KR100481684B1 - Preventives and remedies of neurodegenerative disease containing pinusolide derivatives as active ingredients - Google Patents

Abstract

The present invention relates to the use of a pinusolidide derivative having the structure of the formula (1) as a prophylactic and therapeutic agent for degenerative cranial nervous system diseases, and to a cypress leaf extract having a prophylactic and therapeutic activity for degenerative cranial nervous system diseases.

(One)

In the above formula, R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ₂ is a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms.

Description

Preventive and remedies of neurodegenerative disease containing pinusolide derivatives as active ingredients

The present invention relates to the use of a pinusolidide derivative having the structure of the formula (1) as a prophylactic and therapeutic agent for degenerative cranial nervous system diseases, and to a cypress leaf extract having a prophylactic and therapeutic activity for degenerative cranial nervous system diseases.

(One)

In the above formula, R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ₂ is a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms.

The population distribution in modern society is changing rapidly with the aging trend. Accordingly, the incidence of diseases associated with aging, such as degenerative neurological diseases such as stroke and dementia, is also increasing. Cerebral nervous system disease is a disease in which mortality increases every year, and it is ranked second after cancer in analyzing the causes of death in Korea, and it is a serious disease that occupies the second to third place of death in the world. However, there are no clear medicines or treatments that can cure them, and the social and economic losses are getting worse.

Neurodegenerative diseases can occur when the brain is damaged by various physical and mechanical factors, such as secondary symptoms caused by other adult diseases such as structural degeneration of the neuronal cells due to aging, circulatory disorders, or traffic accidents, industrial accidents, and carbon monoxide poisoning. have .

Damage to the brain tissue results in the death of neurons, neuronal cell death is largely divided into two.

First, it is caused by glutamate, an excitatory neurotransmitter in the brain. Glutamate acts as a neurotransmitter in the normal state, but excessive secretion by various causes causes the death of neurons. Neurotoxicity caused by glutamate is divided into acute toxicity and chronic toxicity. Acute toxicity begins with the extra inflow of extracellular Ca ²⁺ into the cell, followed by the influx of ions such as Na ⁺ , K ⁺ and Cl ^- to maintain the osmotic pressure of the cell, causing the cell to swell and eventually burst and die. appear. Chronic toxicity is that glutamate receptors, N-methyl-D-aspartate (NMDA) and non-NMDA receptors, activate Ca ²⁺ into the cell, resulting in intracellular calcium Calcium-dependent enzymes are overactivated, resulting in cell death.

Second, damage is caused by direct oxidative stress. The ratio of unsaturated fatty acids and consumption of oxygen is high in the brain, which leads to high production of oxygen radicals, but relatively low amounts of antioxidant enzymes compared to other organs, resulting in free radicals containing oxygen radicals. The risk of injury is high, which is also known as the damage mechanism when reperfusion occurs after ischemia.

As described above, researches on neuronal cell death mechanisms related to degenerative cranial nervous system disease have been actively conducted from various angles, and researches to develop medicines that can prevent and treat them based on this are being actively conducted.

Accordingly, the present inventors searched for neuroprotective activity of natural products using the cerebral cortical cells of primary cultured rats that induced neurotoxicity with glutamate to find a substance that can prevent or treat degenerative neurological diseases from natural products. ( Biota orientalis Endl, Thuja orientalis L., Platycladus orientalis L.) It was confirmed that the total methanol extract of the leaves had significant protective activity.

The cypress ( Biota orientalis Endl, Thuja orientalis L., Platycladus orientalis L.) is an evergreen conifer plant belonging to the family Cupressaceae , native to Korea, Japan and China and used for garden trees and hedges. The young branches and leaves of the cypress tree have the effect of bleeding, hemostasis, and giant wind. The baekle leaves are bleeding, non-bleeding and long wind in folk medicine and oriental medicine. (腸 風), customs pain, (濕 鼻 痛), high blood pressure (高血壓) has been used as a therapeutic agent. The chemical components of the cypress are essential oils such as tujene, fenchone, pinene and cayophyllene, biotol, cedrene, cedrol and Sesquiterpenes such as cuparene have been reported. Diterpene and quercetin, such as pinusolide, totarol and 8b-hydroxy-3-oxopimara-15-ene ), Flavonoid components such as quercitrin, myricetin, hinokiflavone and amentoflavone have also been reported. On the other hand, deoxypodophyllotoxine as an anticancer component and pinusolid as a PAF-antagonist have been reported through studies on the bioactivity of cypress leaves. However, no biological activity on the nervous system has been reported.

Accordingly, the present inventors have conducted basic research to develop extracts of cypress leaves, their CH ₂ Cl ₂ fractions, and single substances separated therefrom as preventive and therapeutic agents for degenerative neurological diseases, and degenerative neurological diseases such as stroke and senile dementia. It is suggested as a candidate material that can be developed as a preventive and therapeutic agent for.

It is an object of the present invention to provide a cypress leaf extract having a novel use of a pinusolide derivative having a structure of the formula (1) and a prophylactic and therapeutic activity for degenerative neurological diseases.

The present invention provides a prophylactic and therapeutic agent for degenerative cerebral nervous system diseases comprising a pinusolid derivative having the structure of formula (1) as an active ingredient:

(One)

In the above formula, R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ₂ is a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms.

The present invention also provides a compound having the structure of formula (2):

(2)

Among the compounds having the structure of formula (1), the compound of formula (2) wherein R ₁ is a hydrogen atom and R ₂ is a methoxy group is a novel compound reported for the first time in nature. -methoxypinusolidic acid), wherein R ₁ is a methyl group and R ₂ is a hydrogen atom, the compound of formula (3) is Pinusolide (Pinusolide).

(3)

In another aspect, the present invention is extracted with C _1-4 alcohol, or suspended again with water and fractionated with CH ₂ Cl ₂ , or the CH ₂ Cl ₂ fraction is extracted again with C _1-4 alcohol, or the extracts Chromatography provides a method for producing the extract of cypress leaves. The present invention also provides an agent for preventing and treating neurodegenerative diseases of the cedar leaf extract as an active ingredient.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention provides a novel use of the pinusolide derivative of formula (1) as an agent for the prevention and treatment of degenerative neurological diseases:

(One)

In the above formula, R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ₂ is a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms.

The pinusolid derivative of the formula (1) of the present invention may be synthesized by a conventional synthesis method from the pinusolid, which is a compound of the formula (3), which is isolated or separated and purified from a cypress leaf.

The present invention provides a cypress leaf extract having the activity of preventing and treating degenerative cerebral nervous system disease from which the cypress leaf is extracted with an organic solvent. The organic solvent may be any solvent that can be used conventionally, preferably C _1-4 alcohol, most preferably methanol. In addition to the above process, the present invention provides a water fraction and a dichloromethane fraction in which the crude C _1-4 alcohol extract is suspended in water and then fractionated with dichloromethane (CH ₂ Cl ₂ ). It also provides a C _1~4 alcohol extracted fraction of the CH ₂ Cl ₂ fraction back to the C _1~4 alcohol.

Specifically, the present invention will be described with an example of the extraction process of cypress leaves.

The white leaf is ground, and then ultrasonically extracted with methanol, filtered and concentrated under reduced pressure to obtain a white leaf methanol extract. In addition to this process, the methanol extract is suspended in water and fractionated with dichloromethane (CH ₂ Cl ₂ ) to obtain a water fraction and a dichloromethane fraction. The dichloromethane fraction was further fractionated with methanol and hexane to obtain a secondary methanol fraction. The secondary methanol fraction was, for example, silica gel chromatography using hexane-ethyl acetate-methanol mixed solution, and recrystallized to obtain a compound of formula (2) and a compound of formula (3).

These chemical structures were determined by various physicochemical and spectroscopic analyzes of 15-methoxypinusolide (2) and pinusolide (3), which are pinusolide derivatives. The substance being reported separately.

The physicochemical and spectroscopic properties of the compound of formula (2) and the compound of formula (3) are described in the Examples below.

The cypress leaf extract and the compounds of formula (2) and the compounds of formula (3) isolated and purified therefrom were searched for the prophylactic and therapeutic activity of neurodegenerative diseases.

After culturing the cerebral cortex cells directly isolated from the fetus of the rat, the sample to be measured for activity was treated to the cultured cells by concentration, and then treated with glutamate to induce toxicity to neurons, and after 24 hours The neuronal protective activity of the samples was determined by measuring the survival rate of the cells by MTT assay. In addition, after administering the sample to the cerebral cortical cells cultured for 12 days or more, the cultured cells were cultured for 1-2 hours, and then the survival rate of the cells was measured by MTT assay on cultured cells induced by artificial neuronal cell death with staurosporin. Judging the protective activity against death

As a result, the cypress leaf extract of the present invention, the CH ₂ Cl ₂ fraction, the secondary methanol fraction, the compound of formula (2) and the compound of formula (3) isolated therefrom are effective for preventing and treating neurodegenerative diseases. Indicated.

The cypress leaf extract of the present invention, the CH ₂ Cl ₂ fraction, the secondary methanol fraction or the compound of formula (1) may be formulated by methods known in the pharmaceutical art, and may be used on their own or in a pharmaceutically acceptable carrier, It may be mixed with excipients and the like to be formulated into a pharmaceutically conventionally acceptable pharmaceutical preparation, such as injections, solutions, syrups, tablets, capsules, etc., the compounds of formula (1) of the present invention By means of a pharmaceutically acceptable inorganic or organic salt. They can also be administered orally or parenterally.

The dose of the cypress leaf extract of the present invention, the CH ₂ Cl ₂ fraction, the secondary methanol fraction or the compound of the formula (1) is determined by the absorption rate, excretion rate, age and weight of the patient, sex and condition, Although appropriately selected depending on the severity of the disease to be treated and the like, in general, it is preferable to administer to adults at 0.01 to 500 mg / kg, preferably 0.1 to 200 mg / kg per day, more preferably the cypress leaf extract of the present invention, The CH ₂ Cl ₂ fraction and the secondary methanol fraction may be administered 1 to 500 mg 1-3 times daily. The compound of formula (1) of the present invention may be administered 1 to 200 mg 1-3 times daily. The unit dosage form so formulated may be administered several times at regular time intervals as needed.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for illustrative purposes of the present invention and are not intended to limit the protection scope of the present invention.

The reagents and instruments used in the examples below are as follows.

Kiesgel 60 (Merck Art. 9385), Sephadex LH-20 (Pharmacia) for column chromatography and precoated-Kiesgel 60 F ₂₅₄ (Merck Art. 5715), RP-18 (Merck Art. 15685) Used. Extraction of plant material and solvent for column chromatography were first distilled from reagent (Duksan Chemical Co.), and solvent for semi-preparative HPLC was used as HPLC grade solvent (EM Sciences). Melting points were measured using a Buchi micro melting point measurement device, the optical luminosity was measured using a JASCO DIP-1000, UV for Shimadzu UV-1601PC UV-VIS spectrophotometer, and IR for Perkin Elmer 1710 spectrophotometer. NMR used JEOL GSX 400 spectrometer and JEOL LA 300 spectrometer, and mass was measured using VG Trio 2 spectrometer. HPLC was performed using Hitachi L-6200 pump and Hitachi L-4000 UV detector.

Example 1 Extraction and Fraction of Cucumber Leaf

The cypress leaf was purchased at Gyeongdong Market in June 1997, and after botanical appraisal, corroborated specimens were stored in the herbarium of Seoul National University College of Pharmacy.

9.0 kg of the cypress leaves were ultrasonically extracted three times with 80% methanol for 3 hours, and then concentrated under reduced pressure to obtain a methanol extract (830 g). This was suspended in deionized water, fractionated with dichloromethane and concentrated under reduced pressure to obtain a dichloromethane fraction (400 g), which was then partitioned between 90% methanol and hexane to obtain a 90% methanol fraction (110 g) (see Figure 1).

Example 2

Isolation and Structural Determination of Compounds of Formula (2) and Pinusolide (3) from Cucumber Leaf Extracts

In order to purely separate and purify the neuronal protective active ingredient, the 90% methanol fraction was subjected to silica gel column chromatography using a mixed solution of hexane-ethyl acetate-methanol to 30 fractions (fr.1 to fr.30). Divided. Fraction 10 (fr. 10) of these was carried out again by silica gel column chromatography using a mixed solution of hexane-ethyl acetate was divided into 15 small fractions (fr. 10-1 to fr. 10-15). Eight subfractions (fr.10-8) were divided into ten subfractions (fr.10-8-1 to fr.10-8-10) by C18 reverse phase column chromatography, and fr.10- The 8-7 fractions were subjected to RP-HPLC using a water-methanol-acetonitrile (45:20:35) mixed solvent to separate the compound of formula (2) (65 mg). In addition, fraction 9 (fr. 9) was subjected to conventional silica gel chromatography to separate the compound of formula (3) (see FIG. 2).

The physicochemical and spectroscopic properties of the compound of formula (2) and the compound of formula (3) separated and purified above are as follows.

Compound of formula (2) (15-Methoxy-8 (17), 13-labdadien-16,15-olide-19-oic acid) :

Light yellowish oil,

[a] ²⁵ _D + 43 ° (c 0.83, CHCl ₃ ),

IR (KBr) n _max : 3450, 2923, 1750, 1665, 1110 cm ^-1 ,

¹ H-NMR (CDCl ₃ ) and ¹³ C-NMR (CDCl ₃ ): see Table 1 below,

EIMS (70 eV) m / z (rel. Int.): 362 [M] ⁺ (3), 344 [MH ₂ O] ⁺ (6), 317 [M-COOH] ⁺ (13), 285 (13), 235 (22), 189 (40), 128 (100), 96 (59), 81 (28),

HREIMS m / z [M] ⁺ : 362.2088 (calcd for C ₂₁ H ₃₀ O ₅ , 362.2093),

Chemical structure:

(2)

Table 1. ¹ H and ¹³ C NMR results of a compound of Formula (2) (CDCl ₃ )

Compound of formula (3) (Pinusolide) :

C ₂₁ H ₃₀ O ₄ , white needle,

Rf = 0.11 ( n- hexane: EtOAc = 10: 1), mp 82-83 ° C.,

[a] _D = + 58.5 ° (c 0.1 in CHCl ₃ ),

IR n _max (neat): 2950 (-OH), 1755 (C = O of lactone) cm ^-1 ,

EIMS (70 eV) m / z (rel. Int.): 346 [M ⁺ ] (0.6), 287 [M-COOCH ₃ ] ⁺ (15), 217 (10, 121 (100), 109 (27), 81 (22), 79 (23),

¹ H NMR (300 MHz, CDCl ₃ ) d: 0.51 (3H, s , 20-CH ₃ ), 1.11 (3H, s , 18-CH ₃ ), 3.62 (3H, s , 18-OCH ₃ ), 4.58 (1H , br s , 17a-H), 4.77 (1H, br s , 17b-H), 4.89 (2H, br s , 15-H), 7.10 (1H, br s , 14-H),

¹³ C NMR (75 MHz, CDCl ₃ ), d: 12.59 (20-C), 19.94 (2-C), 21.85 (11-C), 24.64 (12-C), 26.23 (6-C), 28.81 (18 -C), 38.21 (3-C), 38.67 (7-C), 39.22 (1-C), 40.29 (10-C), 44.31 (4-C), 51.14 (21-C), 55.69 (9- C), 56.30 (5-C), 70.11 (15-C), 106.68 (17-C), 134.78 (13-C), 143.82 (14-C), 147.47 (8-C), 174.33 (16-C = O), 177.71 (19-C = O),

Chemical structure:

(3)

Experimental Example 1 Activity Study of Cypress Leaf Extract

Laboratory animals

Sprague-Dawley rats were supplied from the Seoul National University Animal Breeding Farm and were bred in the experimental animal laboratory of the College of Pharmacy, Seoul National University. The environment of the kennel was kept at 22 ± 5 ℃ and the lighting time was fixed from 7 am to 7 pm, and the feed was 23.2% of crude protein, 4.0% of crude fat, 6.0% of crude fiber, 10.0% of crude ash and 0.6% of crude calcium. Solid feed (Seoul, Samyang) containing 0.4% was used.

Isolation and Culture of Cerebral Cortical Cells in Rats

Only the cerebral portion of the rat fetus was extracted, and carefully removed the meninges under the dissecting microscope, and the cerebral tissues were treated with 0.25% trypsin for 30 minutes to soften the tissues and liberated into individual cells. The isolated cerebral cortical cells were transplanted into a culture vessel coated with collagen to 1.0 × 10 ⁶ cells / ml. The culture solution was made up of 90% DMEM, 10% fetal bovine serum, and 1 mM sodium pyruvate, 100 IU / ml penicillin and 100 g / ml streptomycin. Cultivation of the cells was carried out every 3 to 4 days with fresh medium and continuously supplied with a mixture of air (95%) and CO ₂ (5%) in a 37 ° C incubator (Kim YC, Kim SR, Markelonis GJ and Oh). TH (1998) Ginsenosides Rb1 and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration. J. Neurosci. Res. 53: 426-432).

Dosing of the sample

To the cerebral cortical cells of rats cultured for 15 days, total methanol extract and each fraction of the cypress leaf were administered at a concentration of 50 μg / ml, and 100 μM glutamate was applied for 30 minutes and incubated for 24 hours.

Induction of Neurotoxicity by Glutamate

After culturing the cerebral cortical cells directly isolated from the fetus of rats for 14 days, the samples to be measured for activity were treated to the culture cells by concentration, and then treated with 100 μM glutamate one hour later to induce toxicity to neurons. After 24 hours, the neuronal protective activity of the sample was determined by measuring the survival rate of the cells by MTT assay.

MTT assay

MTT (5 mg / mL) was added to 10% of the culture medium in the culture of cerebral cortical cells under cultivation, and after further incubation for 3 hours, the resultant formazan was dissolved in DMSO and absorbance at 540 nm. (Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immuno. Methods 65: 55-61).

Experiment result

Cerebral neuroprotective activity against glutamate toxicity of primary oriented cerebral cortical cells of the white-leafed methanol extract and its fractions of the present invention was measured by MTT assay, and is shown in Table 2 below.

TABLE 2

As shown in Table 2, when glutamate was administered to the primary cerebral cortical cells to induce neurotoxicity, the total extract of the cypress leaf extract, CH ₂ Cl ₂ extract, and 90% methanol extract showed significant neuroprotective activity. Showed. The 90% methanol fraction of the cypress leaf showed the best neuronal protective activity.

Experimental Example 2 Investigation of the Activity of the Compound of Formula (2) and the Compound of Formula (3)

Dosing of the sample

The compound to be searched for the neuroprotective activity was dissolved in DMSO (within 0.1% final concentration), diluted with distilled water, and passed through a millipore membrane (0.22m, Millex-GV, USA). After making it into a state, it was administered to the brain neurons cultured at different concentrations.

Induction of neuronal cell death by staurosporin

The sample was administered to cerebral cortical cells cultured for at least 12 days, incubated for 1 to 2 hours, and then dissolved in distilled water or DMSO (within 0.1% final concentration) at a concentration of 10 μM, followed by millipore membrane (0.22). μm, Millex-GV, USA) to make a sterile state, and then administration of the staurosporin to a concentration of 100 nM to induce apoptosis for 24 hours.

Experiment result

(1) Neuronal Protective Activity against Cerebral Cortical Cells in Rats Induced Toxicity with Glutamate

The neurotoxic blocking effect of two diterpenes of Example 2 was searched as in Experiment 1, using a screening system that induced toxicity of the cerebral cortical cells of primary cultures with glutamate. It is shown in Table 3 below.

TABLE 3

Note: The neuroprotective activity of MK-801 (dizocipline maleate), an NMDA receptor antagonist, used as a positive control was 91.7 ± 3.6 ^*** at a concentration of 10 μM. Significance: ^* p <0.05, ^** p <0.01 , ^*** p <0.001.

As shown in Table 3, it was confirmed that the compound of formula (2) of the present invention significantly blocks neurotoxicity by glutamate. Compared to the 0% survival rate of the cells which induced neurotoxicity by the glutamate which was not treated with the sample, the cell survival rate when the compound of Formula (2) was treated at a concentration of 10 mM was 72.7 ± 4.7%. When the compound of formula (3) was treated at a concentration of 10 mM, the cell viability was 42.3 ± 2.2%, showing a lower activity than the compound of formula (2).

(2) Neuroprotective activity against primary cultured cerebral cortical cells in rats induced by cell death with staurosporin

The neuronal protective effect of the two diterpenes of Example 2 on primary cultured cerebral cortical cells of rats induced by apoptosis with staurosporin was searched, and the results are shown in Table 4 below.

Table 4

compound Cell survival rate (%) 0.1 μM 1.0 μM 5 μM Control 100.0 ± 0.1 100.0 ± 0.1 100 ± 0.1 Staurosphlin treatment 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 15-methoxypinulic acid (2) 23.0 ± 1.4 56`.7 ± 5.4 ** 64.3 ± 6.8 *** Pinusolidic acid (3) 14.8 ± 3.4 23.8 ± 2.3 34.3 ± 2.2 **

The neuroprotective activity of cycloheximide, a protein synthesis inhibitor used as a positive control, was 73.5 ± 6.4 ^*** at a concentration of 1 μM. Significance: ^* p <0.05, ^** p <0.01, ^*** p <0.001.

As shown in Table 4, it was confirmed that the compound of formula (2) of the present invention significantly blocks apoptosis by staurosporin. Compared to 0% survival rate of cells induced by apoptosis by untreated staurosporin, the cell survival rate when the compound of Formula (2) was treated at a concentration of 5 μM was 64.3 ± 6.8%. The cell viability of the compound of formula (3) when treated at a concentration of 5 μM was 34.3 ± 2.2%, showing lower activity than the compound of formula (2).

The compound of formula (2) and the compound of formula (3) isolated from the cypress leaf extract as described above not only show a significant protective activity against excessive neuronal cytotoxicity induced by glutamate, but also brain neurons by staurosporin It was confirmed that the blocking of apoptosis significantly.

Next, the present invention will be described in more detail as formulation examples.

Formulation Example 1

10 mg of compound of formula (2)

Appropriate sterile distilled water for injection

pH adjuster

The compound of formula (2) is dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount is 2 ml, and then filled into a 2 ml ampoule and sterilized to prepare an injection.

Formulation Example 2

2 mg of compound of formula (3)

Appropriate sterile distilled water for injection

pH adjuster

The compound of formula (3) is dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, the total amount is 2 ml, and then filled into 2 ml ampoules to prepare an injection.

Formulation Example 3

10 mg of extract of the side of the extract of Example 1

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 4

10 mg of compound of formula (2)

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 5

5 mg of compound of formula (3)

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 6

5 mg of compound of formula (2)

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 7

50 mg of compound of formula (3)

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml brown bottles and sterilized to prepare a liquid.

Formulation Example 8

100 mg of compound of formula (2)

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml brown bottles and sterilized to prepare a liquid.

As can be seen from the above, the compound of formula (1) such as total methanol extract, CH ₂ Cl ₂ extract, secondary methanol extract, pinusolide, 15-methoxypinusolide, and the like of a cypress leaf include stroke and dementia It can be used as a prophylactic and therapeutic agent for neurodegenerative diseases.

Figure 1 and Figure 2 shows the process of preparing the cypress leaf extract of the present invention,

Figure 3 shows the correlation between HMBC and NOESY of the compound of formula (2) of the present invention.

Claims (6)

A prophylactic and therapeutic agent for degenerative cerebral nervous system diseases comprising a pinusolid derivative having the structure of formula (1) as an active ingredient: (One) In the above formula, R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ₂ is a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms. A compound having the structure of formula (2) (2) delete delete The agent for preventing and treating neurodegenerative neurological diseases according to claim 1, wherein the compound having the structure of formula (1) is a compound of formula (3): (3) An agent for the prevention and treatment of degenerative cerebral nervous system diseases comprising the compound having the structure of formula (2) according to claim 2 as an active ingredient.