observe the effect of persimmon leaf extract on the secretion of inflammatory factors.
  • mice C57 mice, female, 60, weighing 18-22 g; experimental animal license number: SCXK (Beijing) 2014-0004.
  • NXQ Persimmon leaf extract
  • FLX fluoxetine
  • sodium carboxymethyl cellulose open box
  • round swimming cylinder and suspension instrument.
  • mice were randomly divided into five groups: the solvent control group (Veh), the positive drug fluoxetine (FLX, 10 mg/kg), and the persimmon leaf extract.
  • NXQ low dose (20mg/kg) administration group persimmon leaf extract (NXQ) medium dose (40mg/kg) administration group, persimmon leaf extract (NXQ) high dose (80mg/kg) administration group
  • the drug was administered once a day for 7 days, and the solvent control group was simultaneously given the same volume of 0.5% aqueous sodium carboxymethylcellulose solution. Behavioral testing was started 1 h after the last dose.
  • Another 10 mice were used for biochemical indicators as a normal control group.
  • Drug preparation Persimmon leaf extract or fluoxetine was fully dissolved in 0.5% sodium carboxymethylcellulose, and was administered at the dose of 0.1 ml/10 g body weight.
  • the open-box experiment is mainly used to test the spontaneous activity of mice to rule out the effects of drug central excitatory interference.
  • the experiment was carried out in a black wooden box with a bottom of 25 cm x 25 cm and a height of 20 cm, and the bottom surface was 25 squares.
  • the mice were placed in a central square, adapted for 5 min, and taken out. Each mouse was washed with 75% alcohol to remove the residual odor of the previous mouse.
  • mice crossing the grid within 5 minutes of the imaging recording the four claws can be counted into the square to count as the horizontal movement score
  • the number of hind limbs upright the two front paws are vacated or climbing the wall
  • the tail of the mouse (1 cm from the tip of the tail) was adhered with a tape and fixed on the tail-suspended stent to make the mouse into an inverted suspension position, and the eyes of the mice were blocked by the partitions on both sides so as not to interfere with each other.
  • the camera recorded the cumulative immobility time of the mice within 5 min. The criterion for the "immobility" of mice is to stop struggling, and the body is vertically suspended and still.
  • mice On the second day of the tail suspension experiment, the mice were placed in a circular swimming cylinder of 20 cm in height, 12 cm in diameter and 10 cm in water depth for 6 min, and the cumulative immobility time of the mice 4 min after imaging recording.
  • the criterion for the "immobility" of mice is that they stop struggling in the water and are floating, with only small limb movements to keep the head floating on the water surface.
  • the eyeballs were removed and the blood was removed. After standing for 10 minutes in the anticoagulation tube, the cells were centrifuged at 8500 r/min for 15 min, and the serum was separated and stored in a refrigerator at -80 °C for use. Serum corticosterone levels were measured by enzyme-linked immunosorbent assay (ELISA). At the same time, another group of mice (10 rats) without any stress treatment was taken as the normal control group. According to the same method of the experimental group, the eyeballs were removed, blood was separated, and serum corticosterone levels were determined.
  • ELISA enzyme-linked immunosorbent assay
  • the frozen brain tissue was taken, weighed and placed in a glass homogenizer, and pre-cooled 0.1 mol/L perchloric acid solution containing 0.01% EDTA-2Na was added at a ratio of 10 mL/kg, and rapidly homogenized in an ice bath. 2min.
  • the ground homogenate was transferred to a brown centrifuge tube, centrifuged at 14,000 g for 20 min at 4 ° C, and the supernatant was taken.
  • the supernatant was filtered through a 0.22 ⁇ m filter and the monoamine neurotransmitter 5-HT, NE in the brain tissue was detected on the machine. , DA level.
  • Example 1 based on the cell level of Example 1, the whole animal experiment was further used to evaluate the antidepressant pharmacodynamics of the persimmon leaf extract.
  • the forced swimming test (FST) and the mouse tail suspension test (TST) are the most commonly used models of acute behavioral despair.
  • the immobile state of mice in FST and TST reflects a persistent defeat in indescribable behavioral despair, or a passive state of tolerance when dealing with stress stimuli. It is currently believed that this behavioral despair is similar to the component that constitutes clinical depression.
  • the shortening of the duration of the animal's immobility state under this stress state reflects the antidepressant properties of the drug.
  • Most antidepressants reduce their immobility time in mouse FST and TST, and their pharmacodynamics are shown to be associated with clinical efficacy.
  • Acute stress is a systemic non-specific adaptive response that occurs when various internal and external environmental factors and social and psychological factors are stimulated in the body. It can cause brain dysfunction and neuronal damage.
  • the body activates the hypothalamus.
  • the pituitary-adrenal axis (HPA axis) which promotes elevated concentrations of adrenocorticotropic hormone and corticosterone (CORT) in serum, is an important feedback and self-protection pathway against external emergencies.
  • HPA axis pituitary-adrenal axis
  • CORT corticosterone
  • persimmon leaf extract can significantly reduce the level of serum corticosterone in acutely stressed mice, which may play a role in protecting neurons.
  • brain tissue monoamine neurotransmitters 5-HT, DA and NE are closely related to the depression monoamine hypothesis.
  • HPLC HPLC that the persimmon leaf extract can significantly reverse the acute stress mouse brain in time. Tissues are down-regulated in three types of neurotransmitters. This is also one of the important mechanisms by which persimmon leaf extract exerts antidepressant effects.
  • the present invention has confirmed that the persimmon leaf extract has a significant effect on typical depressive disorders, but the persimmon leaf extract contains flavonoids, organic acids, triterpenoids and coumarins. And a variety of components, and the content of flavonoids accounted for 30% (mainly quercetin, kaempferol and its mono- and di-glycosides such as rutin, hyperoside, etc.), has also been reported Quercetin and hyperoside have certain antidepressant activity. However, the persimmon leaf extract of the present invention showed by HPLC that the contents of quercetin, hyperoside, kaempferol and rutin were not more than 1.5%, respectively.
  • the present embodiment further extracts the persimmon leaf extract with a single flavonoid compound (quercetin, kaempferol, rutin, hyperoside) and combinations thereof.
  • the antidepressant efficacy comparison was performed, and the selected typical depressive disorder model was an internationally recognized chronic unpredictable mild stress model.
  • mice Male, 80, weighing 18-22 g, experimental animal license number: SCXK (Yue) 2011-0015; mice were adapted to the feeding environment for one week before the experiment, and all mice were kept at the normal level of temperature 21 ⁇ 2 °C. Experiment with animal rooms and follow the circadian rhythm. The mice were given free access to drinking water.
  • Persimmon leaf extract quercetin, kaempferol, rutin, hyperoside, sodium carboxymethylcellulose, mouse open-box, electronic analytical balance (Sartrious, Germany), frozen desktop high-capacity high-speed centrifugation Machine 5810 (Germany EPPENDORF company), Victor3 multi-function microplate reader (PerkinElmer, USA), ultrasonic tissue disruptor (Sonic, USA), DYY-III electrophoresis (Beijing Liuyi Instrument Factory), vertical plate electrophoresis tank (USA) Bio-Rad), WD-9405 horizontal shaker (Beijing Liuyi Instrument Factory), fluorescent/chemiluminescence imaging system (Sonic, USA), Mill-Q ultrapure water system (Millipore, USA), suspension instrument, Rat tail clips, counters, thermometers, weight scales, etc.
  • ECl luminescent liquid Invitrogen, USA); skim milk powder (Guangzhou Sijia Biotechnology Co., Ltd.); PVDF film (Milipore, USA); X-ray photographic film (Kodak, Japan); corticosterone (CORT) enzyme-linked immunosorbent assay Analytical kit (Enzo, USA); ACTH enzyme-linked immunoassay kit (phoenix pharmaceuticals, USA).
  • mice Adapted to the environment for one week before the experiment, all mice were free to obtain food and water, and followed the circadian rhythm, kept in a constant temperature clean environment, and manually touched daily to adapt to manual operation. After one week of adaptation to the environment, the mice were divided into two groups according to the weight of the mice and the results of the sugar-sweet preference experiment: 10 in the normal control group and 70 in the chronically unpredictable stress (CUMS) group, and the stress was started after the grouping.
  • CUMS chronically unpredictable stress
  • Stress modeling methods include: 1 min tail, 24 h upside down, 24 h of squirrel cage, 5 min of ice swimming at 4 ° C, 1 h of restraint, 24 h of wet cage, 15 min of forced swimming, 24 h of drinking water deprivation, 24 h of food deprivation, electric shock, sole Streak stress, cage stress, cage stress, etc.
  • the syrup preference experiment was conducted at the beginning of the experiment and at the 3rd week after the stress to determine whether the modeling was successful. If the depression model was not successful, the time of CUMS stress would be postponed.
  • the model components were CUMS model group (solvent Veh+CUMS), persimmon leaf extract group (NXQ40mg/kg+CUMS), quercetin group (4.0mg/kg+CUMS), and hyperoside group.
  • CUMS model group solvent Veh+CUMS
  • persimmon leaf extract group NXQ40mg/kg+CUMS
  • quercetin group 4.0mg/kg+CUMS
  • hyperoside group 4.0mg/kg+CUMS
  • kaempferol group 3.0mg/kg+CUMS
  • rutin group 1.5 mg/kg+CUMS
  • persimmon leaf flavonoid composition group [(quercetin 4.0mg/kg+gold) Candidate 4.0 mg/kg + kaempferol 3.0 mg/kg + rutin 1.5 mg/kg) + CUMS]
  • Behavioral testing was performed 4 weeks after administration.
  • the dosages of quercetin, hyperoside, kaempferol and rutin
  • mice were trained to adapt to 1% (w/v) syrup: two bottles of 1% aqueous sugar solution were placed in each cage. After 24 hours, one of the bottles of 1% syrup was replaced with pure water and left for 24 hours. After adaptation, the mice were fasted for 24 h and subjected to a sugar-water preference experiment. In the experiment, each mouse was given two bottles of water freely, one bottle was 25 ml of 1% syrup, and the other bottle was 25 ml of pure water, and the weight of each bottle of water was weighed before the start of the experiment. After 2 h, the remaining weight was measured to obtain the consumption (g) of syrup and pure water. The ratio of the consumption of sugar water to the total liquid consumption (sugar consumption + pure water consumption) indicates the degree of sugar water preference, and the experiment runs through the entire process of the experiment.
  • the experimental method is the same as the "2.4.1 open box experiment" item of the second embodiment.
  • the experimental method is the same as the "2.4.2 hangover experiment" item of Example 2.
  • the experimental method is the same as the "2.4.3 forced swimming experiment" item of the second embodiment.
  • the experimental method was the same as the "2.4.4" item of Example 2, and the level of adrenocorticotropic hormone (ACTH) was measured while detecting corticosterone.
  • ACTH adrenocorticotropic hormone
  • each of the drug-administered groups significantly increased the number of horizontal activities in the mice.
  • the increase in the level of the persimmon leaf extract group was greater, and the difference was statistically significant (P ⁇ 0.05).
  • the order of strength of the group was: persimmon leaf extract group (P ⁇ 0.001)> persimmon leaf flavonoid composition group, quercetin group, hyperoside group and rutin group (P ⁇ 0.01)> kaempferol group (P ⁇ 0.05) (see Figure 11A).
  • persimmon leaf extract significantly (P ⁇ 0.001), persimmon leaf flavonoid composition and hyperoside significantly (P ⁇ 0.05) reduced mouse tail suspension time. There was no statistically significant difference between the other groups and the model group. Compared with the persimmon leaf flavonoid composition group and the hyperoside group, the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the difference was statistically significant (P ⁇ 0.05).
  • persimmon leaf extract significantly (P ⁇ 0.001), persimmon leaf flavonoid composition (P ⁇ 0.01) and hyperoside (P ⁇ 0.05) significantly shortened the immobility time of mice .
  • the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the difference was statistically significant (P ⁇ 0.01, P ⁇ 0.05).
  • persimmon leaf extract significantly reduced serum corticosterone levels (P ⁇ 0.001).
  • Persimmon leaf flavonoid composition and hyperoside can significantly reduce serum corticosterone levels (P ⁇ 0.05).
  • the persimmon leaf extract reduced the serum corticosterone level in CUMS mice more significantly, and the difference was statistically significant (see the left panel of Figure 14).
  • persimmon leaf extract can significantly reduce serum adrenocorticotropic hormone levels (P ⁇ 0.01).
  • the effect of lowering adrenocorticotropic hormone levels in the other groups was not statistically different from the model group. (See the right side of Figure 14).
  • a partial flavonoid composition which is naturally present in the persimmon leaf extract is designed, and the persimmon leaf extract and each flavonoid component monomer (the dosage of the flavonoid monomer used in this part of the study is Parallel drug effects were compared in comparison with the content of persimmon leaf extract in 10 times or more. Unexpectedly, the effect of persimmon leaf extract was significantly stronger in each experiment than the control flavonoid composition and individual monomeric flavonoids.
  • the present invention provides a novel medical use of persimmon leaf extract for the prevention and/or treatment of typical depressive disorders.
  • Animal experiments have demonstrated that persimmon leaf extract has an exact role in improving depressive symptoms and reversing related abnormal biochemical indicators in CUMS mice.
  • the mechanism of anti-depression effect of persimmon leaf extract is achieved by inhibiting nerve inflammation, regulating HPA axis function and monoamine neurotransmitters (5-HT, NE, DA), so it can prevent other non-physical problems (such as heart, Typical depressive disorder caused by cerebrovascular disease, etc.).

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Abstract

The present invention relates to the use of a persimmon leaf extract in preparation of a medicament for prevention and/or treatment of depression. The depression refers to a typical depressive disorder, especially depressive disorders caused by mental and affective disorders.

Description

一种柿叶提取物及其制剂的新医药用途New medical use of persimmon leaf extract and preparation thereof
相关申请的交叉引用Cross-reference to related applications
本申请要求2017年12月12日递交的中国专利申请号为201711218778.7，名称为“一种柿叶提取物及其制剂的新医药用途”的专利申请的权益，在此将其全部内容引入作为参考。The present application claims the benefit of the Japanese Patent Application No. JP-A No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. .
技术领域Technical field
本发明属于医药学领域，具体涉及一种柿叶提取物及其制剂的新医药用途。The invention belongs to the field of medicine, and particularly relates to a new medical use of a persimmon leaf extract and a preparation thereof.
背景技术Background technique
抑郁症(Depression),又称抑郁障碍，是一种慢性、反复性发作的情感精神性疾病，以显著而持久的心境或情绪低落为主要临床特征，常伴有焦虑、思维迟缓、妄想或幻觉、注意力和记忆力下降和睡眠障碍等症状。迄今，抑郁症的病因还不清楚，一般认为生物、心理与社会环境诸多方面因素参与了抑郁症的发病过程。Depression, also known as depressive disorder, is a chronic, recurrent episode of emotional and mental illness characterized by significant and persistent mood or depression, often accompanied by anxiety, slow thinking, delusions or hallucinations. , attention and memory loss and sleep disorders. So far, the cause of depression is still unclear. It is generally believed that many factors in the biological, psychological and social environment are involved in the pathogenesis of depression.
目前全球抑郁症患者发病率高达21％，约15％的患者伴有自杀倾向。世界卫生组织(WHO)预测抑郁症将会在2020年成为继高血压病之后第二大临床慢性疾病，被认为是精神病学中的“感冒”。据调查显示，在中国仅有5％的抑郁症患者接受过治疗，大部分患者由于缺乏对抑郁症的认知，未能及时诊治，导致病情恶化。随着生活节奏加快，生活的工作压力增大，抑郁症已成为严重危害人类健康的常见疾病之一。At present, the incidence of depression in the world is as high as 21%, and about 15% of patients have a suicidal tendency. The World Health Organization (WHO) predicts that depression will become the second largest clinical chronic disease after hypertension in 2020 and is considered a "cold" in psychiatry. According to the survey, only 5% of people with depression in China have been treated. Most patients are not diagnosed due to lack of awareness of depression, which leads to a worsening of the condition. As the pace of life accelerates and the pressure of work in life increases, depression has become one of the most common diseases that seriously endanger human health.
美国精神疾病诊断与统计手册第五版(DSM-5)对抑郁症的分类包括破坏性心境失调、典型抑郁障碍(包括单次、反复发作)、持续抑郁障碍(心境恶劣)、经前期心境恶劣障碍、物质和(或)药物导致的抑郁障碍、由其他躯体问题引起的抑郁障碍、其他特定的抑郁障碍和非特定的抑郁障碍等8种亚型。典型抑郁障碍是抑郁障碍的经典亚型，是现代医学普遍认为的抑郁症，特征为持续至少2周的发作，包括情绪、认知及植物神经系统显著的变化，但是不是由其他躯体问题(如恶性肿瘤、脑血管病等)引起的。The fifth edition of the American Diagnostic and Statistical Manual of Mental Disorders (DSM-5) classifies depression including destructive mood disorders, typical depressive disorders (including single and recurrent episodes), persistent depressive disorder (bad mood), and premenstrual mood. Eight subtypes of disorders, substance and/or drug-induced depressive disorders, depression caused by other physical problems, other specific depressive disorders, and non-specific depressive disorders. Typical depressive disorder is a classic subtype of depressive disorder, a depression commonly recognized in modern medicine, characterized by episodes that last for at least 2 weeks, including significant changes in mood, cognition, and autonomic nervous system, but not by other physical problems (such as Caused by malignant tumors, cerebrovascular diseases, etc.).
1977年提出的血管性抑郁的概念来自对老年期抑郁症的研究，是一种与脑 血管病或血管危险因素密切相关的老年期抑郁综合征。血管性抑郁患者较少有典型的失落感、自罪，抑郁症状较轻，但多易伴注意障碍、淡漠、运动迟滞和以执行功能损害为主的认知损害。因此，血管性抑郁有别于上述典型抑郁障碍，随着血管损伤的减轻，血管性抑郁的抑郁症状也将随之缓解。The concept of vascular depression proposed in 1977 comes from the study of senile depression, an age-related depression syndrome closely related to cerebrovascular disease or vascular risk factors. Patients with vascular depression have less typical loss, self-sin, and milder depressive symptoms, but more likely to be accompanied by attention disorder, apathy, exercise retardation, and cognitive impairment based on executive impairment. Therefore, vascular depression is different from the above-mentioned typical depressive disorder. As the vascular injury is reduced, the depressive symptoms of vascular depression will also be relieved.
药物是目前抑郁症最主要的治疗手段。目前临床上常用的抗抑郁药包括选择性5-羟色胺(5-HT)重摄取抑制剂(SSRIs)、5-HT/去甲肾上腺素(NE)双重重摄取抑制剂(SNRIs)、三环类(TCAs)和单胺氧化酶抑制剂(MAOIs)等化学类药物。由于抑郁症的病因尚未研究清楚，因此对不同类型的抑郁症，这些化学药物的疗效存在较大差异，而且还存在起效延迟(3～8周)、不良反应多、有效率低(50％-70％)、复发率高(35％～60％)、依赖性不强且价格昂贵等亟待解决的问题。化学药物主要存在的副作用包括胃肠道症状(恶心、腹泻、胃出血、消化不良)，肝毒性，体重增加及代谢异常，心血管问题(心率、QT间期延长、高血压、体位性低血压)，泌尿系统症状(尿潴留、尿失禁)，性功能障碍，低钠血症，骨质疏松及骨折风险，出血，中枢神经系统紊乱(癫痫阈降低、锥体外系副作用、认知障碍)，泌汗，睡眠障碍，情感症状(情感平淡、心境转相、反常反应)，眼部症状(青光眼、白内障)，高泌乳素血症。因此，为寻求更安全、有效、低毒、经济的抗抑郁药，越来越多的研究者将目光投向资源丰富的天然植物。Drugs are currently the most important treatment for depression. Currently commonly used antidepressants include selective serotonin (5-HT) reuptake inhibitors (SSRIs), 5-HT/norepinephrine (NE) double reuptake inhibitors (SNRIs), and tricyclics. Chemicals such as (TCAs) and monoamine oxidase inhibitors (MAOIs). Because the cause of depression has not been studied clearly, the efficacy of these chemicals varies greatly for different types of depression, and there are delays in onset (3 to 8 weeks), more adverse reactions, and lower efficiency (50%). -70%), high recurrence rate (35% to 60%), low dependence and high price, etc. The main side effects of chemical drugs include gastrointestinal symptoms (nausea, diarrhea, stomach bleeding, indigestion), liver toxicity, weight gain and metabolic abnormalities, cardiovascular problems (heart rate, QT interval prolongation, hypertension, orthostatic hypotension) ), urinary system symptoms (urinary retention, urinary incontinence), sexual dysfunction, hyponatremia, osteoporosis and fracture risk, bleeding, central nervous system disorders (lower epileptic threshold, extrapyramidal side effects, cognitive impairment), Sweat, sleep disorders, emotional symptoms (emotional dullness, mood shift, abnormal reaction), ocular symptoms (glaucoma, cataract), hyperprolactinemia. Therefore, in search of safer, more effective, less toxic, and economical antidepressants, more and more researchers are turning their attention to resource-rich natural plants.
柿叶(Persimmon leaf)为柿科柿属植物柿(Diospyros kaki Thunb)的新鲜或干燥叶，其入药始载于明代《滇南本草》“经霜叶敷臃疮”，而《本草再新》则记载用于“治咳嗽吐血，止渴生津”。对柿叶化学成分的深入研究发现，柿叶中含有黄酮、有机酸、香豆素、三萜类等多种活性成分和营养物质，对心脑血管疾病具有较好的防治作用。现载于《中国药典》的柿叶提取物，以柿的叶为原料，用水煮醇沉、乙酸乙酯萃取等方法提取精制而成，功能以活血化瘀为主，主治气滞血瘀引起的病症。脑心清片即是以柿叶提取物为原料制成的口服片剂，用于冠心病、脑动脉粥样硬化症等心脑血管疾病。前期研究表明，黄酮类成分可能是柿叶提取物发挥药效的物质基础。《中国药典》将槲皮素(Quercetin)和山柰酚(Kaemferol)作为柿叶提取物总黄酮的质控标准，规定总含量规定不得低于8.6％；但是，黄酮类成分在柿叶提取物中仅占很少的部分，显然该提取物的大部分化学成分仍是不清楚的。Persimmon leaf is a fresh or dried leaf of Diospyros kaki Thunb. It was first used in the Ming Dynasty, "Yinnan Materia Medica", "Frosted Leaf Applying Acne", and "Materia Medica Renewed" It is recorded for "cough, vomiting blood, and quenching thirst." In-depth study on the chemical constituents of persimmon leaves found that persimmon leaves contain various active ingredients and nutrients such as flavonoids, organic acids, coumarins, triterpenoids, and have a good preventive effect on cardiovascular and cerebrovascular diseases. The persimmon leaf extract contained in the Chinese Pharmacopoeia is extracted from the leaves of persimmon, extracted with water, alcohol, and ethyl acetate. The function is mainly to promote blood circulation and remove blood stasis. Illness. Naoxinqing Tablet is an oral tablet made from persimmon leaf extract and used for cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral atherosclerosis. Previous studies have shown that flavonoids may be the material basis for the efficacy of persimmon leaf extracts. "Chinese Pharmacopoeia" quercetin (Quercetin) and kaempferol (Kaemferol) as the quality control standard of total flavonoids in persimmon leaf extract, the total content of the regulation is not less than 8.6%; however, flavonoids in persimmon leaf extract Only a small part of it, it is clear that most of the chemical composition of the extract is still unclear.
严彩英等在《文拉法辛联合中药治疗老年抑郁症的临床研究》(严彩英，裴瑜，等；国际精神病学杂志，2016，43(5)：842-845)一文中报道了文拉 法辛和盐酸川芎嗪、银杏达莫、脑心清片等药物联合治疗老年抑郁症患者。经过连续8周的治疗，同时给予文拉法辛、盐酸川芎嗪(静脉滴注)、银杏达莫(静脉滴注)和脑心清片(口服)的观察组仅在治疗第1周末和第2周末的临床总有效率、HAMD24总评分和焦虑/躯体因子评分优于单纯服用文拉法辛的对照组；但是随着治疗时间的延长，观察组的治疗优势逐渐减弱，与对照组的差异没有统计学意义。该篇文章提示，盐酸川芎嗪、银杏达莫和脑心清片联合文拉法辛缓解老年抑郁症引起的焦虑、抑郁症状，仅起效速度优于单一应用文拉法辛治疗，但是长期并没有治疗优势。因此，基于该研究，可以推测，活血化瘀的盐酸川芎嗪、银杏达莫和脑心清片即使合用对老年抑郁没有显著的改善作用。Yan Caiying et al. reported on the clinical study of venlafaxine combined with traditional Chinese medicine in the treatment of senile depression (Yan Caiying, Yan Yu, et al; International Journal of Psychiatry, 2016, 43(5): 842-845). It is combined with ligustrazine hydrochloride, Yinxingdamo, Naoxinqing tablets and other drugs to treat elderly patients with depression. After 8 consecutive weeks of treatment, the observation group given venlafaxine, ligustrazine hydrochloride (intravenous drip), Yinxingdamo (intravenous drip) and Naoxinqing Tablet (oral) was only treated on the 1st weekend and the first The total clinical effective rate, HAMD24 total score and anxiety/body factor score of the weekend were better than those of the venlafaxine-only control group. However, with the prolongation of treatment time, the therapeutic advantage of the observation group gradually weakened, and the difference with the control group. Not statistically significant. This article suggests that ligustrazine hydrochloride, Yinxingdamo and Naoxinqing tablets combined with venlafaxine relieve anxiety and depressive symptoms caused by depression in the elderly. The onset rate is better than that of single-application venlafaxine treatment, but it has not been used for a long time. Therapeutic advantage. Therefore, based on this study, it can be speculated that the combination of Ligustrazine Hydrochloride, Yinxingdamo and Naoxinqing Tablets for promoting blood circulation and removing blood stasis has no significant improvement effect on geriatric depression.
汪瑾宇等也报道了脑心清片合并艾司西酞普兰治疗血管性抑郁的疗效(汪瑾宇，何影，等；脑心清片合并艾司西酞普兰治疗血管性抑郁临床研究[J]；新中医，2014，46(2)：47-49)。该临床研究纳入标准为：“具有脑血管病和血管危险因素的临床和(或)实验室证据，或者在脑血管事件发生后的6～12月内出现抑郁；Hamilton抑郁量表(HAMD，24项)评分≥20分。”显然，该研究的治疗对象是前述的“由其他躯体问题引起的抑郁障碍”，而不是典型的抑郁障碍。通过8周的观察治疗，该研究发现虽然脑心清片合并艾司西酞普兰组的HAMD评分下降更显著，但是与单用艾司西酞普兰组的有效率差异并不大(P＞0.05)。因此，艾司西酞普兰仍然是治疗抑郁的主要因素。脑心清片在其中的作用更直接的应该在于改善了脑血管病，从而有可能辅助艾司西酞普兰改善抑郁症状。Wang Yuyu et al also reported the efficacy of Naoxinqing tablets combined with escitalopram in the treatment of vascular depression (Wang Yuyu, He Ying, et al; Clinical study of Naoxinqing tablets combined with escitalopram in the treatment of vascular depression] [J]; Chinese Medicine, 2014, 46(2): 47-49). The clinical study included criteria for: "Clinical and/or laboratory evidence of cerebrovascular disease and vascular risk factors, or depression within 6 to 12 months after cerebrovascular events; Hamilton Depression Scale (HAMD, 24) Item) score ≥ 20 points. "Obviously, the subject of the study was the aforementioned "depressive disorder caused by other physical problems" rather than the typical depressive disorder. After 8 weeks of observation and treatment, the study found that although the HAMD score of Naoxinqing tablets combined with escitalopram group decreased more significantly, the difference in efficiency from the escitalopram group alone was not significant (P>0.05). ). Therefore, escitalopram is still a major factor in the treatment of depression. The role of Naoxinqing Tablet in its role should be more direct in improving cerebrovascular disease, which may help escitalopram to improve depressive symptoms.
综合现有技术的报道，柿叶提取物对血管性抑郁或老年性抑郁可能有某种程度的辅助改善作用(实际上并没有明确的证据证明)。但是，至今尚未有发现柿叶提取物可以用于治疗与其他躯体问题无关的典型抑郁障碍的报道。According to the prior art report, persimmon leaf extract may have some degree of auxiliary improvement on vascular depression or senile depression (in fact, there is no clear evidence). However, it has not been found so far that persimmon leaf extract can be used to treat typical depressive disorders unrelated to other physical problems.
发明内容Summary of the invention
针对现有技术的不足，本发明的目的在于提供柿叶提取物在预防和/或治疗抑郁症，尤其是典型抑郁障碍中的新的医药用途。In view of the deficiencies of the prior art, it is an object of the present invention to provide new medical uses of persimmon leaf extracts in the prevention and/or treatment of depression, especially typical depressive disorders.
为了实现上述发明目的，本发明采用了如下的技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
柿叶提取物在制备用于预防和/或治疗抑郁症的药物中的应用。Application of persimmon leaf extract in the preparation of a medicament for preventing and/or treating depression.
优选地，所述抑郁症是指典型抑郁障碍，尤其是精神性、情感障碍所致的抑郁障碍。Preferably, the depression refers to a typical depressive disorder, especially a depression caused by mental and affective disorders.
优选地，所述柿叶提取物作为所述用于预防和/或治疗抑郁症的药物的唯一活性成分。Preferably, the persimmon leaf extract is the sole active ingredient of the drug for preventing and/or treating depression.
优选地，所述用于预防和/或治疗抑郁症的药物包括或不包括药学上可以接受的辅料。Preferably, the medicament for preventing and/or treating depression includes or does not include a pharmaceutically acceptable excipient.
还优选地，所述用于预防和/或治疗抑郁症的药物为口服制剂或非口服制剂。Still preferably, the drug for preventing and/or treating depression is an oral preparation or a non-oral preparation.
所述口服制剂选自散剂、普通口服片剂、分散片、胶囊剂、软胶囊剂、丸剂、滴丸剂、微丸剂、颗粒剂、口腔崩解片和口腔速溶膜剂中的一种或多种。The oral preparation is one or more selected from the group consisting of a powder, a general oral tablet, a dispersible tablet, a capsule, a soft capsule, a pill, a pill, a pellet, a granule, an orally disintegrating tablet, and an oral fast dissolving film. .
所述非口服制剂选自注射剂、冻干粉针和大容量输液剂中的一种或多种。The non-oral preparation is selected from one or more of an injection, a lyophilized powder, and a bulk infusion.
优选地，所述用于预防和/或治疗抑郁症的药物为口服制剂。Preferably, the drug for preventing and/or treating depression is an oral preparation.
作为一个优选的实施方案，所述用于预防和/或治疗抑郁症的药物为脑心清片或脑心清胶囊。As a preferred embodiment, the drug for preventing and/or treating depression is Naoxinqing Tablet or Naoxinqing Capsule.
将所述柿叶提取物，加入或不加入药学上可以接受的辅料，按照本领域常规的方法，即可制备得到上述于预防和/或治疗抑郁症的药物。The above-mentioned persimmon leaf extract can be prepared by the method conventional in the art, with or without the addition of a pharmaceutically acceptable excipient, for the prevention and/or treatment of depression.
上述柿叶提取物通过如下方法制备：The above persimmon leaf extract is prepared by the following method:
取干燥柿叶，加水煎煮2次，每次1～2小时，合并水煎液，滤过，浓缩至相对密度1.12～1.15(60℃)，加乙醇至含醇量达80～90％；静置过夜，滤取上清液，备用；沉淀物用60～70％乙醇洗涤，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取2次以上，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, each time for 1 to 2 hours, combine water decoction, filter, concentrate to a relative density of 1.12 ~ 1.15 (60 ° C), add ethanol to 80-90% alcohol content; After standing overnight, the supernatant was collected by filtration, and the precipitate was washed with 60-70% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and ethanol was added thereto, and an appropriate amount of water was added. Mix and filter, filter the filtrate with ethyl acetate 2 times or more, combine with ethyl acetate solution, recover ethyl acetate and concentrate to thick paste, dry at low temperature, that is.
优选地，上述柿叶提取物通过如下方法制备：Preferably, the above persimmon leaf extract is prepared by the following method:
取干燥柿叶，加水煎煮2次，第1次2小时，第2次1小时，合并滤液，滤过，浓缩汁相对密度1.12～1.15(60℃)，加乙醇至含醇量达85％；静置过夜，滤取上清液，备用；沉淀物用65％乙醇洗涤2次，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取4次，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, the first 2 hours, the second time 1 hour, combine the filtrate, filter, the relative density of concentrated juice is 1.12~1.15 (60 °C), add ethanol to 85% alcohol content After standing overnight, the supernatant was collected by filtration, and the precipitate was washed twice with 65% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and the ethanol was recovered, and the appropriate amount of water was added. , mixing, filtration, the filtrate was extracted with ethyl acetate 4 times, combined with ethyl acetate solution, ethyl acetate was recovered and concentrated into a thick paste, dried at low temperature, that is.
本发明所述用于预防和/或治疗抑郁症的药物施用对象为哺乳动物，优选为人。The pharmaceutical administration object for preventing and/or treating depression according to the present invention is a mammal, preferably a human.
本发明所述用于预防和/或治疗抑郁症的药物施用于有需要的人时，所述柿叶提取物的给药量为每天2～10mg/kg体重。When the medicament for preventing and/or treating depression of the present invention is administered to a person in need thereof, the persimmon leaf extract is administered in an amount of 2 to 10 mg/kg of body weight per day.
本发明还提供一种预防和/或治疗抑郁症的方法，该方法包括向有需要的患者施用柿叶提取物的步骤；The present invention also provides a method of preventing and/or treating depression, the method comprising the step of administering a persimmon leaf extract to a patient in need thereof;
优选地，所述柿叶提取物通过如下方法制备：Preferably, the persimmon leaf extract is prepared by the following method:
取干燥柿叶，加水煎煮2次，每次1～2小时，合并水煎液，滤过，浓缩至相对密度1.12～1.15(60℃)，加乙醇至含醇量达80～90％；静置过夜，滤取上清 液，备用；沉淀物用60～70％乙醇洗涤，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取2次以上，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得；Take dried persimmon leaves, add water for 2 times, each time for 1 to 2 hours, combine water decoction, filter, concentrate to a relative density of 1.12 ~ 1.15 (60 ° C), add ethanol to 80-90% alcohol content; After standing overnight, the supernatant was collected by filtration, and the precipitate was washed with 60-70% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and ethanol was added thereto, and an appropriate amount of water was added. Mix and filter, filter the filtrate with ethyl acetate for more than 2 times, combine with ethyl acetate solution, recover ethyl acetate and concentrate into thick paste, dry at low temperature, that is;
更优选地，所述柿叶提取物通过如下方法制备：More preferably, the persimmon leaf extract is prepared by the following method:
取干燥柿叶，加水煎煮2次，第1次2小时，第2次1小时，合并滤液，滤过，浓缩汁相对密度1.12～1.15(60℃)，加乙醇至含醇量达85％；静置过夜，滤取上清液，备用；沉淀物用65％乙醇洗涤2次，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取4次，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, the first 2 hours, the second time 1 hour, combine the filtrate, filter, the relative density of concentrated juice is 1.12~1.15 (60 °C), add ethanol to 85% alcohol content After standing overnight, the supernatant was collected by filtration, and the precipitate was washed twice with 65% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and the ethanol was recovered, and the appropriate amount of water was added. , mixing, filtration, the filtrate was extracted with ethyl acetate 4 times, combined with ethyl acetate solution, ethyl acetate was recovered and concentrated into a thick paste, dried at low temperature, that is.
优选地，所述抑郁症是指典型抑郁障碍，尤其是精神性、情感障碍所致的抑郁障碍。Preferably, the depression refers to a typical depressive disorder, especially a depression caused by mental and affective disorders.
所述有需要的患者为有需要的哺乳动物，优选为有需要的人。The patient in need is a mammal in need thereof, preferably a human in need thereof.
所述预防和/或治疗抑郁症的方法，包括向有需要的人施用柿叶提取物2～10mg/kg体重/天的步骤。The method for preventing and/or treating depression includes the step of administering a persimmon leaf extract of 2 to 10 mg/kg body weight/day to a person in need thereof.
上述药学上可以接受的辅料，包括但不限于：The above pharmaceutically acceptable excipients include, but are not limited to:
稀释剂：例如选自淀粉、糊精、预胶化淀粉、乳糖、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙、氧化镁、碳酸镁、氢氧化铝凝胶、β-环糊精、甘露醇、山梨醇、甲基纤维素、羟丙基甲基纤维素、聚乙烯吡咯烷酮、羟甲基纤维素钠中的一种或多种。Diluent: for example selected from the group consisting of starch, dextrin, pregelatinized starch, lactose, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, magnesium oxide, magnesium carbonate, aluminum hydroxide gel, β-cyclodextrin One or more of mannitol, sorbitol, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and sodium carboxymethylcellulose.
粘合剂：例如选自蒸馏水、乙醇、淀粉浆、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素和乙基纤维素、羟丙甲纤维素中的一种或多种。Binder: For example, one or more selected from the group consisting of distilled water, ethanol, starch syrup, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and ethyl cellulose, hypromellose.
润滑剂：例如选自十二烷基硫酸镁、聚乙二醇、微粉硅胶、硬脂酸镁、滑石粉中的一种或多种。Lubricant: for example, one or more selected from the group consisting of magnesium lauryl sulfate, polyethylene glycol, micronized silica gel, magnesium stearate, and talc.
崩解剂：例如选自淀粉(玉米、马铃薯)、微晶纤维素、海藻酸、海藻酸钠、离子交换树脂、泡腾酸-碱系统、羟丙基淀粉、羧甲基淀粉钠、交联羧甲基纤维素钠、交联聚维酮、羧甲基纤维素、羧甲基纤维素钙、低取代羟丙基纤维素、部分α化淀粉和微晶纤维素中的一种或多种。Disintegrator: for example selected from starch (corn, potato), microcrystalline cellulose, alginic acid, sodium alginate, ion exchange resin, effervescent-alkali system, hydroxypropyl starch, sodium carboxymethyl starch, cross-linking One or more of sodium carboxymethylcellulose, crospovidone, carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, partially alpha-starch, and microcrystalline cellulose .
成膜材料：明胶、虫胶、阿拉伯胶、琼脂、淀粉、糊精、PVA05-88、PVA17-88和乙烯-醋酸乙烯共聚物(EVA)中的一种或多种。Film forming materials: one or more of gelatin, shellac, gum arabic, agar, starch, dextrin, PVA05-88, PVA17-88, and ethylene-vinyl acetate copolymer (EVA).
矫味剂：例如选自糖精钠、甜蜜素、阿斯巴甜、甜菊苷以及香精类中的一种或多种。Flavoring agent: for example, one or more selected from the group consisting of sodium saccharin, cyclamate, aspartame, stevioside, and flavors.
注射用溶剂：例如选自注射用水、麻油、茶油、花生油、玉米油、橄榄油、棉籽油、豆油、蓖麻油及桃仁油、油酸乙酯、苯甲酸苄酯、丙二醇、聚乙二醇400、二甲基乙酰胺(DMA)、乙醇和甘油中的一种或多种。Solvent for injection: for example, selected from water for injection, sesame oil, tea oil, peanut oil, corn oil, olive oil, cottonseed oil, soybean oil, castor oil and peach oil, ethyl oleate, benzyl benzoate, propylene glycol, polyethylene glycol 400, one or more of dimethylacetamide (DMA), ethanol, and glycerin.
本发明通过细胞体外模型和动物整体模型研究发现，所述柿叶提取物具有良好的抗抑郁作用。通过机理研究，认为本发明所述柿叶提取物通过以下几方面起到预防和/或治疗抑郁症的作用：The present invention finds that the persimmon leaf extract has a good antidepressant effect by a cell in vitro model and an animal model study. Through mechanism research, it is considered that the persimmon leaf extract of the present invention plays a role in preventing and/or treating depression by the following aspects:
1)抗神经炎症作用，可抑制脑内神经炎症因子，如TNF-α、IL-1β，IL-6等的释放。1) Anti-neuroinflammatory effect can inhibit the release of neuroinflammatory factors such as TNF-α, IL-1β, IL-6 in the brain.
2)具备调控下丘脑-垂体-肾上腺轴的作用，可抑制应激引起的血清皮质酮(醇)和促肾上腺皮质激素的分泌。2) It has the function of regulating the hypothalamic-pituitary-adrenal axis, which can inhibit the secretion of serum corticosterone (alcohol) and adrenocorticotropic hormone caused by stress.
3)可调节脑内单胺类神经递质，包括5-羟色胺、多巴胺、去甲肾上腺素的平衡，起到精神情感调控作用。3) It can regulate the balance of monoamine neurotransmitters in the brain, including the balance of serotonin, dopamine and norepinephrine, and play a role in mental and emotional regulation.
虽然现有技术中已经公开柿叶提取物中含有槲皮素、山柰酚和金丝桃苷等黄酮类成分，且这些成分都被报道过具有一定的抗抑郁作用，如①贝伟剑,王德勤，等.HPLC法测定脑心清片浸膏中金丝桃苷的含量[J].广西中医学院学报,2009(02):62-63；②贝伟剑,罗杰，等.HPLC法测定柿叶浸膏中槲皮素和山柰酚[J].中草药,2005(07):59-60；③传娟娟,李燕.贯叶金丝桃(圣约翰草)的国内外研究概况[J].西北药学杂志,2016(03):330-332；④陈箐筠,干信.槲皮素对皮质酮损伤的pc12细胞的保护作用[J].化学与生物工程,2009(01):47-49；⑤刘健翔,方吟荃,等.槲皮素与贯叶连翘提取物合用抗抑郁作用初步研究[J].浙江大学学报(医学版),2013(06):615-619；⑥陈蕾.黄酮类化合物的抗抑郁作用研究[J].江西中医药,2011,42(10):55-57。但是令人意想不到的是，本发明通过实验发现，柿叶提取物抗抑郁的作用不仅显著强于槲皮素、山柰酚、金丝桃苷、芦丁等黄酮单体，而且也明显强于这些黄酮的组合物(模拟柿叶提取物中天然存在的黄酮组合)。说明柿叶提取物中起抗抑郁作用的物质基础不仅仅是槲皮素、山柰酚、金丝桃苷、芦丁及其组合，很有可能是未见报道但是具有抗抑郁活性的其它成分。Although it has been disclosed in the prior art that the persimmon leaf extract contains flavonoids such as quercetin, kaempferol and hyperoside, and these ingredients have been reported to have certain antidepressant effects, such as 1 Bayi Sword, Wang Deqin, et al.Determination of hyperoside in Naoxinqing Tablet Extract by HPLC[J].Journal of Guangxi Traditional Chinese Medical College,2009(02):62-63;2 Bei Weijian, Luo Jie, et al. HPLC Determination of quercetin and kaempferol in persimmon leaf extracts[J].Chinese Traditional and Herbal Drugs,2005(07):59-60;3 Chuan Juanjuan,Li Yan.Overview of Domestic and Foreign Studies of Hypericum perforatum (St. John's Wort) [J]. Northwest Pharmaceutical Journal, 2016 (03): 330-332; 4 Chen Yu, Gan Xin. Protective effect of quercetin on corticosterone-induced pc12 cells[J].Chemistry & Bioengineering,2009(01): 47-49;5 Liu Jianxiang,Fang Wei, et al. Preliminary study on antidepressant effect of quercetin and extract of Hypericum perforatum L.[J].Journal of Zhejiang University(Medical Science),2013(06):615-619;6陈Lei.Study on the antidepressant effect of flavonoids[J].Jiangxi Traditional Chinese Medicine,2011,42(10):55-57. However, it is unexpectedly found that the anti-depressant effect of persimmon leaf extract is not only stronger than that of flavonoids such as quercetin, kaempferol, hyperoside, rutin, but also significantly stronger than that of experiments. A composition of these flavonoids (simulating the combination of flavonoids naturally present in the extract of persimmon leaves). The material basis for the antidepressant effect of persimmon leaf extract is not only quercetin, kaempferol, hyperoside, rutin and combinations thereof, but most likely other ingredients that have not been reported but have antidepressant activity. .
以柿叶提取物为有效成分的制剂，如脑心清片在临床已经应用多年，其安全性已经得到证明。本发明为柿叶提取物提供了一种新的医药用途。基于本发明，将为临床治疗典型抑郁障碍提供一种新的安全性高的治疗选择。Formulations using persimmon leaf extract as an active ingredient, such as Naoxinqing tablets, have been used for many years in the clinic, and their safety has been proven. The present invention provides a new medical use for persimmon leaf extract. Based on the present invention, a new safe treatment option for clinical treatment of typical depressive disorder will be provided.
附图说明DRAWINGS
以下参照附图，对本发明作详细的说明。The invention will now be described in detail with reference to the accompanying drawings.
图1示出的是实施例1中不同浓度柿叶提取物(3.125～400μg/mL)与正常培养的PC12细胞共同孵育24h后，MTT法检测的细胞存活率。Figure 1 shows the cell viability measured by MTT assay after the incubation of different concentrations of persimmon leaf extract (3.125-400 μg/mL) in Example 1 with normal cultured PC12 cells for 24 hours.
图2示出的是实施例1中不同浓度皮质酮(Cort,0～800μM)与正常培养的PC12细胞共同孵育24h后，MTT法检测的细胞存活率(图2A)和LDH检测法检测的LDH活性(图2B)，图中， *P<0.05, **P<0.01, ***P<0.001vs.Cort (0μM)组(正常对照组)。 Figure 2 is a graph showing the cell viability measured by MTT assay (Fig. 2A) and the LDH assay by LDH after incubation with different concentrations of corticosterone (Cort, 0-800 μM) in normalized cells for 24 h. Activity (Fig. 2B), in the figure, * P<0.05, ** P<0.01, *** P<0.001 vs. Cort (0 μM) group (normal control group).
图3示出的是实施例1中不同浓度柿叶提取物(6.25,12.5,25,50,100μg/mL)对皮质酮(Cort,200μM)损伤PC12细胞的保护作用，其中图3A示出的是对细胞存活率的影响，图3B示出的是对LDH活性的影响；图中： *P<0.05, **P<0.01, ***P<0.001vs.Cort组(皮质酮组)， ##P<0.01vs.Veh组(正常对照组)。 Figure 3 shows the protective effect of different concentrations of persimmon leaf extract (6.25, 12.5, 25, 50, 100 μg/mL) on corticosterone (Cort, 200 μM) damage PC12 cells in Example 1, wherein Figure 3A shows Effect on cell viability, Figure 3B shows the effect on LDH activity; in the figure: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Cort group (corticosterone group), # # P<0.01vs.Veh group (normal control group).
图4示出的是实施例1中不同浓度柿叶提取物(6.25,12.5,25,50,100μg/mL)对皮质酮(Cort,200μM)损伤PC12细胞分泌炎症因子(TNF-α、IL-1β和IL-6)的影响；其中， *P<0.05, **P<0.01, ***P<0.001vs.Cort组(皮质酮组)， ##P<0.01vs.Control组(正常对照组)。 Figure 4 is a graph showing that different concentrations of persimmon leaf extract (6.25, 12.5, 25, 50, 100 μg/mL) in Example 1 secreted inflammatory cytokines (TNF-α, IL-1β) in corticosterone (Cort, 200 μM)-injured PC12 cells. And IL-6); among them, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Cort group (corticosterone group), ## P < 0.01 vs. Control group (normal control group) ).
图5示出的是实施例2中敞箱实验的结果，其中图5A显示的是各组小鼠水平得分情况，图5B显示的是各组小鼠垂直得分情况。Fig. 5 shows the results of the open box experiment in Example 2, in which Fig. 5A shows the level scores of the mice in each group, and Fig. 5B shows the vertical scores of the mice in each group.
图6示出的是实施例2中悬尾实验的结果，图中， *P<0.05, **P<0.01, ***P<0.001vs.Veh(溶剂对照组)。 Figure 6 shows the results of the tail suspension experiment in Example 2, in which * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Veh (solvent control group).
图7示出的是实施例2中小鼠强迫游泳实验的结果，图中， *P<0.05, **P<0.01, ***P<0.001vs.Veh(溶剂对照组)。 Fig. 7 shows the results of a forced swimming test of mice in Example 2, in which * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Veh (solvent control group).
图8示出的是实施例2中柿叶提取物对急性应激小鼠血清皮质酮水平的影响的测定结果，图中， *P<0.05, **P<0.01, ***P<0.001vs.Veh2(溶剂对照组)； ##P＜0.01vs.Veh1(正常对照组)。 Figure 8 is a graph showing the results of the effect of persimmon leaf extract of Example 2 on serum corticosterone levels in acutely stressed mice, in which * P < 0.05, ** P < 0.01, *** P < 0.001 Vs. Veh2 (solvent control group); ## P<0.01 vs. Veh1 (normal control group).
图9示出的是实施例2中柿叶提取物对急性应激小鼠大脑单胺类神经递质水平的影响的测定结果。其中，图9A示出的是5-羟色胺(5-HT)的测定结果，图9B示出的是多巴胺(DA)的测定结果，图9C示出的是去甲肾上腺素(NE)的测定结果。图中， *P<0.05, **P<0.01, ***P<0.001vs.Veh(溶剂对照组)； ###P＜0.01vs.Normal(正常对照组)。 Fig. 9 is a graph showing the results of measuring the effect of persimmon leaf extract of Example 2 on brain monoamine neurotransmitter levels in acutely stressed mice. Here, FIG. 9A shows the measurement results of serotonin (5-HT), FIG. 9B shows the measurement results of dopamine (DA), and FIG. 9C shows the measurement results of norepinephrine (NE). . In the figure, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Veh (solvent control group); ### P < 0.01 vs. Normal (normal control group).
图10示出的是实施例3中糖水偏爱实验的结果。图中，1为正常对照组，2为模型组，3为柿叶提取物，4为柿叶黄酮组合物，5为槲皮素，6为山柰酚，7为金丝桃苷，8为芦丁； *P<0.05, **P<0.01, ***P<0.001vs.模型对照组； ###P＜0.01vs.正常对照组； $P<0.05, $$P<0.01vs.柿叶提取物。 Figure 10 shows the results of the sugar water preference experiment in Example 3. In the figure, 1 is the normal control group, 2 is the model group, 3 is the persimmon leaf extract, 4 is the persimmon leaf flavonoid composition, 5 is quercetin, 6 is kaempferol, 7 is hyperoside, 8 is Rutin; * P<0.05, ** P<0.01, *** P<0.001 vs. model control group; ### P<0.01 vs. normal control group; $ P<0.05, $$ P<0.01 vs. Persimmon leaf extract.
图11示出的是实施例3中敞箱实验的结果；其中图11A示出的是小鼠水平运动得分，图11B示出的是小鼠垂直运动得分。图中，1为正常对照组，2为模型组，3为柿叶提取物，4为柿叶黄酮组合物，5为槲皮素，6为山柰酚，7为金丝桃苷，8为芦丁； *P<0.05, **P<0.01, ***P<0.001vs.模型对照组； ###P＜0.01vs.正常对照组； $P<0.05, $$P<0.01vs.柿叶提取物。 Figure 11 shows the results of the open box experiment in Example 3; wherein Figure 11A shows the mouse horizontal motion score and Figure 11B shows the mouse vertical motion score. In the figure, 1 is the normal control group, 2 is the model group, 3 is the persimmon leaf extract, 4 is the persimmon leaf flavonoid composition, 5 is quercetin, 6 is kaempferol, 7 is hyperoside, 8 is Rutin; * P<0.05, ** P<0.01, *** P<0.001 vs. model control group; ### P<0.01 vs. normal control group; $ P<0.05, $$ P<0.01 vs. Persimmon leaf extract.
图12示出的是实施例3中悬尾实验的结果。图中，1为正常对照组，2为模型组，3为柿叶提取物，4为柿叶黄酮组合物，5为槲皮素，6为山柰酚，7为金丝桃苷，8为芦丁； *P<0.05, **P<0.01, ***P<0.001vs.模型对照组； ###P＜ 0.01vs.正常对照组； $P<0.05, $$P<0.01vs.柿叶提取物。 Figure 12 shows the results of the tail suspension experiment in Example 3. In the figure, 1 is the normal control group, 2 is the model group, 3 is the persimmon leaf extract, 4 is the persimmon leaf flavonoid composition, 5 is quercetin, 6 is kaempferol, 7 is hyperoside, 8 is Rutin; * P<0.05, ** P<0.01, *** P<0.001 vs. model control group; ### P< 0.01 vs. normal control group; $ P<0.05, $$ P<0.01 vs. Persimmon leaf extract.
图13示出的是实施例3中小鼠强迫游泳实验的结果，图中，1为正常对照组，2为模型组，3为柿叶提取物，4为柿叶黄酮组合物，5为槲皮素，6为山柰酚，7为金丝桃苷，8为芦丁； *P<0.05, **P<0.01, ***P<0.001vs.模型对照组； ###P＜0.01vs.正常对照组； $P<0.05, $$P<0.01vs.柿叶提取物。 Figure 13 is a graph showing the results of a forced swimming test in a mouse in Example 3. In the figure, 1 is a normal control group, 2 is a model group, 3 is a persimmon leaf extract, 4 is a persimmon leaf flavonoid composition, and 5 is a mink skin. 6, 6 is kaempferol, 7 is hyperoside, 8 is rutin; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. model control group; ### P<0.01vs Normal control group; $ P<0.05, $$ P<0.01 vs. persimmon leaf extract.
图14示出的是实施例3中CUMS小鼠血清皮质酮和促肾上腺皮质激素水平的测定结果；其中，图左侧示出的血清皮质酮水平测定结果，图右侧示出的是血清促肾上腺皮质激素水平测定结果。图中， *P<0.05, **P<0.01, ***P<0.001vs.模型对照组； ###P＜0.01vs.正常对照组； $P<0.05, $$P<0.01vs.柿叶提取物。 Figure 14 is a graph showing the results of measurement of serum corticosterone and adrenocorticotropic hormone levels in CUMS mice in Example 3; wherein the results of serum corticosterone levels shown on the left side of the figure are shown on the right side of the figure. The results of the determination of adrenocortical hormone levels. In the figure, * P<0.05, ** P<0.01, *** P<0.001 vs. model control group; ### P<0.01 vs. normal control group; $ P<0.05, $$ P<0.01 vs. Persimmon leaf extract.
实施发明的最佳方式The best way to implement the invention
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。The invention is described below with reference to specific embodiments. Those skilled in the art can understand that the examples are only intended to illustrate the invention, and the scope of the invention is not limited in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中，部分试剂和仪器购买情况如下：The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagent materials and the like used in the following examples are commercially available products unless otherwise specified. Among them, some reagents and instruments are purchased as follows:
柿叶提取物(NXQ)：广州白云山和记黄埔中药有限公司，批号H16P006，制备方法为：Persimmon Leaf Extract (NXQ): Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd., batch number H16P006, preparation method is:
取干燥柿叶，加水煎煮2次，第1次2小时，第2次1小时，合并滤液，滤过，浓缩汁相对密度1.12～1.15(60℃)，加乙醇至含醇量达85％；静置过夜，滤取上清液，备用；沉淀物用65％乙醇洗涤2次，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取4次，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, the first 2 hours, the second time 1 hour, combine the filtrate, filter, the relative density of concentrated juice is 1.12~1.15 (60 °C), add ethanol to 85% alcohol content After standing overnight, the supernatant was collected by filtration, and the precipitate was washed twice with 65% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and the ethanol was recovered, and the appropriate amount of water was added. , mixing, filtration, the filtrate was extracted with ethyl acetate 4 times, combined with ethyl acetate solution, ethyl acetate was recovered and concentrated into a thick paste, dried at low temperature, that is.
氟西汀、皮质酮、噻唑蓝(MTT)、二甲基亚砜(DMSO)：Sigma公司；Fluoxetine, corticosterone, thiazole blue (MTT), dimethyl sulfoxide (DMSO): Sigma;
DMEM高糖培养基、澳洲胎牛血清、马血清：Gibco公司；DMEM high sugar medium, Australian fetal bovine serum, horse serum: Gibco;
PC12细胞：大鼠来源，中科院细胞库；PC12 cells: rat source, Chinese Academy of Sciences cell bank;
LDH检测试剂盒：南京建成生物科技有限公司；LDH test kit: Nanjing Jiancheng Biotechnology Co., Ltd.;
炎症因子ELISA检测试剂盒：生工生物工程(上海)股份有限公司；ELISA Kit for Inflammatory Factors: Bioengineering (Shanghai) Co., Ltd.;
细胞培养箱：Thermo公司；Cell culture incubator: Thermo Corporation;
酶标仪：Bio-rad公司；Microplate reader: Bio-rad;
敞箱：上海欣软信息科技有限公司；Open box: Shanghai Xinsoft Information Technology Co., Ltd.;
圆形游泳缸：上海移数信息科技有限公司；Round swimming cylinder: Shanghai Shift Information Technology Co., Ltd.;
悬尾仪：上海欣软信息科技有限公司。Suspended instrument: Shanghai Xinsoft Information Technology Co., Ltd.
实施例1 柿叶提取物体外对皮质酮所致神经细胞损伤的保护作用 Example 1 Protective effect of corticosterone-induced nerve cell damage on the extract of persimmon leaves
1.1实验药物配制1.1 experimental drug preparation
柿叶提取物溶于100％DMSO中，配制成400mg/mL的储存液，使用时将其稀释成400、200、100、50、25、12.5、6.25、3.125μg/mL，并保证DMSO的浓度低于0.1％适合进行细胞实验。The persimmon leaf extract was dissolved in 100% DMSO to prepare a 400 mg/mL stock solution, which was diluted to 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 μg/mL, and the concentration of DMSO was ensured. Less than 0.1% is suitable for cell experiments.
1.2数据分析及统计1.2 Data analysis and statistics
所有数据均采用均数±标准差
Figure PCTCN2018120186-appb-000001
表示，用SPSS软件分析数据，方差分析(One-Way ANOVA)。经方差齐性检验，若方差齐性则组间两两比较采用Bonferronic法；若方差不齐将采用Welch法进行分析，多重比较采用Dunnett’s T3法。P＜0.05为存在显著性差异，具有统计学意义。 Mean ± standard deviation for all data
Figure PCTCN2018120186-appb-000001
Said to analyze data with SPSS software, analysis of variance (One-Way ANOVA). After the homogeneity test of variance, if the variance is homogeneous, the Bonferronic method is used for comparison between groups. If the variance is not uniform, the Welch method will be used for analysis, and the multiple comparisons will be performed by Dunnett's T3 method. P < 0.05 was considered to be statistically significant.
1.3不同浓度柿叶提取物对PC12细胞存活率的影响1.3Effects of different concentrations of persimmon leaf extract on the survival rate of PC12 cells
8个不同浓度柿叶醋酸乙酯提取物(3.125～400μg/mL)与正常培养的PC12细胞共同孵育24h后，采用MTT法进行PC12细胞存活率检测。MTT的结果显示(见图1及表1)，与正常组对比，各浓度柿叶提取物处理组的细胞存活率无明显区别。经单因素方差(One-way ANOVA)分析，差异不具统计学意义(P>0.05)。因此，所选择的柿叶提取物浓度范围适用于后续抗抑郁细胞水平的筛选。Eight different concentrations of persimmon leaf ethyl acetate extract (3.125-400μg/mL) were incubated with normal cultured PC12 cells for 24h, and the survival rate of PC12 cells was detected by MTT assay. The results of MTT showed (see Figure 1 and Table 1). Compared with the normal group, there was no significant difference in cell viability between the peripate leaf extract treatment groups. After one-way ANOVA analysis, the difference was not statistically significant (P>0.05). Therefore, the selected persimmon leaf extract concentration range is suitable for screening of subsequent antidepressant cell levels.
表1 柿叶提取物(NXQ)对正常培养的PC12细胞存活率的影响Table 1 Effect of Persimmon Leaf Extract (NXQ) on the Survival Rate of Normal Cultured PC12 Cells
Figure PCTCN2018120186-appb-000002
Figure PCTCN2018120186-appb-000002
1.4神经细胞皮质酮损伤模型的建立1.4 Establishment of a model of cortical ketone damage in nerve cells
细胞培养至对数生长期，用含5％胎牛血清、10％马血清的DMEM培养基(含青霉素钠200kU/L，链霉素100mg/L，pH 7.4)重悬细胞，调细胞密度为1×10 5个/mL.细胞接种于96孔板中，各孔加入100μL细胞液，置于37℃、CO 2培养箱中贴壁孵育24h，待细胞长满孔底后即开始实验。细胞血清剥夺1h后，加入不同浓度的皮质酮(0,25,50,100,200,400μM)，作用24h后，用MTT法和LDH检测法观察皮质酮出现致损伤浓度，选择皮质酮的最佳造模浓度。 The cells were cultured to logarithmic growth phase, and the cells were resuspended in DMEM medium containing 5% fetal bovine serum and 10% horse serum (containing penicillin sodium 200 kU/L, streptomycin 100 mg/L, pH 7.4), and the cell density was adjusted. 1×10 5 /mL. The cells were seeded in a 96-well plate, 100 μL of cell liquid was added to each well, and the cells were incubated at 37 ° C in a CO 2 incubator for 24 hours. The experiment was started after the cells were full. After deprivation of the cells for 1 h, different concentrations of corticosterone (0, 25, 50, 100, 200, 400 μM) were added. After 24 h, the lesion concentration of corticosterone was observed by MTT assay and LDH assay, and the optimal model concentration of corticosterone was selected.
结果见图2和表2。0～800μM的皮质酮对PC12细胞的损伤呈剂量依赖性。经One-way ANOVA分析，200μM皮质酮处理PC12细胞24h后显著性降低细胞活力(P<0.01)，细胞存活率下降30％(图2A)。LDH检测结果也与此相似，给予不同浓度皮质酮处理后，各组的LDH外漏存在显著性差异(P<0.01,图2B)。当皮质酮的浓度在200μM作用24h后，细胞LDH外漏与正常对照组比较显著性增加。根据以上结果，200μM的皮质酮损伤PC12细胞24h可作为后续实验皮质酮诱导细胞浓度。The results are shown in Figure 2 and Table 2. Corticosterone from 0 to 800 μM was dose-dependently damaging PC12 cells. After one-way ANOVA analysis, PC12 cells treated with 200 μM corticosterone significantly reduced cell viability (P<0.01) and cell viability decreased by 30% (Fig. 2A). The LDH test results were similar. After treatment with different concentrations of corticosterone, there was a significant difference in LDH leakage between the groups (P<0.01, Figure 2B). When the concentration of corticosterone was treated at 200 μM for 24 h, the LDH leakage of the cells was significantly increased compared with the normal control group. According to the above results, 200 μM corticosterone damage PC12 cells for 24 h can be used as a subsequent experimental corticosterone-induced cell concentration.
表2 皮质酮(Cort)对PC12细胞存活率和LDH的影响Table 2 Effect of corticosterone (Cort) on PC12 cell survival rate and LDH
Figure PCTCN2018120186-appb-000003
Figure PCTCN2018120186-appb-000003
*P<0.05, **P<0.01, ***P<0.001vs.正常对照组. * P<0.05, ** P<0.01, *** P<0.001 vs. normal control group.
1.5不同浓度柿叶提取物对皮质酮损伤PC12细胞的保护作用1.5The protective effect of different concentrations of persimmon leaf extract on PC12 cells injured by corticosterone
细胞种板96孔板后12h后，不同浓度柿叶提取物(3.125～400μg/mL)预处理细胞1h。除对照孔外，其余各孔加入200μM皮质酮共同孵育24h。收集细胞上清液用于检测乳酸脱氢酶(LDH)活性变化，MTT法检测96孔板内细胞存活率，酶标仪570nm检测各孔吸光度值。以对照组平均吸光度为100％，以各处理组吸收度值与对照组的比值计算存活力和LDH的变化情况。After 12 h of 96-well plates, the cells were pretreated with different concentrations of persimmon leaf extract (3.125-400 μg/mL) for 1 h. Except for the control wells, the other wells were incubated with 200 μM corticosterone for 24 h. The supernatant of the cells was collected for detecting the change of lactate dehydrogenase (LDH) activity, the cell viability of the 96-well plate was measured by MTT assay, and the absorbance of each well was detected by a microplate reader at 570 nm. The average absorbance of the control group was 100%, and the changes of viability and LDH were calculated by the ratio of the absorbance value of each treatment group to the control group.
结果见图3和表3。经One-way ANOVA分析显示，200μM的皮质酮对PC12细胞进行损伤24h后，PC12细胞的细胞活力明显下降而细胞上清液LDH的分泌则明显增加。从6.25～100μg/mL，柿叶提取物呈浓度依赖性的增加细胞存活率和降低上清液LDH分泌，其中25、50、100μg/mL对皮质酮诱导的细胞损伤具有显著抵抗作用(P<0.01)。该结果提示，25μM的柿叶醋酸乙酯提取物在细胞水平即具有明显的抗抑郁活性。The results are shown in Figures 3 and 3. One-way ANOVA analysis showed that after 200μM corticosterone damage PC12 cells for 24h, the cell viability of PC12 cells decreased significantly and the secretion of LDH in cell supernatants increased significantly. From 6.25 to 100 μg/mL, persimmon leaf extract increased cell viability and decreased supernatant LDH secretion in a concentration-dependent manner, of which 25, 50, 100 μg/mL had significant resistance to corticosterone-induced cell damage (P< 0.01). The results suggest that 25 μM persimmon leaf ethyl acetate extract has significant antidepressant activity at the cellular level.
表3 柿叶提取物对皮质酮造成的细胞存活率及乳酸脱氢酶变化的影响Table 3 Effect of Persimmon Leaf Extract on Cellular Survival Rate and Changes of Lactate Dehydrogenase Induced by Corticosterone
Figure PCTCN2018120186-appb-000004
Figure PCTCN2018120186-appb-000004
Figure PCTCN2018120186-appb-000005
Figure PCTCN2018120186-appb-000005
*P<0.05, **P<0.01, ***P<0.001vs.模型对照组， ##P<0.01vs.正常对照组 * P<0.05, ** P<0.01, *** P<0.001 vs. model control group, ## P<0.01 vs. normal control group
1.6柿叶提取物对皮质酮诱导PC12细胞炎症反应的影响Effects of 1.6 Persimmon Leaf Extract on Inflammatory Response of PC12 Cells Induced by Corticosterone
由于200μM皮质酮处理24h引起细胞存活率下降，因此，为了评价柿叶提取物在抗抑郁症过程是否发挥了抗炎效应，选择不同浓度柿叶提取物与皮质酮共培养处理PC12细胞4h，ELISA法检测上清液中炎症因子IL-1β、IL-6和TNF-α的浓度，以观察柿叶提取物对炎症因子分泌的影响。Since the cell viability decreased after treatment with 200 μM corticosterone for 24 h, in order to evaluate whether the extract of persimmon leaves exerted anti-inflammatory effect in the process of anti-depression, PC12 cells were co-cultured with different concentrations of persimmon leaf extract and corticosterone for 4 h, ELISA The concentration of inflammatory factors IL-1β, IL-6 and TNF-α in the supernatant was determined to observe the effect of persimmon leaf extract on the secretion of inflammatory factors.
ELISA的结果显示(见图4及表4)，与空白对照组(Control)比较，200μM皮质酮极显著地增加PC12细胞上清液中TNF-α、IL-1β和IL-6的分泌水平(P<0.001)。与空白对照组比较，柿叶提取物最大浓度单独处理(无皮质酮)并不产生炎症效应(P>0.05)。柿叶提取物处理1h后，各剂量均不同程度地抑制由皮质酮引起的PC12细胞炎症因子的释放。其中，柿叶提取物在剂量为25μg/ml时即对TNF-α、IL-1β和IL-6的抑制产生显著效果，均具有统计学意义(P<0.01)。The results of the ELISA showed (see Figure 4 and Table 4) that compared with the blank control group (Control), 200 μM corticosterone significantly increased the secretion levels of TNF-α, IL-1β and IL-6 in the supernatant of PC12 cells ( P < 0.001). Compared with the blank control group, the maximum concentration of persimmon leaf extract alone (no corticosterone) did not produce inflammatory effects (P>0.05). After treatment with persimmon leaf extract for 1 h, each dose inhibited the release of inflammatory factors in PC12 cells induced by corticosterone. Among them, the persimmon leaf extract had significant effects on the inhibition of TNF-α, IL-1β and IL-6 at a dose of 25 μg/ml, both of which were statistically significant (P<0.01).
表4 柿叶提取物对皮质酮引起的炎症因子分泌的影响Table 4 Effect of Persimmon Leaf Extract on Secretion of Inflammatory Factors Induced by Corticosterone
Figure PCTCN2018120186-appb-000006
Figure PCTCN2018120186-appb-000006
*P<0.05, **P<0.01, ***P<0.001vs.Cort组， ##P<0.01vs.正常对照组. * P<0.05, ** P<0.01, *** P<0.001 vs.Cort group, ## P<0.01 vs. normal control group.
1.7结论1.7 Conclusion
本实施例成功建立了皮质酮损伤PC12细胞抑郁模型。通过研究发现柿叶提取物在3.125～400μM浓度范围对正常培养细胞无毒害作用，但是在PC12细胞抑郁模型中，柿叶提取物在50μM即可产生较好的神经细胞保护作用，其作用机理可能与其抑制皮质酮引起的炎症反应有关。本实施例的结果提示，柿叶提取物在细胞层面上具有明显的抗抑郁活性。This example successfully established a corticosterone-induced PC12 cell depression model. It was found that the persimmon leaf extract had no toxic effect on normal culture cells in the concentration range of 3.125～400μM, but in the PC12 cell depression model, the persimmon leaf extract could produce better neuroprotective effect at 50μM, and its mechanism may be It is related to the inhibition of the inflammatory response caused by corticosterone. The results of this example suggest that the persimmon leaf extract has significant antidepressant activity at the cellular level.
实施例2 柿叶提取物对急性应激诱导小鼠抑郁样行为的作用 Example 2 Effect of Persimmon Leaf Extract on Depression-like Behavior in Mice Induced by Acute Stress
2.1实验动物2.1 experimental animals
C57小鼠，雌性，60只，体重18～22g；实验动物许可证号：SCXK(京)2014-0004。C57 mice, female, 60, weighing 18-22 g; experimental animal license number: SCXK (Beijing) 2014-0004.
2.2实验药物与仪器2.2 Experimental drugs and instruments
柿叶提取物(NXQ)、氟西汀(FLX)、羧甲基纤维素钠、敞箱、圆形游泳缸、悬尾仪。Persimmon leaf extract (NXQ), fluoxetine (FLX), sodium carboxymethyl cellulose, open box, round swimming cylinder, and suspension instrument.
2.3实验分组与给药方法2.3 Experimental grouping and administration methods
实验用小鼠适应饲养环境1周后，将50只小鼠随机分为五组，分别为溶剂对照组(Veh)、阳性药氟西汀(FLX,10mg/kg)给药组、柿叶提取物(NXQ)低剂量(20mg/kg)给药组、柿叶提取物(NXQ)中剂量(40mg/kg)给药组、柿叶提取物(NXQ)高剂量(80mg/kg)给药组；每天灌胃给药一次，连续给药7天，溶剂对照组同时给以同体积的0.5％羧甲基纤维素钠水溶液。末次给药后1h，开始行为学检测。另外10只小鼠用于生化指标检测作为正常对照组。One week after the experimental mice were adapted to the feeding environment, 50 mice were randomly divided into five groups: the solvent control group (Veh), the positive drug fluoxetine (FLX, 10 mg/kg), and the persimmon leaf extract. NXQ low dose (20mg/kg) administration group, persimmon leaf extract (NXQ) medium dose (40mg/kg) administration group, persimmon leaf extract (NXQ) high dose (80mg/kg) administration group The drug was administered once a day for 7 days, and the solvent control group was simultaneously given the same volume of 0.5% aqueous sodium carboxymethylcellulose solution. Behavioral testing was started 1 h after the last dose. Another 10 mice were used for biochemical indicators as a normal control group.
药物配制：柿叶提取物或氟西汀均以0.5％羧甲基纤维素钠充分溶解，按给药剂量现配现用，以0.1ml/10g体重给药体积进行灌胃。Drug preparation: Persimmon leaf extract or fluoxetine was fully dissolved in 0.5% sodium carboxymethylcellulose, and was administered at the dose of 0.1 ml/10 g body weight.
2.4实验方法2.4 Experimental methods
2.4.1敞箱实验2.4.1 Open box experiment
敞箱实验主要用于测试小鼠自发活动能力以排除药物中枢兴奋作用干扰实验结果。实验在一个底部为25cm×25cm，高为20cm的黑色木箱中进行，底面为25个正方格。第6天给药后1h，小鼠放入中央方格，适应5min，拿出。每换一只小鼠，用75％酒精擦洗一遍以去除前一只小鼠残留气味的影响。实验第7天给药后1h，摄像记录5min内小鼠穿越格子数(四爪均进入方格内方可计数，为水平运动得分)、后肢直立次数(两前爪腾空或攀附箱壁，为垂直运动得分)。The open-box experiment is mainly used to test the spontaneous activity of mice to rule out the effects of drug central excitatory interference. The experiment was carried out in a black wooden box with a bottom of 25 cm x 25 cm and a height of 20 cm, and the bottom surface was 25 squares. One hour after the administration on the sixth day, the mice were placed in a central square, adapted for 5 min, and taken out. Each mouse was washed with 75% alcohol to remove the residual odor of the previous mouse. On the 7th day after the experiment, 1 hour after the administration, the number of mice crossing the grid within 5 minutes of the imaging recording (the four claws can be counted into the square to count as the horizontal movement score) and the number of hind limbs upright (the two front paws are vacated or climbing the wall) Vertical motion score).
2.4.2悬尾实验2.4.2 Suspension experiment
用胶布将小鼠尾(距尾尖1cm处)粘住后固定于悬尾支架上，使小鼠成倒悬体位，两侧以隔板挡开小鼠视线，使之不互相干扰。摄像记录5min内小鼠的累计不动时间。小鼠“不动”的判定标准为停止挣扎，身体呈垂直倒悬状态，静止不动。The tail of the mouse (1 cm from the tip of the tail) was adhered with a tape and fixed on the tail-suspended stent to make the mouse into an inverted suspension position, and the eyes of the mice were blocked by the partitions on both sides so as not to interfere with each other. The camera recorded the cumulative immobility time of the mice within 5 min. The criterion for the "immobility" of mice is to stop struggling, and the body is vertically suspended and still.
2.4.3强迫游泳实验2.4.3 Forced swimming experiment
在悬尾实验的第二天，将小鼠逐一放入高20cm、直径12cm、水深10cm的圆形游泳缸中进行强迫游泳6min，摄像记录后4min小鼠的累计不动时间。 小鼠“不动”的判定标准为在水中停止挣扎，呈漂浮状态，仅有细小的肢体运动以保持头部漂浮水面。On the second day of the tail suspension experiment, the mice were placed in a circular swimming cylinder of 20 cm in height, 12 cm in diameter and 10 cm in water depth for 6 min, and the cumulative immobility time of the mice 4 min after imaging recording. The criterion for the "immobility" of mice is that they stop struggling in the water and are floating, with only small limb movements to keep the head floating on the water surface.
2.4.4ELISA检测血清皮质酮2.4.4 ELISA for detection of serum corticosterone
小鼠行为学实验结束后摘除眼球取血，抗凝管中静置10min后，8500r/min离心15min，分离血清，-80℃冰箱保存备用。采用酶联免疫吸附(ELISA)法检测小鼠血清中皮质酮水平。同时，另取一组未经任何应激处理的小鼠(10只)作为正常对照组，按照实验组相同的方法，摘除眼球取血，分离血清，测定血清中皮质酮水平。At the end of the mouse behavioral experiment, the eyeballs were removed and the blood was removed. After standing for 10 minutes in the anticoagulation tube, the cells were centrifuged at 8500 r/min for 15 min, and the serum was separated and stored in a refrigerator at -80 °C for use. Serum corticosterone levels were measured by enzyme-linked immunosorbent assay (ELISA). At the same time, another group of mice (10 rats) without any stress treatment was taken as the normal control group. According to the same method of the experimental group, the eyeballs were removed, blood was separated, and serum corticosterone levels were determined.
2.4.5HPLC检测脑组织中单胺类神经递质2.4.5 HPLC detection of monoamine neurotransmitters in brain tissue
2.4.5.1色谱条件2.4.5.1 Chromatographic conditions
色谱柱:Dikma Diamonsil C18(5μm,4.6mm×250mm)；Column: Dikma Diamonsil C18 (5 μm, 4.6 mm × 250 mm);
流动相:A-B(90:10，体积比),A为磷酸二氢钠水溶液(含25.0mmol/L NaH 2PO 4,1.7mmol/L OSA，0.7mmol/L三乙胺，0.025mmol/L EDTA-2Na，pH＝3.0)，B为乙腈； Mobile phase: AB (90:10, volume ratio), A is aqueous sodium dihydrogen phosphate solution (containing 25.0 mmol/L NaH 2 PO 4 , 1.7 mmol/L OSA, 0.7 mmol/L triethylamine, 0.025 mmol/L EDTA) -2Na, pH = 3.0), B is acetonitrile;
流速：1.0mL/min；Flow rate: 1.0 mL/min;
柱温:32℃；Column temperature: 32 ° C;
检测电势:E1＝-150V,E2＝220V；Detection potential: E1=-150V, E2=220V;
进样量10μLInjection volume 10μL
2.4.5.2样品处理2.4.5.2 Sample Processing
取冻存的脑组织,称重后置于玻璃勻浆器，按10mL/kg的比例加入预冷的含有0.01％EDTA-2Na的0.1mol/L高氯酸溶液，于冰浴下快速匀浆2min。将研磨好的匀浆液移入棕色离心管，于4℃条件下14000g离心20min后取上清，上清液用0.22μm滤器过滤后上机检测脑组织中单胺类神经递质5-HT、NE、DA水平。The frozen brain tissue was taken, weighed and placed in a glass homogenizer, and pre-cooled 0.1 mol/L perchloric acid solution containing 0.01% EDTA-2Na was added at a ratio of 10 mL/kg, and rapidly homogenized in an ice bath. 2min. The ground homogenate was transferred to a brown centrifuge tube, centrifuged at 14,000 g for 20 min at 4 ° C, and the supernatant was taken. The supernatant was filtered through a 0.22 μm filter and the monoamine neurotransmitter 5-HT, NE in the brain tissue was detected on the machine. , DA level.
2.5数据分析及统计2.5 Data Analysis and Statistics
用SPSS软件分析数据，方差分析(One-Way ANOVA)。经方差齐性检验，若方差齐性则组间两两比较采用Bonferronic法；若方差不齐将采用Welch法进行分析，多重比较采用Dunnett’s T3法。P＜0.05为存在显著性差异，具有统计学意义。Data was analyzed using SPSS software, and analysis of variance (One-Way ANOVA). After the homogeneity test of variance, if the variance is homogeneous, the Bonferronic method is used for comparison between groups; if the variance is not uniform, the Welch method is used for analysis, and the multiple comparison is performed by Dunnett's T3 method. P < 0.05 was considered to be statistically significant.
2.6实验结果2.6 Experimental results
2.6.1柿叶提取物连续给药对小鼠自主活动的影响2.6.1 Effect of continuous administration of persimmon leaf extract on spontaneous activity of mice
敞箱实验结果见图5和表5。各组小鼠间的水平运动得分(P>0.05)和垂直运动得分(P>0.05)差异均不具有统计学意义，说明柿叶提取物连续给药均不对小鼠的自主活动产生影响。The results of the open box experiment are shown in Figure 5 and Table 5. There was no statistically significant difference in the horizontal exercise score (P>0.05) and vertical exercise score (P>0.05) between the groups, indicating that continuous administration of persimmon leaf extract did not affect the spontaneous activity of the mice.
表5 敞箱实验实验结果
Figure PCTCN2018120186-appb-000007
Table 5 Open box experiment results
Figure PCTCN2018120186-appb-000007
Figure PCTCN2018120186-appb-000008
Figure PCTCN2018120186-appb-000008
Figure PCTCN2018120186-appb-000009
Figure PCTCN2018120186-appb-000009
2.6.2柿叶提取物对悬尾急性应激小鼠行为学的影响2.6.2 Effect of Persimmon Leaf Extract on Behavior of Suspension Tail Acute Stress Mice
结果见图6和表6。柿叶提取物各剂量较模型对照组，均不同程度缩短小鼠悬尾不动时间且作用呈剂量依赖关系；剂量20、40和80mg/kg均效果显著，差异均有统计学意义(P<0.05，P<0.01)。柿叶提取物中剂量(40mg/kg)的效果与氟西汀(10mg/kg)相当，柿叶提取物高剂量(80mg/kg)缩短小鼠悬尾不动时间效果好于氟西汀。The results are shown in Figures 6 and 6. The doses of persimmon leaf extracts were compared with the model control group, and the duration of mice suspension was reduced in a dose-dependent manner. The doses of 20, 40 and 80 mg/kg were significant, and the differences were statistically significant (P< 0.05, P < 0.01). The effect of the dose of persimmon leaf extract (40mg/kg) was similar to that of fluoxetine (10mg/kg). The high dose (80mg/kg) of persimmon leaf extract shortened the time of suspension of mice and was better than that of fluoxetine.
表6 小鼠悬尾实验结果
Figure PCTCN2018120186-appb-000010
Table 6 Results of mouse tail suspension experiment
Figure PCTCN2018120186-appb-000010
Figure PCTCN2018120186-appb-000011
Figure PCTCN2018120186-appb-000011
*P<0.05, **P<0.01, ***P<0.001vs.溶剂对照组. * P<0.05, ** P<0.01, *** P<0.001 vs. solvent control group.
2.6.3柿叶提取物对强迫游泳急性应激小鼠行为学的影响2.6.3 Effects of Persimmon Leaf Extract on Behavior of Mice with Forced Swimming and Acute Stress
小鼠强迫游泳试验结果见图7与表7。柿叶提取物各剂量较模型对照组，均不同程度缩短小鼠强迫游泳不动时间且作用呈剂量依赖关系，其中剂量40mg/kg和80mg/kg效果显著，差异均有统计学意义(P<0.05，P<0.01)。柿叶提取物高剂量(80mg/kg)缩短小鼠强迫游泳不动时间的效果略好于氟西汀(10mg/kg)。本实验结果与悬尾试验结果相似。The results of the forced swimming test in mice are shown in Figures 7 and 7. Compared with the model control group, the doses of persimmon leaf extracts shortened the forced swimming time and the dose-dependent relationship. The doses of 40mg/kg and 80mg/kg were significant, and the differences were statistically significant (P< 0.05, P < 0.01). The high dose (80 mg/kg) of persimmon leaf extract shortened the forced swimming time of mice slightly better than fluoxetine (10 mg/kg). The results of this experiment are similar to those of the tail suspension test.
表7 小鼠强迫游泳实验结果Table 7 Results of forced swimming test in mice
Figure PCTCN2018120186-appb-000012
Figure PCTCN2018120186-appb-000012
Figure PCTCN2018120186-appb-000013
Figure PCTCN2018120186-appb-000013
*P<0.05, **P<0.01, ***P<0.001vs.溶剂对照组. * P<0.05, ** P<0.01, *** P<0.001 vs. solvent control group.
2.6.4柿叶提取物对急性应激小鼠血清皮质酮的影响2.6.4 Effect of Persimmon Leaf Extract on Serum Corticosterone in Acute Stressed Mice
该部分实验，新增正常无应激小鼠血清进行对比，结果见图8和表8。与对照组比较，模型组应激小鼠血清皮质酮水平显著升高(P<0.001)；与模型组比较，柿叶提取物40、80mg/kg组血清皮质酮水平均明显降低，差异具有统计学意义(P<0.01,P<0.001)。而且，柿叶提取物高剂量(80mg/kg)降低急性应激小鼠血清皮质酮水平的作用显著强于氟西汀(10mg/kg)。In this part of the experiment, the serum of normal unstressed mice was added for comparison. The results are shown in Figure 8 and Table 8. Compared with the control group, serum corticosterone levels in the model group were significantly increased (P<0.001). Compared with the model group, the serum corticosterone levels in the 40 and 80 mg/kg groups were significantly lower. Academic significance (P<0.01, P<0.001). Moreover, the high dose of persimmon leaf extract (80 mg/kg) was significantly stronger than that of fluoxetine (10 mg/kg) in reducing serum corticosterone levels in acutely stressed mice.
表8 柿叶提取物对急性应激小鼠血清皮质酮水平的影响
Figure PCTCN2018120186-appb-000014
Table 8 Effect of Persimmon Leaf Extract on Serum Corticosterone Level in Acute Stressed Mice
Figure PCTCN2018120186-appb-000014
Figure PCTCN2018120186-appb-000015
Figure PCTCN2018120186-appb-000015
*P<0.05, **P<0.01, ***P<0.001vs.溶剂对照组， ##P<0.01vs.正常对照组. * P<0.05, ** P<0.01, *** P<0.001 vs. solvent control group, ## P<0.01 vs. normal control group.
2.6.5柿叶提取物对急性应激小鼠脑组织单胺类神经递质的影响Effects of Persimmon Leaf Extract on Monoamine Neurotransmitters in Brain Tissue of Acute Stress Mice
该部分实验，新增正常对照无应激小鼠脑组织进行对比，结果见图9和表9。与正常对照组比较，模型组应激小鼠5-HT、DA、NE水平显著降低(P<0.01)；与模型组应激比较，柿叶提取物20、40、80mg/kg组5-HT水平明显升高，差异具有统计学意义(P<0.05，P<0.01)，柿叶提取物20、40、80mg/kg组DA、NE水平显著升高，差异具有统计学意义(P<0.05,P<0.01)。此外，柿叶提取物高剂量(80mg/kg)升高急性应激小鼠脑组织中5-HT、DA、NE水平的作用强于氟西汀(10mg/kg)。In this part of the experiment, brain tissue of normal control unstressed mice was added for comparison. The results are shown in Figure 9 and Table 9. Compared with the normal control group, the levels of 5-HT, DA and NE in the stress group of the model group were significantly lower (P<0.01). Compared with the stress in the model group, the 5-HT of the persimmon leaf extract 20, 40, 80 mg/kg group The level was significantly increased, the difference was statistically significant (P<0.05, P<0.01). The levels of DA and NE in the persimmon leaf extracts of 20, 40 and 80 mg/kg were significantly increased, and the difference was statistically significant (P<0.05, P < 0.01). In addition, the high dose (80 mg/kg) of persimmon leaf extract increased the levels of 5-HT, DA and NE in the brain tissue of acute stressed mice more strongly than fluoxetine (10 mg/kg).
表9 柿叶提取物对小鼠大脑单胺类神经递质水平的影响
Figure PCTCN2018120186-appb-000016
Table 9 Effect of Persimmon Leaf Extract on Monoamine Neurotransmitter Levels in Mouse Brain
Figure PCTCN2018120186-appb-000016
Figure PCTCN2018120186-appb-000017
Figure PCTCN2018120186-appb-000017
Figure PCTCN2018120186-appb-000018
Figure PCTCN2018120186-appb-000018
*P<0.05, **P<0.01, ***P<0.001vs.溶剂对照组， ###P<0.001vs.正常对照组。 * P<0.05, ** P<0.01, *** P<0.001 vs. solvent control group, ### P<0.001 vs. normal control group.
2.7实验结论2.7 Experimental conclusion
本实施例在实施例1的细胞水平的基础上，进一步采用整体动物实验进行柿叶提取物抗抑郁药效学的评价。小鼠强迫游泳实验(FST)和小鼠悬尾实验(TST)是最为常用的急性行为绝望模型。小鼠在FST和TST中表现的不动状态反映了其在不可逃避的行为绝望中的一种持续性挫败，或者是处理应激刺激时产生的被动承受状态。目前认为这种行为绝望类似于构成临床抑郁症的成分，这种应激状态下动物不动状态持续时间的缩短反应了药物的抗抑郁特性。多数抗抑郁药在小鼠FST和TST中能减少其不动时间，且其药效学显示与临床药效相关。In the present example, based on the cell level of Example 1, the whole animal experiment was further used to evaluate the antidepressant pharmacodynamics of the persimmon leaf extract. The forced swimming test (FST) and the mouse tail suspension test (TST) are the most commonly used models of acute behavioral despair. The immobile state of mice in FST and TST reflects a persistent defeat in indescribable behavioral despair, or a passive state of tolerance when dealing with stress stimuli. It is currently believed that this behavioral despair is similar to the component that constitutes clinical depression. The shortening of the duration of the animal's immobility state under this stress state reflects the antidepressant properties of the drug. Most antidepressants reduce their immobility time in mouse FST and TST, and their pharmacodynamics are shown to be associated with clinical efficacy.
本实施例的结果显示，柿叶提取物连续给药在不影响小鼠自主活动的情况下，可显著性减少小鼠在强迫游泳和悬尾试验中的不动时间；其中柿叶提取物中剂量减少小鼠悬尾不动时间的作用与氟西汀相当，高剂量减少小鼠强迫游泳不动时间和悬尾不动时间的作用好于氟西汀。该结果证实柿叶提取物单独用药即可产生明显的抗抑郁效应。The results of this example show that continuous administration of persimmon leaf extract can significantly reduce the immobility time of mice in forced swimming and tail suspension test without affecting the spontaneous activity of mice; The effect of dose reduction on the time of suspension of mice was comparable to that of fluoxetine. The high dose reduced the duration of forced swimming and the time of suspension of mice was better than that of fluoxetine. This result confirmed that the persimmon leaf extract alone produced a significant antidepressant effect.
急性应激是机体内各种内外环境因素及社会、心里因素刺激时所出现的全身性非特异性适应反应，可以引起大脑功能减退及神经元损害同时在急性应激情况下，机体激活下丘脑-垂体-肾上腺轴(HPA轴)，促使血清中促肾上腺皮质激素和皮质酮(Corticosterone,CORT)的浓度升高，是对抗外来突发事件的一条重要反馈和自我保护途径。在生化指标检测上，柿叶提取物可显著减少急性应激小鼠血清皮质酮的水平，从而可能起到保护神经元的作用。此外，脑组织单胺类神经递质5-HT、DA和NE与抑郁症单胺类假说密切相关，本实施例通过HPLC检测发现，柿叶提取物能及时显著性逆转急性应激小鼠脑组织三类神经递质的下调。这也是柿叶提取物发挥抗抑郁作用的重要机制之一。Acute stress is a systemic non-specific adaptive response that occurs when various internal and external environmental factors and social and psychological factors are stimulated in the body. It can cause brain dysfunction and neuronal damage. In the case of acute stress, the body activates the hypothalamus. The pituitary-adrenal axis (HPA axis), which promotes elevated concentrations of adrenocorticotropic hormone and corticosterone (CORT) in serum, is an important feedback and self-protection pathway against external emergencies. In the detection of biochemical indicators, persimmon leaf extract can significantly reduce the level of serum corticosterone in acutely stressed mice, which may play a role in protecting neurons. In addition, brain tissue monoamine neurotransmitters 5-HT, DA and NE are closely related to the depression monoamine hypothesis. In this example, it was found by HPLC that the persimmon leaf extract can significantly reverse the acute stress mouse brain in time. Tissues are down-regulated in three types of neurotransmitters. This is also one of the important mechanisms by which persimmon leaf extract exerts antidepressant effects.
实施例3 柿叶提取物抗抑郁作用的物质基础的研究 Example 3 Study on the material basis of anti-depressant effect of persimmon leaf extract
在实施例1和2的实验中，本发明已证实柿叶提取物对典型的抑郁障碍具备显著性的作用，但柿叶提取物中含有黄酮类、有机酸、三萜类及香豆素类等多种组分，而黄酮类成分的含量占30％(主要为槲皮素、山柰酚苷元及其单、 双糖苷类如芦丁、金丝桃苷等)，已有报道也提示槲皮素、金丝桃苷具备一定的抗抑郁活性。但是本发明所述的柿叶提取物经HPLC检测显示，槲皮素、金丝桃苷、山柰酚和芦丁的含量均分别不超过1.5％。此外尚不清楚柿叶提取物中的有机酸、三萜类、香豆素等组分是否存在协同增效的可能。为了初步探索柿叶提取物抗抑郁作用的物质基础，本实施例进一步将柿叶提取物与单一的黄酮类化合物(槲皮素、山柰酚、芦丁、金丝桃苷)及它们的组合物进行抗抑郁药效对比，所选的典型抑郁障碍模型为国际认可的慢性不可预知温和应激模型。In the experiments of Examples 1 and 2, the present invention has confirmed that the persimmon leaf extract has a significant effect on typical depressive disorders, but the persimmon leaf extract contains flavonoids, organic acids, triterpenoids and coumarins. And a variety of components, and the content of flavonoids accounted for 30% (mainly quercetin, kaempferol and its mono- and di-glycosides such as rutin, hyperoside, etc.), has also been reported Quercetin and hyperoside have certain antidepressant activity. However, the persimmon leaf extract of the present invention showed by HPLC that the contents of quercetin, hyperoside, kaempferol and rutin were not more than 1.5%, respectively. In addition, it is not clear whether the organic acid, triterpenoids, coumarin and other components in the persimmon leaf extract have synergistic effects. In order to initially explore the material basis of the antidepressant effect of the persimmon leaf extract, the present embodiment further extracts the persimmon leaf extract with a single flavonoid compound (quercetin, kaempferol, rutin, hyperoside) and combinations thereof. The antidepressant efficacy comparison was performed, and the selected typical depressive disorder model was an internationally recognized chronic unpredictable mild stress model.
3.1实验动物3.1 experimental animals
C57小鼠，雄性，80只，体重18～22g，实验动物许可证号：SCXK(粤)2011-0015；实验前小鼠适应饲养环境一周，所有小鼠饲养在温度21±2℃的普通级实验动物房，并遵循昼夜节律。小鼠可自由摄食饮水。C57 mice, male, 80, weighing 18-22 g, experimental animal license number: SCXK (Yue) 2011-0015; mice were adapted to the feeding environment for one week before the experiment, and all mice were kept at the normal level of temperature 21±2 °C. Experiment with animal rooms and follow the circadian rhythm. The mice were given free access to drinking water.
3.2实验药物与仪器3.2 Experimental drugs and instruments
柿叶提取物、槲皮素、山柰酚、芦丁、金丝桃苷、羧甲基纤维素钠、小鼠开场敞箱、电子分析天平(德国Sartrious公司)、冷冻型台式大容量高速离心机5810(德国EPPENDORF公司)、Victor3多功能酶标仪(美国PerkinElmer公司)、超声组织破碎仪(美国Sonic公司)、DYY-Ⅲ型电泳仪(北京六一仪器厂)、垂直板电泳槽(美国Bio-Rad公司)、WD-9405型水平摇床(北京六一仪器厂)、荧光/化学发光成像系统(美国Sonic公司)、Mill-Q超纯水系统(美国Millipore公司)、悬尾仪，鼠尾夹，计数器，温度计，体重秤等。ECl发光液(Invitrogen公司，美国)；脱脂奶粉(广州斯佳生物科技有限公司)；PVDF膜(Milipore公司，美国)；X线感光胶片(Kodak公司，日本)；皮质酮(CORT)酶联免疫分析试剂盒(美国Enzo公司)；ACTH酶联免疫分析试剂盒(美国phoenix pharmaceuticals公司)。Persimmon leaf extract, quercetin, kaempferol, rutin, hyperoside, sodium carboxymethylcellulose, mouse open-box, electronic analytical balance (Sartrious, Germany), frozen desktop high-capacity high-speed centrifugation Machine 5810 (Germany EPPENDORF company), Victor3 multi-function microplate reader (PerkinElmer, USA), ultrasonic tissue disruptor (Sonic, USA), DYY-III electrophoresis (Beijing Liuyi Instrument Factory), vertical plate electrophoresis tank (USA) Bio-Rad), WD-9405 horizontal shaker (Beijing Liuyi Instrument Factory), fluorescent/chemiluminescence imaging system (Sonic, USA), Mill-Q ultrapure water system (Millipore, USA), suspension instrument, Rat tail clips, counters, thermometers, weight scales, etc. ECl luminescent liquid (Invitrogen, USA); skim milk powder (Guangzhou Sijia Biotechnology Co., Ltd.); PVDF film (Milipore, USA); X-ray photographic film (Kodak, Japan); corticosterone (CORT) enzyme-linked immunosorbent assay Analytical kit (Enzo, USA); ACTH enzyme-linked immunoassay kit (phoenix pharmaceuticals, USA).
3.3实验造模与分组给药3.3 Experimental modeling and group administration
实验前适应环境一周，所有小鼠可自由获得食物和水，并遵循昼夜节律，在温度恒定的清洁级环境中饲养，每日人工抚摸，以适应人工操作。适应环境一周后根据小鼠的体重及糖水偏爱实验结果将小鼠分为两组：正常对照组10只、慢性温和不可预测应激(CUMS)组70只，分组后开始进行应激。应激造模方式有：夹尾1min、昼夜颠倒24h、鼠笼倾斜24h、4℃冰水游泳5min、束缚1h、湿笼24h、强迫游泳15min、饮水剥夺24h、食物剥夺24h、电击足底、频闪应激、独笼应激，合笼应激等。在实验开始、应激后第3周进行糖水偏爱实验用以判定造模是否成功，若抑郁症模型未成功，将顺延CUMS应激的时间。造模成功后将模型组分为CUMS模型组(溶剂Veh+CUMS)、柿叶提取物组(NXQ40mg/kg+CUMS)、槲皮素组(4.0mg/kg+CUMS)、金丝桃苷组(4.0mg/kg+CUMS)、山柰酚组(3.0mg/kg+CUMS)、芦丁组(1.5 mg/kg+CUMS)、柿叶黄酮组合物组[(槲皮素4.0mg/kg+金丝桃苷4.0mg/kg+山柰酚3.0mg/kg+芦丁1.5mg/kg)+CUMS]，每组10只小鼠。给药4周后进行行为学测试。其中槲皮素、金丝桃苷、山柰酚、芦丁的给药剂量均为其在柿叶提取物中含量比例的10倍以上，Adapted to the environment for one week before the experiment, all mice were free to obtain food and water, and followed the circadian rhythm, kept in a constant temperature clean environment, and manually touched daily to adapt to manual operation. After one week of adaptation to the environment, the mice were divided into two groups according to the weight of the mice and the results of the sugar-sweet preference experiment: 10 in the normal control group and 70 in the chronically unpredictable stress (CUMS) group, and the stress was started after the grouping. Stress modeling methods include: 1 min tail, 24 h upside down, 24 h of squirrel cage, 5 min of ice swimming at 4 ° C, 1 h of restraint, 24 h of wet cage, 15 min of forced swimming, 24 h of drinking water deprivation, 24 h of food deprivation, electric shock, sole Streak stress, cage stress, cage stress, etc. The syrup preference experiment was conducted at the beginning of the experiment and at the 3rd week after the stress to determine whether the modeling was successful. If the depression model was not successful, the time of CUMS stress would be postponed. After successful modeling, the model components were CUMS model group (solvent Veh+CUMS), persimmon leaf extract group (NXQ40mg/kg+CUMS), quercetin group (4.0mg/kg+CUMS), and hyperoside group. (4.0mg/kg+CUMS), kaempferol group (3.0mg/kg+CUMS), rutin group (1.5 mg/kg+CUMS), persimmon leaf flavonoid composition group [(quercetin 4.0mg/kg+gold) Candidate 4.0 mg/kg + kaempferol 3.0 mg/kg + rutin 1.5 mg/kg) + CUMS], 10 mice per group. Behavioral testing was performed 4 weeks after administration. The dosages of quercetin, hyperoside, kaempferol and rutin are more than 10 times that of the extract of persimmon leaves.
3.4实验方法3.4 Experimental methods
3.4.1糖水偏爱实验3.4.1 syrup preference experiment
实验前，小鼠训练适应1％(w/v)糖水：每笼放置两瓶1％糖水溶液，24h后其中一瓶1％糖水换成纯水，放置24h。适应后，小鼠禁水禁食24h，进行糖水偏爱实验。实验中，每只小鼠自由饮用两瓶水，一瓶为25ml 1％糖水，另一瓶为25ml纯水，实验开始之前称量每瓶水的重量。2h后，测量剩余的重量，得出糖水和纯水的消耗量(g)。用糖水消耗量与总液体消耗量(糖水消耗量+纯水消耗量)之比表示糖水偏爱度，该实验贯穿于实验的整个过程。Before the experiment, the mice were trained to adapt to 1% (w/v) syrup: two bottles of 1% aqueous sugar solution were placed in each cage. After 24 hours, one of the bottles of 1% syrup was replaced with pure water and left for 24 hours. After adaptation, the mice were fasted for 24 h and subjected to a sugar-water preference experiment. In the experiment, each mouse was given two bottles of water freely, one bottle was 25 ml of 1% syrup, and the other bottle was 25 ml of pure water, and the weight of each bottle of water was weighed before the start of the experiment. After 2 h, the remaining weight was measured to obtain the consumption (g) of syrup and pure water. The ratio of the consumption of sugar water to the total liquid consumption (sugar consumption + pure water consumption) indicates the degree of sugar water preference, and the experiment runs through the entire process of the experiment.
3.4.2敞箱实验3.4.2 open box experiment
实验方法同实施例2的“2.4.1敞箱实验”项。The experimental method is the same as the "2.4.1 open box experiment" item of the second embodiment.
3.4.3悬尾实验3.4.3 Suspension experiment
实验方法同实施例2的“2.4.2悬尾实验”项。The experimental method is the same as the "2.4.2 hangover experiment" item of Example 2.
3.4.4强迫游泳实验3.4.4 forced swimming experiment
实验方法同实施例2的“2.4.3强迫游泳实验”项。The experimental method is the same as the "2.4.3 forced swimming experiment" item of the second embodiment.
3.4.5ELISA检测小鼠血清皮质酮和促肾上腺皮质激素的水平3.4.5 ELISA to detect serum corticosterone and adrenocorticotropic hormone levels in mice
实验方法同实施例2的“2.4.4”项，在检测皮质酮的同时，检测促肾上腺皮质激素(ACTH)水平。The experimental method was the same as the "2.4.4" item of Example 2, and the level of adrenocorticotropic hormone (ACTH) was measured while detecting corticosterone.
3.5数据分析及统计3.5 Data Analysis and Statistics
用SPSS软件分析数据，方差分析(One-Way ANOVA)。经方差齐性检验，若方差齐性则组间两两比较采用Bonferronic法；若方差不齐将采用Welch法进行分析，多重比较采用Dunnett’s T3法。P＜0.05为存在显著性差异，具有统计学意义。Data was analyzed using SPSS software, and analysis of variance (One-Way ANOVA). After the homogeneity test of variance, if the variance is homogeneous, the Bonferronic method is used for comparison between groups; if the variance is not uniform, the Welch method is used for analysis, and the multiple comparison is performed by Dunnett's T3 method. P < 0.05 was considered to be statistically significant.
3.6实验结果3.6 Experimental results
3.6.1CUMS小鼠糖水偏爱实验3.6.1 CUMS mouse sugar water preference experiment
结果见图10。与正常对照组比较，模型组(Veh+CUMS)小鼠糖水偏嗜度显著降低(P<0.001)，说明小鼠抑郁造模成功；与模型组比较，柿叶提取物(40mg/kg)、柿叶黄酮组合物组和金丝桃苷组连续灌胃给药后显著升高小鼠糖水偏爱度(P<0.001,P<0.01，P<0.05)，而槲皮素、山柰酚和芦丁虽然能一定程度上提高CUMS小鼠的糖水偏爱度，但作用无统计学意义。与柿叶黄酮组合物组、金丝桃苷组比较，柿叶提取物组升高CUMS小鼠糖水偏爱度的作用具有统计学意义(P<0.01,P<0.05)。The results are shown in Figure 10. Compared with the normal control group, the saccharide preference in the model group (Veh+CUMS) was significantly lower (P<0.001), indicating that the mice were successful in depression modeling; compared with the model group, the persimmon leaf extract (40 mg/kg), Persimmon leaf flavonoid composition group and hyperoside group significantly increased the saccharide preference of mice after continuous intragastric administration (P<0.001, P<0.01, P<0.05), while quercetin, kaempferol and reed Although Ding can improve the syrup preference of CUMS mice to a certain extent, the effect is not statistically significant. Compared with the flavonoid composition group and the hyperoside group, the persimmon leaf extract group had a statistically significant effect on the saccharide preference of CUMS mice (P<0.01, P<0.05).
该结果提示，柿叶提取物在其有效剂量范围内在蔗糖饮水实验上抗抑郁作用明显优于各黄酮和黄酮组合物。The results suggest that the anti-depressant effect of persimmon leaf extract in the sucrose drinking water experiment is significantly better than that of the flavonoid and flavonoid compositions in its effective dosage range.
3.6.2CUMS小鼠敞箱实验3.6.2 CUMS mouse open box experiment
结果见图11。与正常对照组比较，模型组小鼠在敞箱实验中水平活动次数和垂直活动次数均显著性减少(P<0.001)，提示抑郁症模型建立成功，自主活动减少。The results are shown in Figure 11. Compared with the normal control group, the number of horizontal activities and the number of vertical activities in the open-box experiment were significantly decreased in the model group (P<0.001), suggesting that the depression model was established successfully and the voluntary activity was reduced.
与模型组比较，各给药组均能显著增加小鼠水平活动次数，与其它各给药组比较，柿叶提取物组增加水平作用更大，差异具有统计学意义(P<0.05)；各组作用强度排序为：柿叶提取物组(P<0.001)＞柿叶黄酮组合物组、槲皮素组、金丝桃苷组和芦丁组(P<0.01)＞山柰酚组(P＜0.05)(见图11A)。Compared with the model group, each of the drug-administered groups significantly increased the number of horizontal activities in the mice. Compared with the other drug-administered groups, the increase in the level of the persimmon leaf extract group was greater, and the difference was statistically significant (P<0.05). The order of strength of the group was: persimmon leaf extract group (P<0.001)> persimmon leaf flavonoid composition group, quercetin group, hyperoside group and rutin group (P<0.01)> kaempferol group (P <0.05) (see Figure 11A).
与模型组比较，柿叶提取物、柿叶黄酮组合物组连续给药显著性增加小鼠垂直活动次数(P<0.001，P<0.05)；与柿叶黄酮组合物组相比，柿叶提取物增加垂直活动次数作用更大，差异具有统计学意义(P<0.05)。槲皮素、山柰酚、金丝桃苷和芦丁各组虽然小鼠垂直活动次数有所增加，但是与模型组比较，差异没有统计学意义(见图11B)。Compared with the model group, the continuous administration of persimmon leaf extract and persimmon leaf flavonoid composition significantly increased the number of vertical activities in mice (P<0.001, P<0.05). Compared with the persimmon leaf flavonoid composition group, persimmon leaf extraction The effect of increasing the number of vertical activities was greater, and the difference was statistically significant (P<0.05). Although the number of vertical activities in mice increased in quercetin, kaempferol, hyperoside, and rutin, the difference was not statistically significant compared with the model group (see Figure 11B).
该结果提示，柿叶提取物在其有效剂量范围内在敞箱实验上抗抑郁作用明显优于各黄酮和黄酮组合物。The results suggest that the anti-depressant effect of persimmon leaf extract in the open-box experiment is significantly better than that of the various flavonoid and flavonoid compositions in its effective dose range.
3.6.3CUMS小鼠悬尾实验3.6.3 CUMS mouse tail suspension experiment
结果见图12。与正常对照组比较，模型组小鼠在悬尾实验中不动时间显著性增加(P<0.001)，说明造模成功，CUMS小鼠出现抑郁样症状。The results are shown in Figure 12. Compared with the normal control group, the immobility time of the model group mice was significantly increased in the tail suspension experiment (P<0.001), indicating that the modeling was successful and the CUMS mice developed depression-like symptoms.
与模型组比较，连续给药，柿叶提取物极显著地(P＜0.001)、柿叶黄酮组合物和金丝桃苷显著地(P<0.05)减少小鼠悬尾不动时间。其它组与模型组比较差异没有统计学意义。与柿叶黄酮组合物组、金丝桃苷组相比，柿叶提取物减少CUMS小鼠不动时间作用更大，差异具有统计学意义(P<0.05)。Compared with the model group, continuous administration, persimmon leaf extract significantly (P < 0.001), persimmon leaf flavonoid composition and hyperoside significantly (P < 0.05) reduced mouse tail suspension time. There was no statistically significant difference between the other groups and the model group. Compared with the persimmon leaf flavonoid composition group and the hyperoside group, the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the difference was statistically significant (P<0.05).
该结果提示，柿叶提取物在其有效剂量范围内在小鼠悬尾实验上抗抑郁作用明显优于各黄酮和黄酮组合物。The results suggest that the anti-depressant effect of persimmon leaf extract on the tail suspension experiment in mice is significantly better than that of the various flavonoid and flavonoid compositions.
3.6.4CUMS小鼠强迫游泳实验3.6.4 CUMS mice forced swimming experiment
结果见图13。与正常对照组比较，模型组小鼠在强迫游泳实验中不动时间极显著性增加(P<0.001)，说明造模成功，CUMS小鼠出现抑郁样症状。The result is shown in Figure 13. Compared with the normal control group, the immobility time of the model group mice was significantly increased in the forced swimming test (P<0.001), indicating that the modeling was successful and the CUMS mice developed depression-like symptoms.
与模型组比较，连续给药，柿叶提取物极显著地(P＜0.001)、柿叶黄酮组合物(P<0.01)和金丝桃苷(P<0.05)显著地缩短小鼠不动时间。其它组与模型组比较差异没有统计学意义。与柿叶黄酮组合物组、金丝桃苷组相比，柿叶提取物减少CUMS小鼠不动时间作用更大，差异具有统计学意义(P<0.01，P<0.05)。Compared with the model group, continuous administration, persimmon leaf extract significantly (P < 0.001), persimmon leaf flavonoid composition (P <0.01) and hyperoside (P <0.05) significantly shortened the immobility time of mice . There was no statistically significant difference between the other groups and the model group. Compared with the persimmon leaf flavonoid composition group and the hyperoside group, the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the difference was statistically significant (P<0.01, P<0.05).
该结果提示，柿叶提取物在其有效剂量范围内在小鼠强迫游泳实验上抗抑 郁作用明显优于各黄酮和黄酮组合物。The results suggest that the anti-inhibition effect of persimmon leaf extract on the forced swimming test in mice is significantly better than that of the flavonoid and flavonoid compositions.
3.6.5CUMS小鼠血清皮质酮和促肾上腺皮质激素的水平3.6.5 Serum corticosterone and adrenocorticotropic hormone levels in CUMS mice
结果见图14。与正常对照组相比，模型组小鼠血清皮质酮和促肾上腺素皮质激素水平均极显著升高(P<0.001)。The result is shown in Figure 14. Serum corticosterone and adrenergic corticosteroid levels were significantly increased in the model group compared with the normal control group (P < 0.001).
与模型组比较，柿叶提取物能极显著地减少血清皮质酮水平(P<0.001)。柿叶黄酮组合物和金丝桃苷能够显著降低血清中皮质酮水平(P<0.05)。与柿叶黄酮组合物组和金丝桃苷组比较，柿叶提取物降低CUMS小鼠血清皮质酮水平更为显著，差异具有统计学意义(见图14左侧图)。Compared with the model group, persimmon leaf extract significantly reduced serum corticosterone levels (P < 0.001). Persimmon leaf flavonoid composition and hyperoside can significantly reduce serum corticosterone levels (P<0.05). Compared with the persimmon leaf flavonoid composition group and the hyperoside group, the persimmon leaf extract reduced the serum corticosterone level in CUMS mice more significantly, and the difference was statistically significant (see the left panel of Figure 14).
与模型组比较，柿叶提取物能极显著地降低血清促肾上腺皮质激素水平(P<0.01)。其它各组降低促肾上腺皮质激素水平的作用相较模型组没有统计学差异。(见图14右侧图)。Compared with the model group, persimmon leaf extract can significantly reduce serum adrenocorticotropic hormone levels (P<0.01). The effect of lowering adrenocorticotropic hormone levels in the other groups was not statistically different from the model group. (See the right side of Figure 14).
该结果提示，柿叶提取物在其有效剂量范围内降低抑郁小鼠血清中皮质酮和促肾上腺皮质激素的作用优于各黄酮和黄酮组合物。The results suggest that the persimmon leaf extract is superior to the various flavonoid and flavonoid compositions in reducing the serum corticosterone and adrenocorticotropic hormone in the serum of depressed mice in its effective dose range.
3.7结论3.7 Conclusion
在本实施例中，设计了模拟在柿叶提取物中天然存在的部分黄酮组合物，将其与柿叶提取物及各个黄酮成分单体(该部分研究所采用的黄酮单体剂量是其在柿叶提取物中含量的10倍以上)进行了平行的药效比较。令人意料不到的，柿叶提取物的作用在各项实验中都显著强于对照的黄酮组合物和各单体黄酮。In this embodiment, a partial flavonoid composition which is naturally present in the persimmon leaf extract is designed, and the persimmon leaf extract and each flavonoid component monomer (the dosage of the flavonoid monomer used in this part of the study is Parallel drug effects were compared in comparison with the content of persimmon leaf extract in 10 times or more. Unexpectedly, the effect of persimmon leaf extract was significantly stronger in each experiment than the control flavonoid composition and individual monomeric flavonoids.
总之，本发明提供了柿叶提取物新到的医药用途——用于预防和/或治疗典型抑郁障碍。动物试验证明了柿叶提取物具有确切的改善CUMS小鼠抑郁症状和逆转相关的异常生化指标的作用。柿叶提取物发挥抗抑郁的作用机理是通过抑制神经炎症、调节HPA轴功能和单胺类神经递质(5-HT、NE、DA)实现的，因此可以防治非躯体其它问题(如心、脑血管病变等)引起的典型抑郁障碍。In summary, the present invention provides a novel medical use of persimmon leaf extract for the prevention and/or treatment of typical depressive disorders. Animal experiments have demonstrated that persimmon leaf extract has an exact role in improving depressive symptoms and reversing related abnormal biochemical indicators in CUMS mice. The mechanism of anti-depression effect of persimmon leaf extract is achieved by inhibiting nerve inflammation, regulating HPA axis function and monoamine neurotransmitters (5-HT, NE, DA), so it can prevent other non-physical problems (such as heart, Typical depressive disorder caused by cerebrovascular disease, etc.).

Claims (13)

  1. 柿叶提取物在制备用于预防和/或治疗抑郁症的药物中的应用。Application of persimmon leaf extract in the preparation of a medicament for preventing and/or treating depression.
  2. 根据权利要求1所述的应用，其中，所述抑郁症是指典型抑郁障碍，尤其是精神性、情感障碍所致的抑郁障碍。The use according to claim 1, wherein the depression refers to a typical depressive disorder, particularly a depression caused by a mental or affective disorder.
  3. 根据权利要求1或2所述的应用，其中，所述柿叶提取物作为所述药物的唯一活性成分。The use according to claim 1 or 2, wherein the persimmon leaf extract is the sole active ingredient of the drug.
  4. 根据权利要求1至3中任一项所述的应用，其中，所述用于预防和/或治疗抑郁症的药物包括或不包括药学上可以接受的辅料。The use according to any one of claims 1 to 3, wherein the medicament for preventing and/or treating depression includes or does not include a pharmaceutically acceptable excipient.
  5. 根据权利要求1至4中任一项所述的应用，其中，所述用于预防和/或治疗抑郁症的药物为口服制剂或非口服制剂。The use according to any one of claims 1 to 4, wherein the drug for preventing and/or treating depression is an oral preparation or a non-oral preparation.
  6. 根据权利要求5所述的应用，其中，所述口服制剂选自散剂、普通口服片剂、分散片、胶囊剂、软胶囊剂、丸剂、滴丸剂、微丸剂、颗粒剂、口腔崩解片和口腔速溶膜剂中的一种或多种。The use according to claim 5, wherein the oral preparation is selected from the group consisting of a powder, a general oral tablet, a dispersible tablet, a capsule, a soft capsule, a pill, a pill, a pellet, a granule, an orally disintegrating tablet, and One or more of the instant fast acting films.
  7. 根据权利要求5所述的应用，其中，所述非口服制剂选自注射剂、冻干粉针和大容量输液剂中的一种或多种。The use according to claim 5, wherein the non-oral preparation is selected from one or more of an injection, a lyophilized powder, and a large-volume infusion.
  8. 根据权利要求5所述的应用，其中，所述用于预防和/或治疗抑郁症的药物为口服制剂；更优选为脑心清片或脑心清胶囊。The use according to claim 5, wherein the drug for preventing and/or treating depression is an oral preparation; more preferably, it is a brain heart clearing tablet or a brain heart clearing capsule.
  9. 根据权利要求1至8中任一项所述的应用，其中，所述用于预防和/或治疗抑郁症的药物通过如下方法制备：所述将所述柿叶提取物，加入或不加入药学上可以接受的辅料，按照本领域常规的方法，制备得到临床上可以接受的口服制剂或非口服制剂。The use according to any one of claims 1 to 8, wherein the medicament for preventing and/or treating depression is prepared by the method of extracting the persimmon leaf with or without pharmacy The acceptable acceptable excipients are prepared in accordance with conventional methods in the art to provide clinically acceptable oral or parenteral formulations.
  10. 根据权利要求1至9中任一项所述的应用，其中，所述柿叶提取物通过如下方法制备：The use according to any one of claims 1 to 9, wherein the persimmon leaf extract is prepared by the following method:
    取干燥柿叶，加水煎煮2次，每次1～2小时，合并水煎液，滤过，浓缩至相对密度1.12～1.15(60℃)，加乙醇至含醇量达80～90％；静置过夜，滤取上清液，备用；沉淀物用60～70％乙醇洗涤，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取2次以上，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得；Take dried persimmon leaves, add water for 2 times, each time for 1 to 2 hours, combine water decoction, filter, concentrate to a relative density of 1.12 ~ 1.15 (60 ° C), add ethanol to 80-90% alcohol content; After standing overnight, the supernatant was collected by filtration, and the precipitate was washed with 60-70% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and ethanol was added thereto, and an appropriate amount of water was added. Mix and filter, filter the filtrate with ethyl acetate for more than 2 times, combine with ethyl acetate solution, recover ethyl acetate and concentrate into thick paste, dry at low temperature, that is;
    优选地，上述柿叶提取物通过如下方法制备：Preferably, the above persimmon leaf extract is prepared by the following method:
    取干燥柿叶，加水煎煮2次，第1次2小时，第2次1小时，合并滤液，滤过，浓缩汁相对密度1.12～1.15(60℃)，加乙醇至含醇量达85％；静置过夜，滤取上清液，备用；沉淀物用65％乙醇洗涤2次，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤 液用醋酸乙酯提取4次，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, the first 2 hours, the second time 1 hour, combine the filtrate, filter, the relative density of concentrated juice is 1.12~1.15 (60 °C), add ethanol to 85% alcohol content After standing overnight, the supernatant was collected by filtration, and the precipitate was washed twice with 65% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and the ethanol was recovered, and the appropriate amount of water was added. , mixing, filtration, the filtrate was extracted with ethyl acetate 4 times, combined with ethyl acetate solution, ethyl acetate was recovered and concentrated into a thick paste, dried at low temperature, that is.
  11. 一种预防和/或治疗抑郁症的方法，该方法包括向有需要的患者施用柿叶提取物的步骤；A method for preventing and/or treating depression, the method comprising the step of administering a persimmon leaf extract to a patient in need thereof;
    优选地，所述柿叶提取物通过如下方法制备：Preferably, the persimmon leaf extract is prepared by the following method:
    取干燥柿叶，加水煎煮2次，每次1～2小时，合并水煎液，滤过，浓缩至相对密度1.12～1.15(60℃)，加乙醇至含醇量达80～90％；静置过夜，滤取上清液，备用；沉淀物用60～70％乙醇洗涤，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取2次以上，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得；Take dried persimmon leaves, add water for 2 times, each time for 1 to 2 hours, combine water decoction, filter, concentrate to a relative density of 1.12 ~ 1.15 (60 ° C), add ethanol to 80-90% alcohol content; After standing overnight, the supernatant was collected by filtration, and the precipitate was washed with 60-70% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and ethanol was added thereto, and an appropriate amount of water was added. Mix and filter, filter the filtrate with ethyl acetate for more than 2 times, combine with ethyl acetate solution, recover ethyl acetate and concentrate into thick paste, dry at low temperature, that is;
    更优选地，所述柿叶提取物通过如下方法制备：More preferably, the persimmon leaf extract is prepared by the following method:
    取干燥柿叶，加水煎煮2次，第1次2小时，第2次1小时，合并滤液，滤过，浓缩汁相对密度1.12～1.15(60℃)，加乙醇至含醇量达85％；静置过夜，滤取上清液，备用；沉淀物用65％乙醇洗涤2次，合并洗涤液，静置过夜，滤取上清液，与备用上清液合并，回收乙醇，加入适量水，混匀，滤过，滤液用醋酸乙酯提取4次，合并醋酸乙酯液，回收醋酸乙酯并浓缩成稠膏，低温干燥，即得。Take dried persimmon leaves, add water for 2 times, the first 2 hours, the second time 1 hour, combine the filtrate, filter, the relative density of concentrated juice is 1.12~1.15 (60 °C), add ethanol to 85% alcohol content After standing overnight, the supernatant was collected by filtration, and the precipitate was washed twice with 65% ethanol, and the washing liquid was combined, allowed to stand overnight, and the supernatant was collected by filtration, combined with the supernatant, and the ethanol was recovered, and the appropriate amount of water was added. , mixing, filtration, the filtrate was extracted with ethyl acetate 4 times, combined with ethyl acetate solution, ethyl acetate was recovered and concentrated into a thick paste, dried at low temperature, that is.
  12. 根据权利要求11所述的方法，其中，所述抑郁症是指典型抑郁障碍，尤其是精神性、情感障碍所致的抑郁障碍；The method according to claim 11, wherein said depression refers to a typical depressive disorder, especially a depression caused by mental and affective disorders;
    优选地，所述有需要的患者为有需要的哺乳动物，更优选为有需要的人。Preferably, the patient in need is a mammal in need thereof, more preferably a human in need thereof.
  13. 根据权利要求11或12所述的方法，其中，所述方法包括向有需要的人施用柿叶提取物2～10mg/kg体重/天的步骤。The method according to claim 11 or 12, wherein the method comprises the step of administering a persimmon leaf extract of 2 to 10 mg/kg body weight/day to a person in need thereof.
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