阅读说明：本技术 一种柏子或柏子提取物及其用途 (Semen Platycladi or semen Platycladi extract and its application ) 是由 叶阳 唐希灿 章海燕 姚胜 王维 季萍 于 2013-08-12 设计创作，主要内容包括：本发明提供了一种柏子或柏子提取物的用途。具体地,本发明柏子或柏子提取物用于具有良好的抗疲劳效果,可用于制备抗疲劳的组合物,其中,所述的柏子提取物具有以下一个或多个特征：(a)所述提取物的分子量分布为280-310；(b)所述提取物中不饱和脂肪酸总含量≥80wt％；和/或(c)所述提取物中来源于柏子的二萜类物质总含量≤0.5wt％。本发明柏子或柏子提取物在食品及保健品领域具有广泛应用。(The invention provides an application of cedar seed or cedar seed extract. Specifically, the cedar seed or cedar seed extract disclosed by the invention has a good anti-fatigue effect and can be used for preparing an anti-fatigue composition, wherein the cedar seed extract has one or more of the following characteristics: (a) the molecular weight distribution of the extract is 280-310; (b) the total content of unsaturated fatty acid in the extract is more than or equal to 80 wt%; and/or (c) the total content of diterpenoid substances derived from the cedar seeds in the extract is less than or equal to 0.5 wt%. The cedar seed or cedar seed extract of the invention has wide application in the fields of food and health care products.)

1. The application of the cedar seed extract is characterized in that the cedar seed extract is used for preparing an anti-fatigue composition; the cedar seeds are shelled cedar seeds; the content of unsaturated fatty acid in the cedar seed extract is more than or equal to 80 wt%, and the content of diterpenoid substances is less than or equal to 0.5 wt%;

the cedar seed extract is obtained by CO₂The preparation method comprises supercritical extraction.

2. The use according to claim 1, wherein the composition is a pharmaceutical composition, a food composition or a nutraceutical composition.

3. The use as claimed in claim 1, wherein the cedar seed extract has the following characteristics:

(a) the molecular weight distribution of the extract was 280-310.

4. The use as claimed in claim 1, wherein the cedar seed extract is further characterized by one or more of the following:

(d) the total content of diterpenes in the extract is less than 0.5 wt% or none;

(e) the extract also contains 10-14 wt% of saturated fatty acid.

5. The use of claim 1, wherein the cedar seed extract is formulated for daily administration in an amount of 0.01-50mg/kg body weight.

6. The use according to claim 3 or 4, wherein the diterpenoids comprise pinosylvin, 15, 16-bisnor-13-oxohelianth-8 (17) -en-19 acid, 15, 16-bisnor-13-oxohelianth-8 (17), 11E-diene-19-acid, dihydroxyhelianthatrienoic acid, aragonic acid, 13-epithumic acid (13-epicupressic acid).

7. The use according to claim 4, wherein the saturated fatty acids comprise palmitic acid, stearic acid;

the unsaturated fatty acid comprises oleic acid, linoleic acid, linolenic acid, 9-eicosenoic acid, 11-eicosenoic acid, 5, 11-eicosadienoic acid, 12, 17-eicosadienoic acid, 8,11, 14-eicosatrienoic acid, and 5,11,14, 17-arachidonic acid.

8. The use of claim 1, wherein the cedar seed extract is prepared by the following method:

(i) pulverizing the obtained mature semen Platycladi to obtain semen Platycladi powder;

(ii) (ii) extracting the cedar seed powder obtained in step (i) to form a cedar seed extract.

9. The use according to claim 1, wherein said composition consists of: (i) cedar seed extract as an active ingredient, and (ii) a pharmaceutically or food acceptable carrier.

10. The use of claim 1, wherein said composition is in the form of an oral dosage form.

11. Use of an extract of cedar seed for the preparation of an anti-fatigue composition, said composition being a nootropic composition;

the cedar seed is cedar seed with shell, the content of unsaturated fatty acid in the cedar seed extract is more than or equal to 80 wt%, and the content of diterpenoid substances is less than or equal to 0.5 wt%;

the cedar seed extract is obtained by CO₂The preparation method comprises supercritical extraction.

12. The use as claimed in claim 1 or 11, wherein the cedar seed extract is prepared by the following method:

288g of shelled cedar seeds are crushed and put into SFC, and the extraction conditions are as follows: temperature of the extraction kettle: temperature and pressure of the extraction vessel at 32 ℃: 25 MPa; temperature of the separation kettle: temperature and pressure of the separation vessel at 38 ℃: 6 MPa; supercritical CO₂A flow rate; 30-40L/h; extraction time: and (5) obtaining the cedar seed extract after 1.5 h.

Technical Field

The invention relates to the field of traditional Chinese medicine extracts, in particular to application of a high-activity component extracted from platycladi seeds as a medicine and a functional food for improving cognitive ability, cognitive dysfunction and fatigue.

Background

Cognitive function refers to the ability of the human brain to process, store and retrieve information, including memory, language, visual space, execution, calculation, and understanding of judgment^[1]. Cognitive dysfunction refers to impairment of one or more of the above abilities, and is common in patients with various types of Dementia, the most common of which are Alzheimer's Disease (AD) and vascular Dementia (VaD).

AD is a neurodegenerative disease that occurs in the early senile stage or the elderly, with progressive dementia and cognitive impairment as the main symptoms, accompanied by psychomotor abnormalities. The characteristic clinical manifestations are severe decline of learning and memory function, decline of analysis and judgment ability, emotional change, behavior disorder and even confusion of consciousness. With the aging of the age structure of the human society, the incidence of senile dementia is increasing, and the senile dementia becomes the third major health killer after cardiovascular and cerebrovascular diseases and tumors. It has been reported that in the elderly population over 65 years of age, the prevalence of AD is 4 wt% to 7 wt% of the total population, and that in the elderly over 80 years of age can reach 20 wt%; by 2015, the elderly population in China will exceed 2 hundred million, and in the later 40 th century, 4 hundred million may be broken through. The incidence of AD tends to increase year by year with the aging population, and finding effective drugs for treating AD has become one of the research hotspots in the medical field.

The clinical manifestations of AD are mainly intellectual and cognitive dysfunction, and belong to the categories of traditional Chinese medicine, such as dull disease, amnesia, dementia, and dementia. The traditional Chinese medicine has the characteristics of exact effectiveness, safety and low toxicity, and has greater potential in the prevention and treatment of cognitive dysfunction diseases represented by AD.

Arborvitae seed (arboruitae seed), also known as semen Platycladi, is a dried mature seed of the gymnosperm family, Platycladus orientalis (L.) Franco. Mainly produced in Shandong, Henan and Hebei provinces. Baizi ren is sweet in taste and neutral in nature, and has the functions of nourishing heart, tranquilizing mind, arresting sweating, moistening intestines and relaxing bowels. Bai Zi ren has a long history of application, which was recorded in Shen nong Ben Cao Jing, listed as the first-grade. The record of Shennong Ben Cao Jing records that the traditional Chinese medicine composition has the effects of treating palpitation due to fright, regulating five internal organs, tonifying qi, removing rheumatism, moistening and beautifying the color of a person after being taken for a long time, improving hearing and eyesight, not hunger, not fatigue, losing weight and prolonging life; the compendium of materia Medica records: semen Platycladi has effects of nourishing heart qi, moistening kidney dryness, calming soul, benefiting intelligence, tranquilizing mind, removing heat from hair, treating scabies, and can be used for treating palpitation due to fright, insomnia, amnesia, night sweat, and constipation due to intestinal dryness. Ming Dynasty Nippon desert mentioned in Ben Cao Hui Yan, that is, the Bai Zi ren is sweet and has a mild flavor … …, so the Feng is good for the spleen and stomach; for the middle energizer and moistening, it is good at liver and kidney. Therefore, ancient recipes for nourishing the five internal organs, inducing palpitation, calming the heart, pleasing the color and improving hearing and vision, are also superior agents for prolonging life and relieving pain. "

In recent years, chemical researches show that platycladi seed contains fatty acid, cedrol, sitosterol and diterpenoid components, wherein the diterpenoid components mainly comprise pinusolide (pinusolide), 15, 16-bisnor-13-oxosemiaquifolium-8 (17) -alkene-19 acid (15,16-bisnor-13-oxo-8(17) -labdene-19-oic acid), and 15, 16-bisnor-13-oxosemiaquifolium-8 (17); 11E-diene-19-oic acid, dihydroxy-labdane-trienic acid (12R,13 RS-dihydroycneminic acid). In addition, alpha-cedrol (alpha-cedrol), arborvitae seed diol (platydiol), and 5-hydroxyconiferoic acid (5-hydroxyppensolidine acid) are also included. Phenolic acidic compounds include: (+) -Catechin [ (+) -Catechin ], (-) -epicatechin [ (-) -epicatechin ], and its polymer propionitrile B1, B3(Proeyanidin Bl, B3), 5-hydroxy-7, 4-dimethoxyflavone (5-hydroxy-7, 4-dimethoxyflavone), lignin, etc.

Pharmacological research in recent years shows that the platycladi seed has the effects of promoting intelligence, tranquilizing, moistening and relaxing bowels. The Japanese scholars West shan Xin Hao (West shan Xin Hao et al, TCM book of foreign medicine, 1992,14(4), 53-54.) in 1992 studied the effect of arborvitae seed on the damage of amygdala mouse learning disorder through mouse passive learning avoidance test, and light and dark room test showed that 250,500mg/kg of arborvitae seed ethanol extract is not effective on the learning disorder of mice with damaged amygdala, but can obviously improve, maintain and recover memory in diving platform test, but has no influence on the acetylcholinesterase activity of cerebral cortex, hippocampus and hypothalamus. The Japanese scholars, Broussonetia papyrifera (L.) Nepalusto Jiang, et al, Japan medical science and medicine, book of academic records, 1993,15(2),40-41, found when studying the influence of arborvitae kernel on the passive avoidance of learning disorder of mice with damage of forebrain basal ganglia, dark avoidance and diving tower tests show that 250,500mg/kg of arborvitae kernel ethanol extract has an improvement effect on the passive learning disorder of mice with damage of forebrain basal ganglia, but has no antagonism on hypokinesia. Nishiyama et al [ Nishiyama N, et al, Phytotherapy Research,1992,6,289-293 ].

The research shows that 250,500mg/kg oral administration of the arborvitae seed ethanol extract has the effect of improving the memory acquisition disorder caused by tonsil injury, but fails to improve the memory retention, has no effect on the activity of choline acetate transferase in cortex, hippocampus and hypothalamus, and has no effect on the microscopic pathological change caused by tonsil injury.

Therefore, there is an urgent need in the art to develop an extract and a composition thereof having good effects of improving cognitive ability and cognitive dysfunction, high active ingredients, and being easily available.

Disclosure of Invention

The cedar seed extract provided by the invention has the advantages of high active dose, cognitive ability improvement, cognitive dysfunction improvement and obvious anti-fatigue effect.

The invention provides an application of cedar seed or cedar seed extract in preparing an anti-fatigue composition.

In another preferred embodiment, the composition comprises a pharmaceutical composition, a food composition and/or a nutraceutical composition.

In another preferred embodiment, the cedar seed extract has one or more of the following characteristics:

(a) the molecular weight distribution of the extract is 280-310;

(b) the total content of unsaturated fatty acid in the extract is more than or equal to 80 wt%; and/or

(c) The total content of diterpene substances derived from semen Platycladi in the extract is less than or equal to 0.5 wt%.

In another preferred example, the cedar seed extract also has one or more of the following characteristics:

(d) the diterpene content in the extract is less than 0.5 wt% or none;

(e) the extract also contains 10-14 wt% of saturated fatty acid.

In another preferred embodiment, said extract has both of said characteristics (a), (b), (c), (d) and (e).

In another preferred embodiment, the extract is also used for preparing nootropic compositions.

In another preferred embodiment, the content of the cedar seed extract is formulated to be administered in a daily dose of 0.01-50mg/kg body weight; more preferably, it is 30-40mg/kg body weight (mouse), about 0.3-45mg/kg body weight (human).

In another preferred embodiment, the diterpenoids include pinus koraiensis lactone, 15, 16-bisnor-13-oxoflos Lonicerae-8 (17) -ene-19 acid, 15, 16-bisnor-13-oxoflos Lonicerae-8 (17); 11E-diene-19-oic acid, dihydroxy laburninonic acid, aragonic acid, 13-thujalic acid (13-epicupressic acid);

in another preferred embodiment, the content of aragonic acid in the extract is less than or equal to 0.5 wt%, and the content of 13-epithulic acid (13-epicupressic acid) is less than or equal to 0.5 wt%.

In another preferred embodiment, the saturated fatty acid includes palmitic acid, stearic acid;

the unsaturated fatty acid comprises oleic acid, linoleic acid, linolenic acid, 9-eicosenoic acid, 11-eicosenoic acid, 5, 11-eicosadienoic acid, 12, 17-eicosadienoic acid, 8,11, 14-eicosatrienoic acid, and 5,11,14, 17-arachidonic acid.

In another preferred embodiment, the linolenic acid content is 25 wt%;

in another preferred embodiment, the cedar seed extract is prepared by the following method:

(i) pulverizing the obtained mature semen Platycladi to obtain semen Platycladi powder;

(ii) (ii) extracting the cedar seed powder obtained in step (i) to form a cedar seed extract.

In another preferred embodiment, the extraction comprises supercritical extraction and petroleum ether extraction.

In another preferred embodiment, the composition comprises: (i) cedar seed extract as an active ingredient, and (ii) a pharmaceutically or food acceptable carrier.

In another preferred embodiment, the cedar seed extract as the active ingredient has one or more of the following characteristics:

(a) the molecular weight distribution of the extract is 280-310;

(b) the total content of unsaturated fatty acid in the extract is more than or equal to 80 wt%;

(c) the total content of diterpene substances derived from semen Platycladi in the extract is less than or equal to 0.5 wt%.

In another preferred embodiment, the dosage form of the composition is an oral dosage form (tablet, granule, capsule, oral liquid).

In a second aspect of the invention, there is provided a method of combating fatigue by administering to a subject in need thereof an effective amount of cedar seed or a cedar seed extract.

In another preferred embodiment, the administration amount is 0.01-50mg/kg body weight per daily administration dose; more preferably, it is 30-40mg/kg body weight (mouse), about 0.3-50mg/kg body weight (human).

In another preferred embodiment, the subject is a mammal, more preferably a mouse, rat, human.

In another preferred embodiment, the subject includes a child, an adolescent, an elderly person, a person prone to fatigue.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIGS. 1A-B show that the active sites of semen Platycladi obtained by different extraction methods and commercially available fish oil have different memory improving effects on mouse dysmnesia caused by scopolamine, wherein the effect of the extract BZ-1-SFC of semen Platycladi with shell is better than that of the extract obtained by other methods, and the extract is equivalent to that of the extract BZR-E of semen Platycladi with ethanol, but the dosage of BZR-E is 10 times of that of BZ-1-SFC.

FIG. 2A shows that the BZ-1-SFC extract in multiple doses reduces the time to the escape plateau to various degrees, while 6mg/kg shows the best improvement; FIG. 2B shows that multiple doses of BZ-1-SFC reduced the number of errors in entry of mice into the dead end, with the effect being most pronounced at 6 mg/kg. The action intensity is equivalent to that of positive control huperzine A.

FIG. 3 shows that the anti-fatigue effect of BZ-1-SFC is enhanced with the increase of the tested concentration. Wherein 36mg/kg can obviously improve the physical fatigue phenomenon of mice, and has significant statistical significance.

Detailed Description

The inventor of the invention discovers for the first time that the extraction of the cypress seeds with the shell has good effects of improving cognitive ability and cognitive dysfunction under a very low dosage through extensive and intensive research, and the inventor of the invention simultaneously proves that the extract can also effectively generate an anti-fatigue effect through experiments, thereby having wide application prospects in the fields of food and health care products. On the basis of this, the present invention has been completed.

Term(s) for

As used herein, the terms "Cupressus orientalis extract of the present invention", "Cupressus orientalis active fraction of the present invention", "BZ-1-SFC", which are used interchangeably, refer to Cupressus orientalis extract obtained by extracting Cupressus orientalis with husk, and the Cupressus orientalis extract has an unsaturated fatty acid content of 80 wt% or more and a diterpenoid substance content of 0.5 wt% or less, and has cognitive ability improving and cognitive dysfunction improving properties at low doses.

The terms "nootropic" and "nootropic activity" are used interchangeably and refer to the effect of enhancing cognitive ability and improving cognitive dysfunction after administration of a drug to a human or animal.

Cypress seed extract

The cypress seed extract provided by the invention is a high-activity extract obtained by carrying out supercritical extraction, petroleum ether extraction and other methods on cypress seeds with shells. The semen Platycladi extract has extremely high content (more than 80 wt%) of unsaturated fatty acid, and has extremely low content of substances with no activity for improving cognitive ability and cognitive dysfunction, wherein the content of diterpene substances is less than or equal to 0.5 wt%. Thus, the cedar seed extract of the present invention can be used at an extremely low dose (0.01-50mg/kg body weight, preferably 2-10mg/kg body weight (mouse), more preferably 4-6mg/kg body weight (mouse), more preferably 0.04-6.6mg/kg body weight (human)) to improve cognitive ability and cognitive dysfunction.

The cedar seed extract can be extracted by methods such as supercritical extraction, petroleum ether extraction and the like.

Wherein, the supercritical extraction method comprises the following steps: pulverizing shelled or shelled semen Platycladi, placing in supercritical extraction kettle (HA 221-40-11 supercritical extraction device) of supercritical extraction device, and extracting at 32 deg.C and 25MPa in the extraction kettle with CO₂Extracting at flow rate of 30-34L/h for 1.5 hr, transferring the extractive solution to a separation kettle, and separating semen Platycladi SFC extract at temperature of 38 deg.C and pressure of 6 MPa.

The petroleum ether extraction method comprises the following steps: pulverizing shelled or shelled semen Platycladi, placing into 3000ml conical flask, adding equivalent petroleum ether (boiling point 60-90 deg.C), and ultrasonic extracting for 3 times, each time for 1 hr; mixing the extractive solutions, and evaporating under reduced pressure to obtain semen Platycladi petroleum ether extract.

In the cedar seed extract, the content of unsaturated fatty acid is more than or equal to 80 wt%, and the unsaturated fatty acid comprises oleic acid, linoleic acid, linolenic acid, 9-eicosenoic acid, 11-eicosenoic acid, 5, 11-eicosadienoic acid, 12, 17-eicosadienoic acid, 8,11, 14-eicosatrienoic acid I, 5,11,14, 17-arachidonic acid;

the content of diterpenoid substances in the cedar seed extract is less than or equal to 0.5 wt%, and the cedar seed extract comprises pinus koraiensis lactone, 15, 16-bisnor-13-oxo-flos scutellariae-8 (17) -alkene-19 acid, and 15, 16-bisnor-13-oxo-flos scutellariae-8 (17); 11E-diene-19-oic acid, dihydroxy laburninonic acid, aragonic acid, 13-thujalic acid (13-epicupressic acid); representative diterpenoid substances include aragonic acid and 13-thujalic acid (13-epicupressic acid).

The cedar seed extract also comprises 10-15 wt% of saturated fatty acid, wherein the saturated fatty acid comprises palmitic acid and stearic acid.

The cedar seed extract has the functions of improving memory and promoting intelligence under low dosage (0.01-50mg/kg, preferably 2-10mg (mouse), 0.02-11mg/kg (human), more preferably 4-6mg/kg (mouse), 0.04-6.6mg/kg (human)), and also has certain anti-fatigue effect. Accordingly, the present invention also provides a method for preventing, ameliorating or treating the disease by administering an effective amount of the cedar seed extract to a subject in need thereof.

Composition comprising a metal oxide and a metal oxide

The invention provides a composition, which contains 1-99 wt% of the cedar seed extract as an active ingredient and a pharmaceutically or dietetically acceptable carrier based on the total weight of the composition.

In the present invention, various compositions can be formulated according to methods well known in the art, and the cedar seed extract can be formulated in admixture with a pharmaceutically or dietetically acceptable carrier. Ingredients of pharmaceutically or dietetically acceptable carriers are substances which are suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response), i.e. with a reasonable benefit/risk ratio. The pharmaceutically or dietetically acceptable carrier can also contain natural extract such as licorice extract, nutrient supplements such as vitamins and trace elements, dietary fiber and dextrin.

"pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art and may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers.

In another preferred embodiment of the present invention, the aforementioned dietetically acceptable carrier or excipient may comprise: fillers, disintegrants, lubricants, glidants, effervescent agents, flavoring agents, coating materials, dietary preparations, or sustained release agents.

The dosage form of the composition of the present invention is not particularly limited, and may be any dosage form suitable for administration to a mammal; preferably, the dosage form can be selected from capsules, soft capsules, powder, tablets, granules, oral liquid, spray, cream, emulsion, water or paste and the like.

The composition of the present invention includes a pharmaceutical composition, a food composition, a nutraceutical composition, a food ingredient composition, a dietary supplement composition, a natural pharmaceutical raw material composition, or a cosmetic functional ingredient composition; it can also be health beverage, wine, etc. As long as they contain or consist essentially of cedar seed extract.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

Summary of intelligence-promoting activity of BZ-1-SFC extracted from Cupressus orientalis and conversion of adult dose

The equivalent dose ratio of mouse and human body surface was 387.9 according to the tabulated data in pharmacological testing methodology (national institutes of health, fourth edition). Taking the mouse 1mg/kg dose as an example, the human dose of 0.02kg (mouse average body weight) 387.9/70kg (human average body weight) of 0.11mg/kg was calculated.

In another preferred embodiment, according to the present invention data, the effective dose range of the Cupressus Sempervirens extract is 1-50mg/kg, and the dose is 0.1-5mg/kg in terms of adult. However, the drug effect may fluctuate due to differences in metabolism of the drug in different species of animals and differences in molecular biology of different species of animals. Therefore, the dosage can be respectively floated by 10 times above and below the calculated dosage, and the dosage is converted into adult dosage which is 0.01-50 mg/kg.

Similarly, after the optimal dose for use in mice is determined, the optimal dose for use in adults can also be easily predicted by this method.

The invention has the beneficial effects that:

1. the cedar seed extract has high activity and small application dose: the cedar seed extract with shell provided by the invention has the effects of improving cognitive ability and cognitive dysfunction under low dosage (0.01-50mg/kg, preferably 2-10mg/kg (mouse), 0.02-11mg (human), more preferably 4-6mg/kg (mouse), 0.04-6.6mg/kg (human)), and the activity is greatly increased (250, 500mg/kg body weight (mouse)) compared with the traditional cedar seed extract, so that the cedar seed extract is easier to apply, and is helpful for improving the administration compliance of an application subject.

3. The extraction method has sufficient sources, is economical and convenient: the method adopts extraction of shelled cedar seeds to replace the traditional extraction of shelled cedar seed, has better effect than the shelled cedar seed, and greatly improves the convenience and the economy of raw materials, thereby reducing the production cost and being more beneficial to industrial production and popularization.

Example 1: preparation of cedar seed extract

1.1SFC extraction: 288g of shelled cedar seeds are crushed and put into an SFC (SFC instrument model HA221-40-11, Jiangsu Nantong Huaan supercritical extraction Co., Ltd.), and the extraction conditions are as follows: temperature of the extraction kettle: temperature and pressure of the extraction vessel at 32 ℃: 25 MPa; temperature of the separation kettle: temperature and pressure of the separation vessel at 38 ℃: 6 MPa; supercritical CO₂A flow rate; 30-40L/h; extraction time: and (5) h. Cacumen Platycladi23.2g of extract BZ-1-SFC, the yield is 8.0 wt%.

1.2 extraction of petroleum ether: pulverizing 5.2g of shelled cedar seed, placing into 500ml conical flask, adding 250ml petroleum ether, and ultrasonic extracting for 3 times each for 1 hr. Mixing the petroleum ether extractive solutions, and distilling under reduced pressure to obtain active site BZR-1-PE 1.2g with yield of 23.1 wt%.

Comparative example 1 preparation of arborvitae seed extract

1.1c SFC extraction: 270g of shelled cedar seed is taken out, crushed and put into SFC (SFC instrument model HA221-40-11, Jiangsu Nantong Huaan supercritical extraction Co., Ltd.), and the extraction conditions are as follows: temperature of the extraction kettle: temperature and pressure of the extraction vessel at 32 ℃: 25 MPa; temperature of the separation kettle: temperature and pressure of the separation vessel at 38 ℃: 6 MPa; supercritical CO₂A flow rate; 30-40L/h; extraction time: and (5) h. 23.0g of BZR-SFC of the arborvitae seed extract is obtained, and the yield is 8.5 wt%.

1.2c Petroleum Ether extraction: pulverizing 5g shelled semen Platycladi, placing into 500ml conical flask, adding 250ml petroleum ether, and ultrasonic extracting for 3 times each for 1 hr. Mixing the petroleum ether extractive solutions, and distilling under reduced pressure to obtain active site BZR-PE 1.2g with yield of 24 wt%.

1.3c ethanol extraction: 20.45g of shelled cedar seed was taken to prepare the cedar seed extract BZR-E2.11g with a yield of 10.3 wt% according to the method provided in the literature (Nishiyama N, et al., Phytotherapy Research,1992,6, 289-293).

Example 2: determination of content of labdane diterpene arasequoic acid and 13-epiphellanic acid (13-epicupressic acid) in semen Platycladi or semen Platycladi extract

2.1. Separation and purification of labdane diterpene arasequoia acid (arboricid) and 13-epiaretic acid (13-epicuprisic acid) in arborvitae seed extract:

crushing dried semen Platycladi (10.2kg) by the method of Wang YZ.et al, Phytochemistry,2008,69, 518-526-; to obtain 2.54g of compound 13-thujalic acid (13-epicupressic acid). The spectral data of the compounds aragonic acid and 13-thujalic acid (13-epicupressic acid) are as follows:

aragonic acid, an arboricic acid

White powder, [ alpha ]]_D(20℃)+50(c 1.5,CHCl3)；¹H-NMR(CDCl₃,300MHz):δ0.60(3H,s,CH₃-20),0.90(3H,d,J＝6.4Hz,CH₃-16),1.22(3H,s,CH₃-18),3.68(2H,m,H-15),4.50(1H,s,H-17),4.84(1H,s,H-17).¹³C-NMR(CDCl₃75MHz Δ 12.7(q, C-20),19.8(q, C-16),19.9(t, C-2),21.1(t, C-11),26.0(t, C-6),28.9(q, C-18),30.2(t, C-13),36.4(t, C-12),38.0(t, C-3),38.7(t, C-7),39.1(t, C-1),39.5(t, C-14),40.5(s, C-10),44.1(s, C-4),56.3(d, C-5),56.6(d, C-9),61.2(t, C-15),106.3(t, C-17),148.2(s, C-8),183.3(s, C-19). The above data are reported in the literature [ Su, W.C., Fang, J.M., Cheng, Y.S.Labdanes from Cryptomeria japonica. phytochemistry,1994,37, 1109-.]And (5) the consistency is achieved.

13-Epimeric acid (13-epicupressic acid)

White powder, [ alpha ]]_D(20℃)+57(c 4.5,CHCl₃)；¹H-NMR(CDCl₃,300MHz):δ0.58(3H,s,CH₃-20),1.22(3H,s,CH₃-18),1.26(3H,s,CH₃-16),4.43(1H,s,H-17),4.82(1H,s,H-17),5.02(1H,dd,J＝10.7,1.0Hz,H-15),5.20(1H,dd,J＝17.3,1.0Hz,H-15),5.90(1H,dd,J＝17.3,10.7Hz,H-14)；¹³C-NMR(CDCl₃75MHz Δ 12.7(q, C-20),17.8(t, C-11),19.8(t, C-2),26.0(t, C-6),27.9(q, C-16),28.9(q, C-18),37.9(t, C-3),38.7(t, C-7),39.1(t, C-1),40.6(s, C-10),41.3(s, C-12),44.1(s, C-4),56.3(d, C-5),56.4(d, C-9),73.8(s, C-13),106.5(t, C-17),111.7(t, C-15),144.7(d, C-14),148.0(s, C-8),183.5(s, C-19). The above data are reported in the literature [ Su, W.C., Fang, J.M., Cheng, Y.S.Labdanes from Cryptomeria japonica. phytochemistry,1994,37, 1109-.]And (5) the consistency is achieved.

2.2. Determination of content of kameric acid and 13-thujalic acid (13-epicupressic acid) in cedar seed extract

The content of labdane diterpene arasequoic acid and 13-thujalic acid (13-epicupressic acid) in the cedar seed active site is determined by adopting a High Performance Liquid Chromatography (HPLC) method, and the liquid phase conditions are as follows:

mobile phase: dichloromethane: gradient elution with acetonitrile 30:70

A detector: evaporative Light Scattering Detector (ELSD)

Flow rate: 1ml/min

Column chromatography: waters Acquity Column BEH RP-C18,1.7 μm, 2.1X 100mm

Column temperature: 40 deg.C

The determination method comprises the following steps:

preparation of a test solution: accurately weighing 20.0mg of active site, dissolving dichloromethane, transferring to a volumetric flask of 100ml, and fixing the volume to a scale to obtain a test solution;

preparation of control solutions: respectively weighing labdane type diterpene arasequoic acid and 13-epibatic acid (13-epicuprisic acid) 10mg each, precisely weighing, and adding dichloromethane to obtain 1ml solution containing 200 μ g.

Respectively and precisely measuring 20 μ l of reference substance and sample solution, adding into automatic sample injector of high performance liquid chromatograph, and measuring.

2.3. The results of the content measurement are shown in Table 1

TABLE 1 content of diterpene in the active fraction of Platycladi seed or Platycladi seed

Example 3: determination of intelligence promoting activity of semen Platycladi/semen Platycladi extract series

Comparison of semen Platycladi/semen Platycladi extract series and fish oil as health product on the market for improving mouse dysmnesia model caused by scopolamine (scopolamine)

3.1 Experimental animals

KM mice, 24-30g male and female.

3.2 sample information

The arborvitae seed SFC extract (BZ-1-SFC), arborvitae seed SFC extract (BZR-SFC), arborvitae seed petroleum ether extract (BZR-PE) and arborvitae seed ethanol extract (BZR-E) are extracted from arborvitae seed by the method in example 1; fish oil was purchased from General Nutrition Corporation, GNC, USA.

3.3 Experimental Equipment

Channel type mouse water maze (80cm X50 cm X20 cm)

3.4 sample treatment

1) The compounds were dissolved in 20 wt% aqueous glycerol and 2mg/kg of 5 other compounds were measured except for 20mg/kg of BZR-E and administered orally at 0.1ml/10g of body weight. (the dissolution method and the concentration were determined by reference to the conventional protocol, and the concentration was determined by reference to BZR-E and then was reduced by one order of magnitude for preliminary experiments.)

2) Scopolamine (Scopolamine-damaging agent): 4.5mg/kg, administered by intraperitoneal injection at a volume of 0.1ml/10g body weight.

3) HupA (huperzine A-positive control): 0.1mg/kg, administered orally at 0.1ml/10g volume body weight.

3.5 Experimental methods

The water maze was trained for 4 days, once in the morning and afternoon. And selecting mice with standard training performance after the training on the fourth day, and carrying out drug test on the fifth day. And (3) testing: after 40min of oral administration of the various doses of the compound, 4.5mg/kg of scopolamine (Saline was given to the control group) was intraperitoneally injected, and 20min later, the time to reach the plateau and the number of times of entry into the cul-de-sac of the test mice were within 1 min.

3.6 results of the experiment

The effect of the active sites BZR-SFC and BZ-1-SFC on scopolamine-induced learning and memory impairment in the water maze model is shown in Table 2 and FIG. 1.

TABLE 2 influence of BZR-SFC, BZ-1-SFC, BZR-E on scopolamine induced learning and memory disorders in mice

P <0.01, P <0.005vs 20 wt% aqueous glycerol solution; # P <0.01, # P <0.005vs scop

3.7 conclusion of the experiment

The active sites of the platycladi seeds obtained by different extraction methods and the commercially available fish oil have different memory improvement effects on mouse dysmnesia caused by scopolamine, wherein the effect of the platycladi seed extract BZ-1-SFC with shell is superior to that of the extract obtained by other methods, the platycladi seed extract BZR-E with shell has equivalent effect to that of the extract obtained by other methods, but the dosage of the BZR-E is 10 times of that of the BZ-1-SFC.

Example 4 determination of the optimal amount of BZ-1-SFC nootropic Effect of the Platycladi extract

Based on the results of example 3, the nootropic optimal dose of the cypress seed extract BZ-1-SFC was further determined.

4.1 Experimental animals

KM mice, 24-30g male and female.

4.2 sample information

The cedar seed extract BZ-1-SFC was obtained by extracting cedar seeds in the same manner as in example 1; flaxseed oil is a commercially available drug.

4.3 Experimental Equipment

Channel type mouse water maze (80cm X50 cm X20 cm)

4.4 sample treatment

1) BZ-1-SFC was dissolved in 20 wt% aqueous glycerol and the concentrations of BZ-1-SFC measured were 1mg/kg, 2mg/kg, 4mg/kg, 6mg/kg and 8mg/kg, and administered orally at a volume of 0.1ml/10g of body weight.

2) Scopolamine (Scopolamine-damaging agent): 4.5mg/kg, administered by intraperitoneal injection at a volume of 0.1ml/10g body weight.

3) HupA (huperzine A-positive control): 0.1mg/kg, administered orally at 0.1ml/10g volume body weight.

4.5 Experimental methods

The water maze was trained for 4 days, once in the morning and afternoon. And selecting mice with standard training performance after the training on the fourth day, and carrying out drug test on the fifth day. And (3) testing: after 40min of oral administration of the various doses of the compound, 4.5mg/kg of scopolamine (Saline was given to the control group) was intraperitoneally injected, and 20min later, the time to reach the plateau and the number of times of entry into the cul-de-sac of the test mice were within 1 min.

4.6 results of the experiment

The effect of different doses of BZ-1-SFC on scopolamine-induced learning and memory impairment in the water maze model is shown in table 3 and figure 2:

TABLE 3 Effect of different doses of the active site BZ-1-SFC on scopolamine induced learning and memory disorders in mice

P <0.005vs 20 wt% glycerol solution; # P <0.01, # P <0.005vs scop

As can be seen from Table 3 and FIG. 2, the BZ-1-SFC extract in multiple doses reduced the time to the escape plateau of mice to various degrees, while 6mg/kg showed the best improvement; in addition, the error frequency of entering the blind end of the mice can be reduced under multiple doses of BZ-1-SFC, and the effect is most obvious at 6 mg/kg. The action intensity is equivalent to that of positive control huperzine A.

4.7 conclusion of the experiment

From the above results, the arborvitae seed extract BZ-1-SFC has an effect of improving the learning and memory disorder caused by scopolamine in the dosage range of 1-8mg/kg, wherein the effect is the best when the dosage is 6 mg/kg.

Summarizing the above experimental results, it was found that in a mouse model, the cupressus extract with husk provided by the present invention has the effects of improving cognitive ability and cognitive dysfunction at a low dose (0.01-50mg/kg, preferably 2-10mg/kg (mouse), 0.02-11mg/kg (human), more preferably 4-6mg/kg (mouse), 0.04-6.6mg/kg (human)), and the activity is greatly increased (250, 500mg/kg body weight (mouse)) compared with the traditional cupressus extract, so that the application is easier, thereby being helpful for improving the administration compliance of the administration subjects.

Example 5: determination of anti-fatigue activity of active part of platycladi seed

Whether the test compound can improve physical fatigue on a running exhaustion model of a mouse or not is tested; and simultaneously compared with Flaxseeds oil.

5.1 Experimental animals

C57/BL6 mice, 18-25g male and female combined.

5.2 Experimental Equipment

Mouse plane running machine (6 channel)

5.3 sample treatment

BZ-1-SFC, linseed oil (available from Nature Made Nutritional Products, USA), dissolved with 20 wt% glycerol in water. And (3) measuring the concentration: the BZ-1-SFC adopts 36mg/kg, 6mg/kg and 1mg/kg, and is orally administered according to the volume of 0.1ml/10g body weight.

5.4 Experimental methods

The experimental period for mouse exhaustion was 10 days. Running adaptively on day 1 (15m/min, 10min, no shock); mice were at 15m/min, 5min on day 2; 20m/min, 5 min; 25m/min, 5 min; screening physical ability at a slowly increasing speed of 30m/min and 45min (with electric shock: 0.4mA), and recording the exhaustion time and the electric shock frequency of the mouse within 30m/min and 45 min; continuous oral administration of test compound on days 3-9; the physical performance test was performed on day 10 at a rate of 20m/min, the last oral administration (maximum time limit was 120 minutes) was performed 1 hour before the test, and the time spent and number of shocks were recorded for the mice.

5.5 results of the experiment

The results of the improvement of physical fatigue of mice by the arborvitae seed active site BZ-1-SFC are shown in Table 4 and FIG. 3.

TABLE 4 improvement of physical fatigue in mice by different doses of the active site BZ-1-SFC

5.6 conclusion of the experiment

According to the mouse exhaustion time (figure 3), the BZ-1-SFC is found to have enhanced anti-fatigue effect along with the increase of the tested concentration. Wherein 36mg/kg can obviously improve the physical fatigue phenomenon of mice, and has significant statistical significance.

Example 6 determination of the optimum dose Range of the anti-fatigue Effect of BZ-1-SFC extract of Cupressus orientalis

Based on the results of example 6, the fatigue-resistant optimum dose range of the cypress seed extract BZ-1-SFC was further determined.

6.1 Experimental animals

C57/BL6 mice, 18-25g male and female combined.

6.2 Experimental Equipment

Home-made mouse running machine (6 channel)

6.3 sample treatment

Both BZ-1-SFC and Flaxseeds oil were dissolved with 20 wt% aqueous glycerol. And (3) measuring the concentration: 54mg/kg, 36mg/kg, 18mg/kg, 6mg/kg, 2mg/kg and 1mg/kg of BZ-1-SFC are selected; flaxseeds oil was administered orally at 2mg/kg, at 0.1ml/10g volume body weight.

6.4 Experimental methods

The experimental period for mouse exhaustion was 10 days. Running adaptively on day 1 (15m/min, 10min, no shock); mice were at 15m/min, 5min on day 2; 20m/min, 5 min; 25m/min, 5 min; screening physical ability at a slowly increasing speed of 30m/min and 45min (with electric shock: 0.4mA), and recording the exhaustion time and the electric shock frequency of the mouse within 30m/min and 45 min; continuous oral administration of test compound on days 3-9; the physical performance test was performed on day 10 at a rate of 20m/min, the last oral administration (maximum time limit was 120 minutes) was performed 1 hour before the test, and the time spent and number of shocks were recorded for the mice.

6.5 conclusion of the experiment

It is found that the anti-fatigue effect of BZ-1-SFC is enhanced along with the increase of the tested concentration. Wherein the dosage range of 18-36mg/kg can obviously improve the physical fatigue phenomenon of mice, and has significant statistical significance.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.