AU2018386145A1 - New medical use of persimmon leaf extract and of preparation of persimmon leaf extract - Google Patents

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

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NEW MEDICAL USE OF PERSIMMON LEAF EXTRACT AND OF PREPARATION OF PERSIMMON LEAF EXTRACT TECHNICAL FIELD

The present invention belongs to the medical field, and specifically relates to a new medical use of a persimmon leaf extract and of the preparation of persimmon leaf extract.

BACKGROUND

Depression, also known as depressive disorder, is a chronic and recurrent affective and mental disease, with a significant and persistent mood or emotion stepping down as the main clinical character, often accompanied by symptoms such as anxiety, inhibition of thought, delusions or hallucinations, attention and memory decline, and sleep disorders. So far, the etiology of depression is still unclear. It is generally believed that many factors such as biological, psychological and social environments are involved in the pathogenesis of depression. At present, the incidence of depression in the world is as high as 21%, and about % of patients have suicidal tendencies. The World Health Organization (WHO) predicts that depression will become the second largest clinical chronic disease after hypertension in 2020, which is considered to be a "cold" in psychiatry. According to the survey, only 5% of patients having depression in China have received treatment, while most patients fail to be diagnosed and treated in time due to lack of awareness of depression, leading to worsening of their condition. With the acceleration of life rhythm and the increase of work pressure in life, depression has become one of the common diseases that seriously threaten human health. The fifth edition of the American Diagnostic and Statistical Manual of Mental Disorders (DSM-5) classifies depression into 8 subtypes including destructive mood dysregulation disorder, major depressive disorder (including single episode and recurrent episode), persistent depressive disorder (dysthymia), premenstrual dysphoric disorder, substance / medication-induced depressive disorder, depressive disorder due to another medical condition, other specific depressive disorder and unspecified depressive disorder. Major depressive disorder represents the classic subtype of depressive disorders, which is generally regarded as depression by modern medicine. It is characterized by discrete episodes of at least 2 weeks' duration involving clear-cut changes in affect, cognition, and neurovegetative functions , but not caused by other physical problems (such as malignant tumors, cerebrovascular diseases, and the like). The concept of vascular depression proposed in 1977 comes from the study of senile depression. It is a depression syndrome in old age which associated with cerebrovascular disease or vascular risk factors. Patients having vascular depression are less likely to have typical feelings of loss and delusion of sin, and they have mild depressive symptoms, but they are more likely to be accompanied by attention deficits, indifference, motor delays, and cognitive impairments mainly in executive function. Therefore, vascular depression is different from the above-mentioned typical depressive disorder. With the alleviation of vascular injury, the depressive symptoms of vascular depression will also be relieved. Drugs are currently the main treatment for depression. Antidepressants commonly used in clinical practice include selective serotonin (5-HT) reuptake inhibitors (SSRIs), serotonin-norepinephrine (NE) reuptake inhibitors (SNRIs), tricyclics antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs) and other chemical drugs. Because the etiology of depression has not been studied clearly, there are large differences in the efficacy of these chemical drugs for different subtypes of depression, and there are also problems to be solved such as delayed onset (3-8 weeks), more side effects, low efficiency (50% -70%), high recurrence rate (35% - 60%), low dependence and high price, and the like. The main side effects of the chemical drugs include gastrointestinal symptoms (nausea, diarrhea, stomach bleeding, indigestion), liver toxicity, weight gain and metabolic abnormalities, cardiovascular problems (heart rate, prolonged QT interval, hypertension, orthostatic hypotension), urinary system symptoms (urinary retention, urinary incontinence), sexual dysfunction, hyponatremia, osteoporosis and fracture risk, bleeding, central nervous system disorders (declined epilepsy threshold, extrapyramidal side effects, cognitive impairment), sweating, sleep disorders, emotional symptoms (indifference, mood-incongruence, abnormal reactions), eye symptoms (glaucoma, cataracts), hyperprolactinemia. Therefore, in order to seek safer, more effective, lower toxicity and more economic antidepressants, more and more researchers are turning their attention to natural plants with abundant resources. Persimmon leaf is a fresh or dried leaf of Diospyros kaki Thunb, a plant of Diospyros of Ebenaceae family. It can be used as a medicament, as first described "the frosted leaves can be used for bloated sores" in the "Dian Nan Ben Cao" in Ming Dynasty. It can be used for "treating cough and vomiting blood, quenching thirst and regenerating body fluid" as described in "Ben Cao Zai Xin". With deeper research on the chemical composition, it has been found that persimmon leaves contain a variety of active ingredients and nutrients such as flavonoids, organic acids, coumarins and triterpenes, which have a good preventive effect on cardiovascular and cerebrovascular diseases. The persimmon leaf extract, now listed in the "Pharmacopoeiaof the People's Republic of China", is made from persimmon leaves as raw materials, prepared by hot water extraction and ethanol precipitation, and further purified with ethyl acetate extraction and other methods. The persimmon leaf extract mainly functions to promote blood circulation and remove blood stasis, and is mainly used to treat diseases caused by qi stagnation and blood stasis. Naoxinqing tablets are oral tablets made from persimmon leaf extract as raw materials, and are used for treating cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral atherosclerosis. Earlier studies have shown that flavonoids may be the material basis for persimmon leaf extracts to exert their medicinal effects. In the "Pharmacopoeia of the People's Republic of China", Quercetin and Kaemferol are used as the quality control standard for the total flavonoids of persimmon leaf extracts, which stipulates that the total content should not be less than 8.6%;.However, flavonoids account for only a small part of persimmon leaf extract, and it is apparent that most of the chemical composition of the extract is still unclear. YAN Caiying, et al. reported that venlafaxine were combined with ligustrazine-hydrochloride, ginkgo dipyridolum and Naoxinqing tablet in treatment of senile depression in "Clinical study of venlafaxine combined with traditional Chinese medicine in the treatment of senile depression" (YAN Caiying, PEI Yu, et al; Journal of International Psychiatry, 2016, 43 (5): 842-845). During 8 weeks' treatment, the total clinical effective ratio, HAMD24 total score and anxiety/somatization factor scores of the observation group who were simultaneously given venlafaxine, ligustrazine hydrochloride (intravenous infusion), ginkgo dipyridolum (intravenous infusion), and Naoxinqing tablets (oral) were better than the control group of venlafaxine alone only after 1 week's amd 2 week's treatment; however, with the treatment lasted, the therapeutic advantage of the observation group gradually weakens, the differences from the control group were not significant. This article indicates that compared to with the single use of Venlafaxine treatment, ligustrazine hydrochloride, ginkgo dipyridolum, and Naoxinqing tablets combined with venlafaxine to relieve the anxiety and depression symptoms caused by senile depression only effects faster in short-term treatment, but has no advantages in the long-term treatment. Therefore, based on this study, it may be speculated that the combination of ligustrazine hydrochloride, ginkgo dipyridolum, and Naoxinqing tablets, which promote blood circulation and remove blood stasis, cannot significantly improve the senile depression. WANG Jinyu et al. also reported the efficacy of Naoxinqing tablets combined with escitalopram in the treatment of vascular depression (WANG Jinyu, HE Ying, et al.; Clinical study of Naoxinqing tablets combined with escitalopram in the treatment of vascular depression [J]; Journal of New Chinese Medicine, 2014, 46 (2): 47-49). The inclusion criteria for this clinical study were: "Depression with clinical and/or laboratory evidence with cerebrovascular disease and vascular risk factors, or within 6-12 months after the occurrence of cerebrovascular events; Hamilton Depression Scale (HAMD, 24 items) score >20." Obviously, the subjects treated in this study were the aforementioned "depressive disorder due to another medical condition", instead of "major depressive disorder". After 8 weeks' observation and treatment, it was found that although the group of Naoxinqing tablets combined with escitalopram showed greater decrease in HAMD score, there was no significant difference in the effective rate compared with the group of escitalopram alone (P>0.05). Therefore, escitalopram is still the main factor in the treatment of depression. The role of Naoxinqing tablets in the combination should be more directly in improving cerebrovascular disease, which may help escitalopram to improve depressive symptoms. According to reports in the prior art, the persimmon leaf extract may have a certain degree of auxiliary improvement on vascular depression or senile depression (in fact, there is no clear evidence to prove). However, so far there has been no report that persimmon leaf extract can be used to treat major depressive disorder that is not related to another medical condition.

SUMMARY In view of the deficiencies of the prior art, the object of the present invention is to provide a new medical use of a persimmon leaf extract for the prevention and/or treatment of depression, especially major depressive disorder. In order to achieve the above object of the invention, the present invention adopts the following technical solutions: Use of a persimmon leaf extract in preparing drugs for prevention and/or treatment of depression. Preferably, the depression refers to major depressive disorder, especially depressive disorders caused by mental and affective disorders. Preferably, the persimmon leaf extract is the only active ingredient of the drugs for prevention and/or treatment of depression. Preferably, the drugs for the prevention and/or treatment of depression include or exclude pharmaceutically acceptable excipients. Further preferably, the drug for prevention and/or treatment of depression is an oral preparation or a non-oral preparation. The oral preparation is one or more selected from the group consisting of powders, ordinary oral tablets, dispersible tablets, capsules, soft capsules, pills, dripping pills, pellets, granules, orally disintegrating tablets, and oral fast dissolving films. The non-oral preparation is one or more selected from the group consisting of injections, lyophilized powder for injection and large volume injections. Preferably, the drug for the prevention and/or treatment of depression is an oral preparation. As a preferred embodiment, the drug for prevention and/or treatment of depression is Naoxinqing tablets or Naoxinqing capsules.

The persimmon leaf extract, with or without the addition of pharmaceutically acceptable excipients, can be prepared according to conventional methods in the art to obtain the above drugs for prevention and/or treatment of depression. The above persimmon leaf extract is prepared by the following method: The dried persimmon leaves was decocted with water twice, for 1 to 2 hours each time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of -90%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed with 60-70% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate more than 2 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature. Preferably, the above persimmon leaf extract is prepared by the following method: The dried persimmon leaves was decocted with water twice, for 2 hours for the first time and 1 hour for the second time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 85%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed twice with 65% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate 4 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature. The subject of administration of the drug for the prevention and/or treatment of depression according to the present invention is a mammal, preferably a human. When the drug for the prevention and/or treatment of depression according to the present invention is administered to a human in need, the dosage of the persimmon leaf extract is 2 to 10 mg/kg body weight per day. The present invention also provides a method for preventing and/or treating depression, comprising the step of administering a persimmon leaf extract to a patient in need; Preferably, the persimmon leaf extract is prepared by the following method: The dried persimmon leaves was decocted with water twice, for 1 to 2 hours each time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of -90%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed with 60-70% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate more than 2 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature. Preferably, the above persimmon leaf extract is prepared by the following method: The dried persimmon leaves was decocted with water twice, for 2 hours for the first time and 1 hour for the second time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 85%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed twice with 65% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate 4 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature. Preferably, the depression refers to major depressive disorder, especially depressive disorders caused by mental and affective disorders. The patient in need is a mammal in need, preferably a human in need. The method for preventing and/or treating depression comprises the step of administering the persimmon leaf extract to a human in need with 2 to 10 mg/kg body weight /day. The above pharmaceutically acceptable excipients include but are not limited to: Diluent: for example, one or more selected from the group consisting of starch, dextrin, pregelatinized starch, lactose, microcrystalline cellulose, calcium sulfate, calcium hydrophosphate, calcium carbonate, magnesium oxide, magnesium carbonate, aluminum hydroxide gel, $-cyclodextrin, mannitol, sorbitol, methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, and hydroxymethyl cellulose sodium. Binder: for example, one or more selected from the group consisting of distilled water, ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, and hydroxypropyl methylcellulose. Lubricant: for example, one or more selected from the group consisting of magnesium lauryl sulfate, polyethylene glycol, micronized silica gel, magnesium stearate, and talc. Disintegrant: for example, one or more selected from the group consisting of starch (corn, potato), microcrystalline cellulose, alginic acid, sodium alginate, ion exchange resin, effervescent acid-base system, hydroxypropyl starch, sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, crospovidone, carboxymethyl cellulose, calcium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, partially alpha starch, and microcrystalline cellulose. Film-forming materials: one or more of gelatin, shellac, acacia, 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, one or more selected from the group consisting of water for injection, sesame oil, tea oil, peanut oil, corn oil, olive oil, cottonseed oil, soybean oil, castor oil and peach kernel oil, ethyl oleate, benzyl benzoate, propylene glycol, polyethylene glycol 400, dimethylacetamide (DMA), ethanol, and glycerin. It has been found that the persimmon leaf extract has a good antidepressant effect by the study of the cell models in vitro and the overall animal model according to the present invention. Through mechanism research, it is believed that the persimmon leaf extract of the present invention plays a role in preventing and/or treating depression by the following ways: 1) Anti-neuroinflammation, which can inhibit the release of neuroinflammation factors such as TNF-a, IL-IP, IL-6 in the brain. 2) It can regulate the hypothalamic-pituitary-adrenal axis and can inhibit the secretion of serum corticosterone (cortisol) and adrenocorticotropic hormone induced by stress.

3) It can regulate the balance of monoamine neurotransmitters in the brain, including serotonin, dopamine, and norepinephrine, and plays a role in the regulation of mental emotion. Although it has been disclosed that the persimmon leaf extract contains quercetin, kaempferol and hyperin and other flavonoid components in the prior art, and these components have been reported to have certain antidepressant effects, such as b BEI Weijian, WANG Deqin, et al., Determination of the content of hyperin in Naoxining tablet extract by HPLC [J]. Journal of Guangxi Traditional Chinese Medical University, 2009(02):62-63; 0 BEI Weijian, LUO Jie et al., Determination of quercetin and kaempfetol in Diospyros kaki leaf extract by HPLC [J]. Chinese Traditional and Herbal Drugs, 2005(07):59-60; 1 CHUAN Juanjuan, LI Yan, Research review of Hypericum perforatum (St. John's wort) [J]. Northwest Pharmaceutical Journal, 2016(03):330-332; a CHEN Qingyun, GAN Xin, The protective effect of quercetin on the cultured pcl2 cells lesioned by corticosterone [J]. Chemistry & Bioengineering, 2009(01):47-49; 5 LIU Jianxiang, FANG Yinquan, et al., Synergic antidepressive effect of quercetin and hypericum perforatum extract in mice [J]. Journal of Zhejiang University (Medical Sciences), 2013(06):615-619; CHEN Lei, Study on antidepressant effect of flavonoids [J]. Jiangxi Journal of Traditional Chinese Medicine, 2011, 42(10):55-57. But unexpectedly, through experiments of the present invention, it has been found that the anti-depressant effect of persimmon leaf extract is not only significantly stronger than flavonoid monomers such as quercetin, kaempferol, hyperin, and rutin, but also significantly stronger than the composition of these flavonoids (simulating the combination of flavonoids naturally present in persimmon leaf extract). It indicates that the material basis of persimmon leaf extract for antidepressant effect is not only quercetin, kaempferol, hyperin, rutin and combinations thereof; it is probably other components that have not been reported but have antidepressant activity. .

The preparation with the persimmon leaf extract as the active ingredient, such as Naoxinqing tablets, has been used clinically for many years, and its safety has been proven. The present invention provides a new medical use for the persimmon leaf extract. Based on the present invention, a new and safe treatment choice will be provided for clinical treatment of major depression.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described in detail below with reference to the drawings. Figure 1 shows the cell survival rate of normally cultured PCi2 cells detected by MTT method after incubation with different concentrations of persimmon leaf extracts (3.125-400 pg/mL) for 24 h in Example 1. Figure 2 shows the cell survival rate of normally cultured PCl2 cells detected by MTT method (Figure 2A) and the LDH activity of the cells detected by LDH method (Figure 2B) after incubation with different concentrations of corticosterone (Cort, -800 pM) for 24 h in Example 1; in the Figure, *P< 0.05, **P<0.01,***P< 0.001 vs. Cort (0 pM) group (normal control group). Figure 3 shows the protective effects of different concentrations of persimmon leaf extracts (6.25, 12.5, 25, 50, 100 pg/mL) on corticosterone (Cort, 200 pM)-injured PC12 cells in Example 1, wherein Figure 3A shows the effect on cell survival rate, 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<0.01 vs. Veh group (normal control group). Figure 4 shows the effects of different concentrations of persimmon leaf extracts (6.25, 12.5, 25, 50, 100 pg/mL) on the secretion of inflammatory factors (TNF-a, IL- IPand IL-6) of PC12 cells injured by corticosterone (Cort, 200 pM); in the fugure, *P< 0.05,**P<0.01 ,*P < 0.001 vs. Cort group (corticosterone group), ##P<0.01 vs. Control group (normal control group). Figure 5 shows the results of the open field test in Example 2, wherein Figure A shows the horizontal exercise scores of the mice in each group, and Figure 5B shows the vertical exercise scored of the mice in each group. Figure 6 shows the results of the tail suspension test in Example 2, in the Figure, P < 0.05, *P<0.01,*P < 0.001 vs. Veh (Vehicle control group). Figure 7 shows the results of the forced swim test in mice in Example 2, in the Figure, *P< 0.05, **P<0.01,***P < 0.001 vs. Veh (Vehicle control group). Figure 8 shows the measurement results of the effect of persimmon leaf extract on serum corticosterone levels of the mice with acute stress in Example 2, in the figure, *P< 0.05, **P<0.01,***P< 0.001 vs. Veh2 (Vehicle control group); ##P < 0.01 vs. Vehl (Normal control group). Figure 9 shows the measurement results of the effect of persimmon leaf extract on the level of monoamine neurotransmitters in the brain of the mice with acute stress in Example 2. Among them, Figure 9A shows the measurement result of serotonin (5-HT), Figure 9B shows the measurement result of dopamine (DA), and Figure 9C shows the measurement result of norepinephrine (NE). In the Figure, *P < 0.05, **P<0.01, ***P < 0.001 vs. Veh (Vehicle control group); ###P < 0.01 vs. Normal (Normal control group). Figure 10 shows the results of the scurose preference test in Example 3. In the Figure, 1 represents the normal control group, 2 represents the model group, represents the persimmon leaf extract, 4 represents the persimmon leaf flavonoid composition, 5 represents quercetin, 6 represents kaempferol, 7 represents hyperin, and 8 represents rutin; *P< 0.05, **P<0.01,***P< 0.001 vs. Model control group; ###P < 0.01 vs. Normal control group; $P<0.05,$$P<0.01vs. Persimmon leaf extract. Figure 11 shows the results of the open field test in Example 3; wherein Figure 1lA shows the horizontal exercise scores of the mice, and Figure 11B shows the vertical exercise scores of the mice. In the Figure, 1 represents the normal control group, 2 represents the model group, represents the persimmon leaf extract, 4 represents the persimmon leaf flavonoid composition, 5 represents quercetin, 6 represents kaempferol, 7 represents hyperin, and 8 represents rutin; *P < 0.05, **P<0.01,***P < 0.001 vs. Model control group; ###P < 0.01 vs. Normal control group; $P<0.05,$$P<0.01vs. Persimmon leaf extract. Figure 12 shows the results of the tail suspension test in Example 3. In the Figure, 1 represents the normal control group, 2 represents the model group, represents the persimmon leaf extract, 4 represents the persimmon leaf flavonoid composition, 5 represents quercetin, 6 represents kaempferol, 7 represents hyperin, and 8 represents rutin; *P< 0.05, **P<0.01,***P< 0.001 vs. Model control group; ###P < 0.01 vs. Normal control group; $P<0.05,$$P<0.01vs. Persimmon leaf extract. Figure 13 shows the results of the forced swimming test of the mice in Example 3. In the Figure, 1 represents the normal control group, 2 represents the model group, represents the persimmon leaf extract, 4 represents the persimmon leaf flavonoid composition, 5 represents quercetin, 6 represents kaempferol, 7 represents hyperin, and 8 represents rutin; *P< 0.05, **P<0.01,***P< 0.001 vs. Model control group; ###P < 0.01 vs. Normal control group; $P<0.05,$$P<0.01vs. Persimmon leaf extract. Figure 14 shows the measurement results of the levels of corticosterone and adrenocorticotropic hormone in the serum of CUMS mice in Example 3; wherein, the measurement results of serum corticosterone level is shown on the left panel of the Figure, and the measurement results of serum adrenocorticotropic hormone level is shown on the right panel of the Figure. 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.

DETAILED DESCRIPTION

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only for illustrating the present invention, and they do not limit the scope of the present invention in any way. Unless otherwise indicated, the experimental methods in the following examples are conventional methods. Unless otherwise indicated, the medicinal materials and reagent materials used in the following examples are all commercially available products. Among them, some reagents and instruments are purchased as follows: Persimmon Leaf Extract (NXQ): prepared in Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Company Limited, Batch No. H16P006, the preparation method was: The dried persimmon leaves was decocted with water twice, for 2 hours for the first time and 1 hour for the second time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 85%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed twice with 65% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate 4 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature. Fluoxetine, corticosterone, thiazole blue (MTT), dimethyl sulfoxide (DMSO): Sigma; DMEM high glucose medium, Australian fetal bovine serum, horse serum: Gibco; PC12 cells: rat derived, Chinese Academy of Sciences Cell Bank; LDH test kit: Nanjing Jiancheng Biological Technology Co., Ltd.; Inflammatory factor ELISA test kit: Sangon Biotech (Shanghai) Co., Ltd.; Cell incubator: Thermo; Microplate reader: Bio-rad; Open field box: Shanghai XinRuan Information Technology Co., Ltd.;

Round swimming tank: Shanghai Mobile Datum Information Technology Co., Ltd.; Tail suspension apparatus: Shanghai XinRuan Information Technology Co., Ltd.

Example 1 Protective effect of the persimmon leaf extract on corticosterone-injured nerve cells in vitro 1.1 Preparation of drugs for experiment The persimmon leaf extract was dissolved in 100% DMSO and formulated into a 400 mg/mL stock solution, which was diluted to 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 pg/mL when used with the concentration of DMSO less than 0.1% which was suitable for cell experiments. 1.2 Data analysis and statistics All data were represented as mean standard deviation (x SD), SPSS software was used to analyze the data and calculate variance (One-Way ANOVA). After the homogeneity test of variance, if the variance was homogenous, the Bonferronic method would be used to compare the two groups; if the variance was unequal, the Welch method would be used for analysis, and the Dunnett's T3 method would be used for multiple comparisons. P<0.05 indicated that there was a significant difference, which was statistically significant. 1.3 Effect of different concentrations of the persimmon leaf extract on the survival rate of PC12 cells Normally cultured PC12 cells were incubated with 8 different concentrations of ethyl acetate extract (3.125-400 pg/mL) of persimmon leaves for 24 h, and then the survival rate of PC12 cells was detected by MTT method. The results of MTT showed that (see Figure 1 and Table 1), compared with the normal group, there was no significant difference in cell survival rate among groups treated by different concentration of the persimmon leaf extract. By one-way ANOVA analysis, the difference was not statistically significant (P>0.05). Thus, the selected concentration range of the persimmon leaf extract was suitable for the subsequent screening of antidepressant activity at the cellular level. Table 1 Effect of the persimmon leaf extract (NXQ) on the survival rate of normally cultured PC12 cells Groups Dosage ( g/nL) Sample No. Cell survival rate(%) Normal control group - 6 100 ±7.277 NXQ 3.125 6 97.450 ±3.091

Groups Dosage ( g/nL) Sample No. Cell survival rate (%) NXQ 6.25 6 97.728 ±5.399 NXQ 12.5 6 96.430 ±6.308 NXQ 25 6 99.397 ±4.833 NXQ 50 6 96.708 ±3.634 NXQ 100 6 97.960 ±4.725 NXQ 200 6 97.728 ±3.141 NXQ 400 6 97.311 ±7.794

1.4 Establishment of corticosterone-injured nerve cells model After cultured to the logarithmic growth phase, the cells were resuspended in DMEM medium containing 5% fetal bovine serum and 10% horse serum (containing 200 kU/L penicillin sodium, 100 mg/L streptomycin, pH 7.4), and the cell density was adjusted to 1x105 cells/mL. Cells were seeded in 96-well plates with 100 pL of cell solution each well, and placed in a C02 incubator at 37°C for adherent incubation for 24 h. The experiment was started immediately after the cells covered the bottom of the well. After 1 h of cell serum deprivation, different concentrations of corticosterone (0, 25, 50, 100, 200, 400 M) was added. After 24 h, the injury concentration of corticosterone was observed by MTT method and LDH detection method, and the optimal modeling concentration of corticosterone was selected. The results were shown in Figure 2 and Table 2. 0-800 pM of corticosterone injured PC12 cells in a dose-dependent manner. One-way ANOVA analysis showed that, viability of PC12 cells significantly decreased (P<0.01) after treated with 200 pM of corticosterone for 24 h, and the cell survival rate decreased by 30% (Figure 2A). The same is true in LDH detection, wherein after treatment of different concentrations of corticosterone, there were significant differences in LDH leakage among the groups (P<0.01, Figure 2B). When the concentration of corticosterone was 200 pM, the LDH leakage of the cells increased significantly after 24 h compared with normal control group. Based on the above results, 200 pM of corticosterone treating PC2 cells for 24 h could be used as the corticosterone-induced cell concentration in subsequent experiments.

Table2 Effects of corticosterone (Cort) onPC12 cell survival rate and LDH release Groups Dosage (PM) Sample No. Cell survival rate(%) Supernatant LDH Normal control 0 6 100 ±3.693 100 ±21.119 group

Groups Dosage (PM) Sample No. Cell survival rate (%) Supernatant LDH Cort 25 6 89.729± 2.942 119.025 ±20.491 Cort 50 6 83.887±2.452 125.916 ±23.867

Cort 100 6 79.136 ±2.464 147.798 ± 15.871 Cort 200 6 60.778 2.891** 201.647 33.261** Cort 400 6 53.183 2.733*** 262.286 26.984*** Cort 800 6 41.744 2.562*** 304 ±28.839***

*P< 0.05, **P<0.01,***P< 0.001 vs. Normal control group.

1.5 Protective effect of different concentrations of the persimmon leaf extract on the corticosterone-injured PC12 cells After the cells were seeded in 96-well plate for12 h, the cells were pretreated with different concentrations of the persimmon leaf extract (3.125-400 pg/mL) for 1 h. Except the control wells, 200 pM corticosterone was added to all the wells and co-incubated for 24 h. The cell supernatant was collected to detect the change of lactate dehydrogenase (LDH) activity. The cell survival rate in 96-well plate was detected by the MTT method. The absorbance of each well was detected by microplate reader at 570nm. The average absorbance of the control group was 100%, and the change of cell viability and LDH was calculated by the ratio of the absorbance value of each treatment group to the control group. The results were shown in Figure 3 and Table 3. One-way ANOVA analysis showed that after injured by 200 pM of corticosterone for 24 h, the cell viability of PC12 cells decreased significantly while the LDH secretion in cell supernatant increased significantly. From 6.25 to 100 pg/mL, the persimmon leaf extract increased cell survival rate and decreased LDH secretion in supernatant in a concentration-dependent manner, wherein 25, 50, and 100 pg/mL of the persimmon leaf extract had significant resistance to corticosterone-induced cell injury (P<0.01). The results suggested that the 25 pM ethyl acetate extract of persimmon leaves already exhibited significant antidepressant activity at the cellular level.

Table 3 Effect of the persimmon leaf extract on cell survival rate and lactate dehydrogenase changes indcued by corticosterone Groups Dosage Sample No. Cell survival rate(%) Supernatant LDH Veh - 6 100 ±3.411 537.833± 78.451

Groups Dosage Sample No. Cell survival rate (%) Supernatant LDH Cort+Veh 200pM 6 58.009± 7.749"" 939.667 ±111.946"" Cort+NXQ 200pM+6.25ptg/mL 6 63.452 ±6.133 874.167 ±56.778 Cort+NXQ 200pM+12.5ptg/mL 6 68.896 ±5.226 768 ±95.102 Cort+NXQ 200pM+25ptg/mL 6 88.025 ±3.988** 676.167 ±93.728** Cort+NXQ 200pM+50ptg/mL 6 89.736 ±2.317*** 616.833 ±109.379*** Cort+NXQ 200pM+100[tg/mL 6 93.312 ±2.044*** 509.5 ±39.480*** *P< 0.05, **P<0.01,***P< 0.001 vs. Model control group, #P<0.01 vs. Normal control group 1.6 Effect of the persimmon leaf extract on corticosterone-induced inflammation in PCl2 cells The treatment with 200 pM corticosterone for 24 h caused a decrease in cell survival rate. Therefore, in order to evaluate whether the persimmon leaf extract exerted an anti-inflammatory effect during its antidepressant process, corticosterone co-cultured PCi2 cells were treated with different concentrations of the persimmon leaf extract for 4 h. The concentration of inflammatory factors IL-IP, IL-6 and TNF-a in supernatant were detected by ELISA method to observe the effect of the persimmon leaf extract on the secretion of inflammatory factors. ELISA results showed that (see Figure 4 and Table 4), compared with the control group (Control), 200 pM corticosterone very significantly increased the secretion levels of TNF-a, IL-l$ and IL-6 in the supernatant of PCl2 cell (P<0.001). Compared with the blank control group, the maximum concentration of persimmon leaf extract alone (without corticosterone) did not produce any inflammatory effects (P > 0.05). After treatment with persimmon leaf extract for 1 h, each dosage thereof inhibited the release of inflammatory factors of PC12 cells induced by corticosterone in different degrees. Among them, the persimmon leaf extract already exhibited a significant inhibitive effect on TNF-a, IL- IPand IL-6 at dosage of 25 pg/ml, all with statistical significance (P <0.01). Table 4 Effect of the persimmon leaf extract on secretion of inflammatory factors induced by corticosterone Groups Dosage SampleNo. TNF-a(pg/mL) IL- I(pg/mL) I16(pg/mL)

Cotioi 3 52.733±13.911 56.033±11.120 43.767±9.582

NXQ 100pg/mL 3 42.2337±14.367 54.367±11.381 39.667±7.991

Coit 200pM 3 423.067.+87.168e 403.067±92.858 302.233±84.565w

Coit+NXQ 2OOpM+25ptg/mL 3 299.333±72278* 307.90068.056* 257.833±24.607*

Groups Dosage SampleNo. TNF-a(pg/nL) IL-I(pg/L) I6(pg/nL)

Coit+NXQ 2OpM+50pg/mL 3 228.700t71.399* 261.267±60.640* 222.867+38.394*

Coit+NXQ 2OpM+100[g/mL 3 171.800t45.238* 221.067±50.947* 182.7144.899W

*P< 0.05, **P<0.01,***P< 0.001 vs. Cort group, #P<0.01 vs. Normal control group. 1.7 Conclusion In this example, a depression model of PC12 cells injured by corticosterone was successfully established. Through research, it was found that the persimmon leaf extract had no toxic effects on normally cultured cells in the concentration range of 3.125-400 pM, but in the depression model of PC12 cells, the persimmon leaf extract could produce better protection to nerve cells at 50 pM, and its mechanism might be related to its inhibition of the inflammatory response induced by corticosterone. The results of this example suggested that persimmon leaf extract had obvious antidepressant activity at the cellular level.

Example 2 Effect of the persimmon leaf extract on depression-like behavior in mice induced by acute stress 2.1 Animals C57 mice, 60 females, weighing 18-22 g; experimental animal license number: SCXK (Beijing) 2014-0004. 2.2 Drugs and instruments Persimmon leaf extract (NXQ), fluoxetine (FLX), sodium carboxymethyl cellulose, open field box, round swimming tank, tail suspension apparatus. 2.3 Grouping and administrating methods After the mice adapted to the feeding environment for one week, 50 mice were randomly divided into five groups, namely the vehicle control group (Veh), the positive control fluoxetine (FLX, 10 mg/kg) group, persimmon leaf extract (NXQ) low dosage (20 mg/kg) group, persimmon leaf extract (NXQ) medium dosage (40 mg/kg) group, and persimmon leaf extract (NXQ) high dosage (80 mg/kg) group. The mice were gavaged once a day for 7 consecutive days. The vehicle control group was given the same volume of 0.5% sodium carboxymethyl cellulose aqueous solution. 1 h after the last administration, behavioral test was carried out. The other 10 mice were used for the detection of biochemical indicators as normal control group. Drug preparation: persimmon leaf extract or fluoxetine was fully dissolved with 0.5% sodium carboxymethyl cellulose, prepared when using according to the administration dosage, and administered intragastrically with a volume of 0.1 ml/10 g body weight. 2.4 Method 2.4.1 Open field test The open field test was mainly used to test the spontaneous activity of mice to exclude the interfering of the central excitatory effect of drugs to the experimental results. The experiment was performed in a black wooden box with a bottom of 25 cm x 25 cm and a height of 20 cm, on the bottom of which there are 25 squares. 1 h after the administration on the 6th day, the mouse was placed in the central square, adapted for 5 min, and then taken out. The bottom was scrubbed with 75% alcohol to remove the influence of the residual odor from the previous mouse. 1 h after the administration on the 7th day, the square number of mouse crossed (square in which all the four paws entered can be counted, the number of counted squares was the score of horizontal exercises), and the upright number of hind leg (two front paws vacated or climbed the wall, which was the score of vertical exercises) were recorded by video over 5 min. 2.4.2 Tail suspension test The mouse tail was stacked (1 cm away from the tail tip) with adhesive tape and fixed on the tail suspension bracket, so that the mouse was in an inverted suspension position, and the sides of the mouse were blocked by partitions to prevent them from interfering with each other. The cumulative immobility time of the mouse within 5 min was recorded by video. The criteria for the "immobility" of the mouse include that the mouse stops struggling, its body suspends vertically and hangs still. 2.4.3 Forced swim test On the second day of the tail suspension test, the mice were placed one by one in a 20 cm-high and 12 cm-diameter cylindrical swimming tank with 10 cm depth of water, and forced to swim for 6 min, the cumulative immobility time of the mouse was recorded by video over the later 4 min. The criteria for the "immobility" of the mouse include that the mouse stops struggling in the water, and maintains a floating state, with only small limb movements to keep the head above the water. 2.4.4 Detection of serum corticosterone by ELISA. After the mouse behavior experiment was completed, the eyeballs were removed to collect blood. After standing in the anticoagulation tube for 10 min, the blood was centrifuged at 8500 r/min for 15 min to separate the serum which was stored in the refrigerator at -80°C for later use. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum corticosterone level in the mouse. At the same time, another group of mice (10 mice) without any stress treatment was taken as the normal control group. In accordance with the same method as the experimental group, the eyeballs were removed to collect blood, the serum was separated, and the serum corticosterone level was determined. 2.4.5 Detection of monoamine neurotransmitters in brain tissue by HPLC 2.4.5.1 Chromatographic conditions Chromatographic column: Dikma Diamonsil C18 (5 pm, 4.6 mmx250 mm); Mobile phase: A-B (90:10, Volume ratio), A is an aqueous solution of sodium dihydrogen phosphate (containing 25.0 mmol/L NaH2PO4,1.7 mmol/L OSA, 0.7 mmol/L triethylamine, 0.025 mmol/L EDTA-2Na, pH=3.0), B is acetonitrile; Flow rate: 1.0 mL/min; Column temperature: 32°C; Detection potential: E=- 150V, E2=220V; Injection volume 10 pL 2.4.5.2 Sample pretreatment The frozen brain tissue was weighed and placed in a glass homogenizer, a pre-chilled 0.1 mol/L perchloric acid solution containing 0.01% EDTA-2Na was added at a ratio of 10 mL/kg, and the mixture was homogenized quickly in an ice bath for 2 min. The ground homogenate was transferred into a brown centrifuge tube and centrifuged at 14000 g for 20 min at 4°C, and then the supernatant was collected. After filtered with a 0.22 pm filter, the supernatant was injected into the machine to detect the levels of the monoamine neurotransmitters, such as 5-HT, NE and DA. 2.5 Data analysis and statistics SPSS software was used to analyze the data and calculate variance(One-Way ANOVA). After the homogeneity test of variance, if the variance was homogenous, the Bonferronic method would be used to compare the two groups; if the variance was unequal, the Welch method would be used for analysis, and the Dunnett's T3 method would be used for multiple comparisons. P<0.05 indicated that there was a significant difference, which was statistically significant. 2.6 Results 2.6.1 Effect of continuous administration of the persimmon leaf extract on autonomous activity in mice The results of the open field test were shown in Figure 5 and Table 5. There were no statistically significant differences in horizontal exercise scores (P >0.05) and vertical exercise scores (P>0.05) among all groups, indicating that continuous administration of the persimmon leaf extract did not affect the autonomous activity of the mice. Table 5 Experimental results of the open field test (T±SD) Groups Dosage(mg/kg) SampleNo. horizontalexerisescores veiticalexerisescores Veh --- 10 144.75 17.903 36.125 ±8.043 NXQ (low) 20 10 144.375 18.205 37.375 ±8.434 NXQ (medium) 40 10 144.25 19.404 38.875 ±7.680 NXQ (high) 80 10 146.625 18.943 37.625 ±8.228 FLX 10 10 145.125 18.924 36.750 ±8.730

2.6.2 Effect of the persimmon leaf extract on the behavior of mice with acute stress of tail suspension The results were shown in Figure 6 and Table 6. Compared with the model control group, each dosage of the persimmon leaf extract shortened the tail suspension immobility time of mice in different degrees and the effects were dosage dependent; all dosage of 20, 40 and 80 mg/kg had significant effects, and the differences were statistically significant (P<0.05, P<0.01). The effect of the middle dosage (40 mg/kg) of the persimmon leaf extract was comparable to that of fluoxetine (10 mg/kg), and the high dosage (80 mg/kg) of the persimmon leaf extract was better than fluoxetine in shortening the tail suspension immobility time of mice. Table 6 Experimental results of the tail suspension test in mice (T±SD)

Groups Dosage (mg/kg) Sample No. Immobility time (s) Veh --- 10 190.355 22.969 NXQ (low) 20 10 166.629 27.638* NXQ (medium) 40 10 145.079 ±17.277** NXQ (high) 80 10 120.884 ±22.216** FLX 10 10 143.006± 19.673**

P < 0.05, **P<0.01,***P< 0.001 vs. Vehicle control group.

2.6.3 Effect of the persimmon leaf extract on the behavior of the mice with acute stress of forced swim The results of forced swim test in mice were shown in Figure 7 and Table 7. Compared with the model control group, each dosage of the persimmon leaf extract shortened the immobility time of forced swimming in mice in different degrees, which was dosage dependent. Among them, the effects of dosage of 40 mg/kg and 80 mg/kg were significant, and the differences were statistically significant (P<0.05, P<0.01). The effect of high dosage (80 mg/kg) of the persimmon leaf extract shortening the immobility time was slightly better than that of fluoxetine (10 mg/kg). The experimental results were similar to the results of the tail suspension test. Table 7 Results of forced swim test in mice

Groups Dosage (mg/kg) Sample No. Immobility time (s) Veh --- 10 186.330 ±22.508 NXQ (low) 20 10 172.174 ±22.604

NXQ (medium) 40 10 154.453 31.481*

NXQ (high) 80 10 136.274 33.263** FLX 10 10 141.326 23.237**

P < 0.05, **P<0.01,***P< 0.001 vs. Vehicle control group.

2.6.4 Effect of the persimmon leaf extract on serum corticosterone in mice with acute stress In this part of the experiment, the serum of normal mice with no stress was added for comparison. The results were shown in Figure 8 and Table 8. Compared with the control group, the serum corticosterone level in the mice with stress in the model group significantly increased (P<0.001); compared with the model group, the serum corticosterone levels in 40 and 80 mg/kg persimmon leaf extract groups significantly decreased, and the differences were statistically significant (P<0.01, P<0.001). Moreover, the high dosage of the persimmon leaf extract (80 mg/kg) had a significantly stronger effect on reducing serum corticosterone level in the mice with acute stress than fluoxetine (10 mg/kg). Table 8 Effect of the persimmon leaf extract on serum corticosterone level in mice with acute stress (i±SD) Groups Dosage (mg/kg) Sample No. Serum Corticosterone (tg/L)

Normal (Normal control group) --- 10 71.363 ±8.728

Veh (Vehicle control group) 0 10 188.007 ±7.436#

NXQ (low) 20 10 170.297 ±11.431

NXQ (medium) 40 10 122.733 ±5.098**

NXQ (high) 80 10 91.487 ±15.351*** FLX 10 10 108.473 ±11.262**

*P < 0.05, **P<0.01,***P< 0.001 vs. Vehicle control group, 44P<0.0 vs. Normal control group.

2.6.5 Effects of the persimmon leaf extract on monoamine neurotransmitters in brain tissue of mice with acute stress In this part of the experiment, the brain tissues of normal control mice with no stress were added for comparison. The results were shown in Figure 9 and Table 9. Compared with the normal control group, the levels of 5-HT, DA and NE of the mice with stress in the model group significantly decreased (P<0.01); compared with the stress model group, the 5-HT level in the groups of 20, 40, 80 mg/kg persimmon leaf extract significantly increased, the differences were statistically significant (P<0.05, P<0.01), the levels of DA and NE in the groups of 20, 40, 80 mg/kg persimmon leaf extract significantly increased, the differences were statistically significant (P<0.05, P<0.01). In addition, the high dosage (80 mg/kg) of persimmon leaf extract had better effects on increasing the levels of 5-HT, DA and NE in the brain tissue of the mice with acute stress than fluoxetine (10 mg/kg). Table 9 Effect of the persimmon leaf extract on the levels of monoamine neurotransmitters in brain of mice (T±SD) Groups Dosage Sample 5-HT (ng/g) DA (ng/g) NE (ng/g) (mg/kg) No.

Normal (Normal control --- 10 247.987±11.266 132.947±6.929 206.933±14.258

group) Veh (Model control --- 10 145.587±9.310444 72.077±10.630444 103.230±10.972444

group) NXQ (low) 20 10 176.503±13.309* 102.950±6.832** 154.547±12.986*

NXQ (medium) 40 10 196.830±11.771** 107.403±4.788** 157.217±17.245*

NXQ (high) 80 10 239.677±18.757*** 127.973±8.749*** 197.937±22.251***

FLX 10 10 221.260±17.495*** 119.437±9.001*** 179.233±14.416***

*P< 0.05, **P<0.01,***P< 0.001 vs. Vehicle control group, ##P<0.001 vs. Normal control group. 2.7 Conclusion In this Example, based on the cellular level of Example 1, the anti-depressant efficacy of the persimmon leaf extract was further evaluated utilizing whole animal models. Mouse forced swim test (FST) and mouse tail suspension test (TST) are the most commonly used models of acute behavioral despair. The immobility of the mice in FST and TST reflectes a persistent frustration in the inescapable behavioral despair, or a passive bearing state when dealing with stress stimuli. It is currently believed that this behavioral despair is similar to the components that constitute clinical depression. The shortened duration of the animal's immobility under the stress reflects the antidepressant properties of the drug. Most antidepressant drugs can reduce the immobility time of mice in FST and TST, and their pharmacodynamic is related to their clinical efficacy. The results of this Example showed that continuous administration of the persimmon leaf extract could significantly reduce the immobility time of mice in the forced swim and tail suspension test without affecting the autonomous activity of mice. The effect of medium dosage of the persimmon leaf extract on reducing tail suspension immobility time of mice was similar to that of fluoxetine, and the effect of high dosage on reducing forced swim immobility time of mice and tail suspension immobility time of mice was better than that of fluoxetine. The results confirm that the persimmon leaf extract alone can produce a significant antidepressant effect. Acute stress is a systemic non-specific adaptive response that occurs when various internal and external environmental factors as well as social and psychological factors stimulate the body. It can cause hypofunction of the brain and neuronal damage. In the case of acute stress, the body activates the hypothalamus-pituitary-adrenal axis (HPA axis), so as to increase the concentration of adrenocorticotropic hormone and corticosterone (CORT) in the serum, which is an important feedback and self-protection approach against foreign emergencies. In the detection of biochemical indicators, the persimmon leaf extract can significantly decrease the serum corticosterone level in the mice with acute stress, which may play a role in protecting neurons. In addition, the monoamine neurotransmitters, such as -HT, DA and NE in the brain tissue are closely related to the depression monoamine hypothesis. It is found by HPLC detection in this Example that the persimmon leaf extract can rapidly and significantly reverse the downregulation of three types of neurotransmitters in the brain tissue of the mice with acute stress. This may be one of the important mechanisms by which persimmon the leaf extract exerts its antidepressanteffects.

Example 3 Study on the material basis of the antidepressant effect of the persimmon leaf extract In the experiments in Example 1 and Example 2, the present invention has confirmed that the persimmon leaf extract has a significant effect on major depressive disorder. The persimmon leaf extract contains various components such as flavonoids, organic acids, triterpenes and coumarins, wherein the content of flavonoids accounts for 30% (mainly quercetin, kaempferol aglycone and its mono- and di-glycosides such as rutin, hyperin and the like), and it has been reported that quercetin and hyperin have certain antidepressant activities. However, detected by HPLC, the persimmon leaf extract according to the present invention contains quercetin, hyperin, kaempferol and rutin not exceeding 1.5% respectively. It is unclear whether the other components such as organic acids, triterpenes and coumarin in the persimmon leaf extract may have synergistic effects. In order to preliminarily explore the material basis of the anti-depressant effect of the persimmon leaf extract, in this Example, the anti-depressant efficacy of persimmon leaf extract was further compared with single flavonoids (quercetin, kaempferol, rutin, hyperin) and composition thereof. The selected model of major depressive disorder is an internationally recognized chronic unpredictable mild stress model. 3.1 Animals C57 mice, 80 males, weighing 18-22 g, experimental animal license number: SCXK (Guangdong) 2011-0015; mice were adapted to the feeding environment for one week before the experiment, kept in the general laboratory animal room at a temperature of 21±2C, and followed the circadian rhythm. The animals were normally fed. Drinking water was available ad libitum. 3.2 Drugs and instruments Persimmon leaf extract, quercetin, kaempferol, rutin, hyperin, sodium carboxymethylcellulose, mouse open field box, electronic analytical balance (Sartrious, Germany), frozen desktop large-capacity high-speed centrifuge 5810 (EPPENDORF, Germany), Victor3 multi-functional microplate reader (PerkinElmer, U.S.A), ultrasonic tissue breaker (Sonic, U.S.A), DYY-III electrophoresis instrument (Beijing Liuyi Instrument Factory), vertical plate electrophoresis tank (Bio-Rad, U.S.A), WD-9405 horizontal shaker (Beijing Liuyi Instrument Factory), fluorescence/chemiluminescence imaging system (Sonic, U.S.A), Mill-Q ultrapure water system (Millipore, U.S.A), tail suspension instrument, Rat tail clips, counters, thermometers, scales, and the like ECl Luminescent Solution (Invitrogen, U.S.A); Skimmed Milk Powder (Guangzhou Sijia Biotechnology Co., Ltd.); PVDF membrane (Milipore, U.S.A); X-ray Photographic Film (Kodak, Japan); Corticosterone (CORT); enzyme-linked immunoassay kit (Enzo, U.S.A); ACTH enzyme-linked immunoassay kit (phoenix pharmaceuticals, U.S.A). 3.3 Modeling, grouping and administrating Before the experiment, all mice were adapted to the environment for one week. The animals could access food and water freely, following the circadian rhythm. They were kept in a clean environment at a constant temperature, and manually stroked daily to adapt to manual operation. After adapting to the environment for one week, the mice were divided into two groups according to their weight and results of sucrose preference test: normal control group (n=10) and chronic mild unpredictable stress (CUMS) group (n=70), then stress started to be carried out. Stress modeling methods included: tail suspension for 1 min, day and night reversal for 24 h, cage tilting for 24 h, ice water swimming at 4°C for 5 min, constraint for 1 h, wet cage for 24 h, forced swimming for 15 min, water fasting for 24 h, food fasting for 24 h, electric shock to the sole of the foot, stroboscopic stress, single cage stress, combined cage stress, and the like. At the beginning of the experiment and the 3rd week after stress, the scurose preference test was performed to determine whether the modeling was successful. If the depression model was not successful, the time of CUMS stress would be postponed. After successfully modeling, the model groups were divided into CUMS model group (vehicle Veh + CUMS), persimmon leaf extract group (NXQ 40 mg/kg + CUMS), quercetin group (4.0 mg/kg + CUMS), hyperin group (4.0 mg/kg+CUMS), kaempferol group (3.0 mg/kg+CUMS), rutin group (1.5 mg/kg+CUMS), and persimmon leaf flavonoid composition group [(quercetin 4.0 mg/kg+hyperin 4.0 mg/kg + kaempferol 3.0 mg/kg + rutin 1.5 mg/kg) + CUMS], 10 mice each group. 4 weeks after administration, the behavioral tests were performed.The administrating dosage of quercetin, hyperin, kaempferol and rutin was more than 10 times of their respective content ratio in persimmon leaf extract. 3.4 Method 3.4.1 Scurose preference test Before the experiment, the mice were trained to adapt to 1% (w/v) sugar water: two bottles of 1% sugar water solution were placed in each cage. After 24 h, one bottle of 1% sugar water was replaced with pure water and placed for 24 h. After adapting, the mice were water fasted and food fasted for 24 hours, then the scurose preference test was performed. In the experiment, each mouse was free to access two bottles of water, one bottle was 25 ml 1% sugar water, and the other bottle was 25 ml pure water, which were weighed before the experiment. After 2 h, the remaining weight was measured to obtain the consumption of sugar water and pure water (g).

The ratio of sugar water consumption to total liquid consumption (sugar water consumption + pure water consumption) was used to represent the preference for sugar water. This test ran through the whole process of the experiment. 3.4.2 Open field test Experimental method was the same as "2.4.1 Open field test" in Example 2. 3.4.3 Tail suspension test Experimental method was the same as "2.4.2 Tail suspension test" in Example 2. 3.4.4 Forced swim test Experimental method was the same as "2.4.3 Forced swim test" in Example 2. 3.4.5 Detection of serum corticosterone and adrenocorticotropic hormone level in mice by ELISA Experimental method was the same as "2.4.4" in Example 2, along with the detection of corticosterone, the level of adrenocorticotropic hormone (ACTH) was measured. 3.5 Data analysis and statistics SPSS software was used to analyze the data and calculate variance (One-Way ANOVA). After the homogeneity test of variance, if the variance was homogenous, the Bonferronic method would be used to compare the two groups; if the variance was unequal, the Welch method would be used for analysis, and the Dunnett's T3 method would be used for multiple comparisons. P<0.05 indicated that there was a significant difference, which was statistically significant. 3.6 Results 3.6.1 Scurose preference test of CUMS mice The results were shown in Figure 10. Compared with the normal control group, the mice in the model group (Veh + CUMS) had a significantly reduced preference for sugar water (P<0.001), indicating that the mice depression modeling was successful. Compared with the model group, the persimmon leaf extract group (40 mg/kg), the persimmon leaf flavonoid composition group and hyperin group significantly increased the preference of mice for sugar water after continuous intragastric administration(P<0.001, P<0.01, P<0.05), whereas quercetin, kaempferol and rutin increased the preference of CUMS mice for sugar water to a certain extent, but the effects were not statistically significant. Compared with the persimmon leaf flavonoid composition group and hyperin group, the effect of increasing the preference of CUMS mice for sugar water in the persimmon leaf extract group was statistically significant (P<0.01,P<0.05).

The results suggested that the persimmon leaf extract within its effective dosage range had a significantly better antidepressant effect than single flavonoid and flavonoid composition in the sucrose preference test. 3.6.2 Open field test of CUMS mice The results were shown in Figure 11. Compared with the normal control group, the scores of horizontal exercises and vertical exercises of mice in the model group in open field test were significantly reduced (P<0.001), indicating that the depression model was successfully established and the voluntary activities of animal were reduced. Compared with the model group, the scores of horizontal exercises of mice in each administration group significantly increased. Compared with other administration groups, the increasing effect was greater in the persimmon leaf extract group, and the differences were statistically significant (P<0.05). The order of the effect in each group was: persimmon leaf extract group (P<0.001)> persimmon leaf flavonoid composition group, quercetin group, hyperin group and rutin group (P<0.01)> kaempferol group (P<0.05) (see Figure 11A). Compared with the model group, continuous administration significantly increased the number of vertical exercises of mice in the persimmon leaf extract and persimmon leaf flavonoid composition group (P<0.001, P<0.05); compared with persimmon leaf flavonoid composition group, the increasing effect of the persimmon leaf extract was greater, and the differences were statistically significant (P<0.05). Although the vertical exercises of mice in quercetin group, kaempferol group, hyperin group and rutin group increased, the differences were not statistically significant, compared with the model group (see Figure 11B). The results suggested that the persimmon leaf extract within its effective dosage range had a significantly better antidepressant effect than single flavonoid and flavonoid composition in the open field test. 3.6.3 Tail suspension test of CUMS mice The results were shown in Figure 12. Compared with the normal control group, the immobility time of mice in the model group significantly increased in the tail suspension test (P<0.001), indicating that the modeling was successful and CUMS mice showed depression-like symptoms. Compared with the model group, continuous administration of the persimmon leaf extract very significantly (P<0.001) reduced the tail suspension immobility time of mice, while the persimmon leaf flavonoid composition and hyperin significantly

(P<0.05) reduced the immobility time. There were no statistically significant differences among the other groups and the model group. Compared with the persimmon leaf flavonoid composition group and hyperin group, the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the differences were statistically significant (P<0.05). The results suggested that the persimmon leaf extract within its effective dosage range had a significantly better antidepressant effect than single flavonoid and flavonoid composition in the tail suspension test. 3.6.4 Forced swim test of CUMS mice The results were shown in Figure 13. Compared with the normal control group, the immobility time of the mice in the model group increased very significantly in the forced swim test (P<0.001), indicating that the modeling was successful and the CUMS mice showed depression-like symptoms. Compared with the model group, continuous administration of the persimmon leaf extract very significantly (P<0.001)reduced the immobility time of mice, while the persimmon leaf flavonoid composition (P<0.01) and hyperin (P<0.05) significantly reduced the immobility time. There were no statistically significant differences among the other groups and the model group. Compared with the persimmon leaf flavonoid composition group and hyperin group, the persimmon leaf extract had a greater effect on reducing the immobility time of CUMS mice, and the differences were statistically significant (P<0.01, P<0.05). The results suggested that the persimmon leaf extract within its effective dosage range had a significantly better antidepressant effect than single flavonoid and flavonoid composition in the forced swim test of mice. 3.6.5 Levels of serum corticosterone and adrenocorticotrophic hormone in CUMS mice The results were shown in Figure 14. Compared with the normal control group, the levels of serum corticosterone and adrenocorticotrophic hormone in the model group both very significantly increased (P<O.001). Compared with the model group, persimmon leaf extract could very significantly reduce serum corticosterone level (P<0.001). Persimmon leaf flavonoid composition and hyperin could significantly reduce serum corticosterone level (P<0.05). Compared with the persimmon leaf flavonoid composition group and the hyperin group, the persimmon leaf extract decreased the serum corticosterone level in CUMS mice more significantly, and the differences were statistically significant (see the left panel of Figure 14). Compared with the model group, persimmon leaf extract could very significantly reduce serum adrenocorticotropic hormone level (P<0.01). There were no statistical differences among the other groups in reducing the adrenocorticotropic hormone level compared with the model group. (See the right panel of Figure 14). The results suggested that the persimmon leaf extract within its effective dosage range had a significantly better effect on reducing serum corticosterone and adrenocorticotropic hormone in the depressed mice than single flavonoid and flavonoid composition. 3.7 Conclusion In this Example, a mimetic composition of flavonoids which are part of flavonoids already exist in the persimmon leaf extract was designed, and its efficacy was compared with the persimmon leaf extract and single flavonoid in parallel (the dosage of each single flavonoid applied in the study is more than 10 times of its content ratio in persimmon leaf extract). Unexpectedly, the effects of persimmon leaf extract were significantly stronger in each test than the flavonoid composition and each single flavonoid as control. In summary, the present invention provides a new medical use of persimmon leaf extract for prevention and/or treatment of major depressive disorder. Animal experiments have demonstrated that the persimmon leaf extract has a positive effect on improving the depression symptoms and reversing abnormal biochemical indicators of CUMS mice. The mechanism of persimmon leaf extract exerting its antidepressant effect may involve inhibiting neuroinflammation, regulating HPA axis function and monoamine neurotransmitters (5-HT, NE and DA), so it can prevent major depressive disorder induced by non-physical problems (such as cardio cerebrovascular disease, and the like).

Claims (13)

1. Claims 1. Use of a persimmon leaf extract in preparing drugs for prevention and/or treatment of depression.
2. 2. The use according to claim 1, wherein the depression refers to major depressive disorder, especially depressive disorders caused by mental and affective disorders.
3. 3. The use according to claim 1 or 2, wherein the persimmon leaf extract is the only active ingredient of the drug.
4. 4. The use according to any one of claims 1 to 3, wherein the drug for prevention and/or treatment of depression includes or excludes pharmaceutically acceptable excipients.
5. 5. The use according to any one of claims 1 to 4, wherein the drug for prevention and/or treatment of depression is an oral preparation or a non-oral preparation.
6. 6. The use according to claim 5, wherein the oral preparation is one or more selected from the group consisting of powders, ordinary oral tablets, dispersible tablets, capsules, soft capsules, pills, dripping pills, pellets, granules, orally disintegrating tablets, and oral fast dissolving film.
7. 7. The use according to claim 5, wherein the non-oral preparation is one or more selected from the group consisting of injections, lyophilized powder for injection and large-volume injections.
8. 8. The use according to claim 5, wherein drug for prevention and/or treatment of depression is an oral preparation; more preferably Naoxinqing tablets or Naoxinqing capsules.
9. 9. The use according to any one of claims 1 to 8, wherein the drug for prevention and/or treatment of depression is prepared by the following method: the persimmon leaf extract, with or without the addition of pharmaceutically acceptable excipients, is prepared according to conventional methods in the art to obtain clinically acceptable oral preparations or non-oral preparations.
10. 10. The use according to any one of claims 1 to 9, wherein the persimmon leaf extract is prepared by the following method: the dried persimmon leaves was decocted with water twice, for 1 to 2 hours each time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 80-90%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed with 60-70% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate more than 2 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature; preferably, the persimmon leaf extract is prepared by the following method: the dried persimmon leaves was decocted with water twice, for 2 hours for the first time and 1 hour for the second time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 85%, after standingovernight,the supernatant was collected by filtration and set aside; the precipitate was washed twice with 65% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate 4 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature.
11. 11. A method for preventing and/or treating depression, comprising the step of administering a persimmon leaf extract to a patient in need; preferably, the persimmon leaf extract is prepared by the following method: the dried persimmon leaves was decocted with water twice, for 1 to 2 hours each time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 80-90%, after standing overnight, the supernatant was collected by filtration and set aside; the precipitate was washed with 60-70% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate more than 2 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature; more preferably, the persimmon leaf extract is prepared by the following method: the dried persimmon leaves was decocted with water twice, for 2 hours for the first time and 1 hour for the second time; the water decoction was combined, filtered, concentrated to a relative density of 1.12-1.15 (60°C), and then precipitated by ethanol at the concentration of 85%, after standingovernight,the supernatant was collected by filtration and set aside; the precipitate was washed twice with 65% ethanol, the ethanol solution was combined, after standing overnight, the supernatant was collected by filtration, and combined with the spare supernatant; after recovery of ethanol, appropriate amount of water was added to the concentrated supernatant, mixed well and then filtered; the filtrate was extracted with ethyl acetate 4 times, and the ethyl acetate extracts were combined; after recovery of ethyl acetate, the combined solution was concentrated into a thick paste, and dried at low temperature.
12. 12. The method according to claim 11, wherein the depression refers to major depressive disorder, especially depressive disorders caused by mental and affective disorders; preferably, the patient in need is a mammal in need, preferably a human in need.
13. 13. The method according to claim 11 or 12, wherein the method comprises the step of administering the persimmon leaf extract to a human in need with 2 to 10 mg/kg body weight /day.