WO2004103386A1

WO2004103386A1 - New use of baicalin for treating anxiety

Info

Publication number: WO2004103386A1
Application number: PCT/CN2004/000470
Authority: WO
Inventors: Hong Xue; Hui Zheng; Zihui Wang
Original assignee: Pharmaco Genetics Limited
Priority date: 2003-05-23
Filing date: 2004-05-12
Publication date: 2004-12-02
Also published as: CN1548053A

Abstract

The invention discloses use of baicalin, one medicinal active component extracted from the roots of Scutellaria Baicalensis, for treating various anxieties. It is confirmed that when the oral dose for mouse is in the range of 7.5-30mg/kg, baicalin shows significant effect of antianxiety. According to the radio of body weight of mouse to that of human, the individual dosage of baicalin for oral administration is 0.8-3.5mg/kg. Baicalin, extracted from the roots of Scutellaria Baicalensis and of not less than 97% pure, can be formulated as tablets, capsules, dropping pills, oral liquid and injection, etc. The formulations described above comprising baicalin as the substantial component show excellent effect of antianxiety. A lot of experiments prove that: as a C.N.S. regulating agent, baicalin shows credible ability of antianxiety. The invention not only has prominent pharmacological action, specific administration dosage and significant effect, but also is of safety, hypotoxicity, low cost and resourceful. In view of all respects, the effect of the invention is expected to precede the existing medicines.

Description

New use of baicalin as medicine for treating anxiety

The present invention relates to a new use of baicalin, an effective medicinal ingredient in rhizomes of Scutellaria baicalensis for preparing various medicines for treating various anxiety disorders. Background technique

Traditional Chinese medicine believes that Scutellaria baicalensis Georgi is the dried root of plants in the Labiatae family. Cold, taste bitter, return to the lungs; gall; spleen; large and small intestine meridians. Its medicinal functions are clearing away heat and dampness; purging fire and detoxifying; stopping bleeding; Traditional Chinese medicine is mostly used for damp heat, summer heat, chest tightness and nausea, fullness of damp heat, jaundice of diarrhea, lung fever, cough, fever, thirst, blood heat, vomiting, edema, soreness and fetal movement. (Chinese Pharmacopoeia, the first part of 1995, pp.270) Modern medical research proves that: The main medicinal ingredients contained in the rhizome of Scutellaria baicalensis are: Baicalin, whose content is about 3-15% of the weight of Scutellaria baicalensis plant. The baicalein (Wogonin) content is about 0.5-1.3% of the weight of the baicalein plant. Synonyms of baicalin: Begarin, English name: Baiccdin, Chemical name:

β— D— Glucopyranosiduronic acid, 5,6― dihydroxy— 4— oxo— 2— phenyl― 4H- 1 benzopyran- 7 ~ yl; its chemical structure is shown in Figure 1. The molecular formula and molecular weight are: C ₂₁ H ₁₈ O „and 446.35, physical property is yellow crystal, melting point is 223-225 ° C, UV absorption spectrum is: 242 (4.12); 271 (4.50); 310 (4.22) ] (See Figure 2). Its infrared spectrum is shown in Figure 3.-The pharmacological effects of baicalin currently studied and reported include-

1. Anti-inflammatory and anti-allergic effects;

2. Antibacterial and antiviral effects;

3. Hepatoprotective and choleretic effect;

4. Antihypertensive diuretic effect;

5. Sedative effect on the central nervous system;

6. Antipyretic effect;

7. Antispasmodic and antioxidant effects;

8. The therapeutic effect on diabetic patients;

9. Effects on cardiovascular and blood pressure;

10. Reduces fat and cholesterol in the blood.

[Modern Research and Application of Traditional Chinese Medicine, Volume 4: pp. 3943-3977; Editor of Modern Chinese Medicine Library Editing Committee, published by Xueyuan Press].

Until now, most commonly used anti-anxiety medications in the clinic have been chemical ingredients, such as: nitrostilbam; methazolam; flutezolam; etizolam and etifoxine hydrochloride. [Dade Yin: Manual of Practical New Drugs and Special Medicines, pp. 424-434; published by People's Military Medical Press] These drugs have a strong central nervous system inhibitory effect, and will produce drug dependence effects. Long-term large-scale use will produce more severe nerves. Inhibition and side effects such as drug dependence. At present, there are no reports about the pharmacodynamic effects of baicalin on various anxiety disorders at home and abroad. There are no clinically seen drugs or preparations containing baicalin as the main medicinal raw material to control and reduce various anxiety symptoms. Summary of the Invention

The object of the present invention is to provide a new anti-anxiety drug with low toxicity, low drug side effects, abundant drug sources, and effective treatment and alleviation of symptoms of various anxiety patients.

Baicalin compounds are the main medicinal ingredients in the rhizome of Scutellaria baicalensis. Experiments have shown that baicalin can competitively bind to γ-aminobutyric acid receptor (GABA receptor) functional sites in brain tissues, regulate central nervous system dysfunction, and thus achieve pharmacological effects of treating and reducing various anxiety symptoms . Currently, the GABA receptor is considered to be an amino acid conductor that plays a major inhibitory role in the central nervous system of lactating animals. GABA transmits many of its information activities through GABA receptors confined to the cell body and nerve endings [Tallinan, JF. Gallager, DW. 1985. The GABA-ergic system: a locus of benzodiazepine action. Annu Rev Neurosci.; 8: 21 — 44]. The literature states that the GABA receptor in the human brain has many functional ranges and ion channels, as well as benzodiazepine (BDZ), barbiturates; tetrogen toxin and binding sites for narcotic steroids, [Smith, GB. Olsen, RW. 1995. Trends Pharmacol Sci. 16 (5): 162—8]; [Pritchett, DB. 1989.. Nature; 338 (6216): 582— 5].

Benzodiazepine (BDZ) species include methyldiazepine (triazine); trizzolam and fiunitrazepam. In the mammalian central nervous system, the main behavioral functions of traditional benzodiazepines are related to anxiolytic, anticonvulsant, sedative, and muscle relaxation effects. The chemical drugs diazepam, nitroazepam, trizolam, and benzodiazepine are all GABA receptor competitive binding substances. These compounds competitively bind to the corresponding functional sites of the GABA receptor, thereby effectively blocking anxiety and convulsions. Waiting for the release of related substances.

The pharmaceutical concept of baicalin as an anti-anxiety drug has not been reported at home and abroad. However, a large number of in vitro and overall pharmacodynamic experimental research results of the present inventors have confirmed that the traditional Chinese medicine monomer compound has the function of competitively binding to the G A B A receptor B D Z functional site, and is a G A B A receptor B

DZ function binding site stimulants have obvious anxiolytic and sedative effects. Baicalin as an anti-anxiety drug has the characteristics of low toxicity and low side effects after administration compared with other chemical ingredients. The lethal dose of baicalin injected subcutaneously in mice was 6g / kg; mice were injected intraperitoneally with LD _{5 of} baicalin. The dose was 3. 081g / kg [Wang Baokui et al .: Chinese Hospital Pharmaceutical Journal 1993: 13 (8) 354-355], [Lin Jiqiang et al .: Acta Physiologica Sinica, 1958; 22 (3) 249]; [Kumazaki Equality: Central Medical Journal, 1959; 145: 907]. The dose of oral baicalin LD _5Q determined by the inventors was 8.937 + 1.146g / kg ₀

Baicalin has low toxicity, no obvious central nervous system inhibitory effect and muscle relaxation effect, and within the effective anxiolytic dose range, it can avoid the central nervous system inhibitory effect caused by other chemical ingredients anxiolytic drugs. It is a safe and reliable A new anti-anxiety drug that can be taken for a long time.

The results of preclinical animal experiments showed that the optimal anti-anxiety dose of oral baicalin in mice was 7 ~ 30 mg I kg once. According to the commonly used method of drug dose design, the effective dose per unit weight of human oral baicalin is estimated to range from about 0.8 to 3.5 mg / kg / times. And, depending on the severity of the patient's anxiety symptoms and the elimination of anxiety symptoms after taking the drug, one or three times a day can be chosen.

When baicalin is used as an anti-anxiety drug, baicalin can be used as the main medicinal ingredient to make various dosage forms, such as tablets, capsules, drip pills, oral liquids, and injections. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the molecular structural formula of baicalin;

Figure 2 is a scan of absorbance of baicalin;

Figure 3 is the infrared spectrum of baicalin;

Figure 4 is a curve showing the concentration of baicalin that competitively binds to the GABA receptor, with a 50% inhibitory diazepam concentration (IC _{5 ()} ). detailed description

I. Gamma-aminobutyric acid receptor (GABA) competitively binds to flavonoids assay results:

A radioimmunocompetitive binding method was used to determine the compound baicalin's 50% stable inhibitory binding concentration (IC _5a ) and the competitive inhibitory stable binding constant () at the GABA receptor. In the experiment, isolated rat brain cells were used to extract and purify GABA receptor protein from rat brain nerves at low temperature. [N-methyl-3H] Flunitrazepam provided by Amersham Phamacia Biotech UK Limited was used as an immunological marker and a baicalin compound with a purity of> 98% as the competitive junction 组合。 The compound. Application of competitive combined radioimmunoassay methods (Viola, H. 1999 Biochem Biophys Res Commun 262 (3): 643-6); [Villar, HO. 1989. Mol Pharmacol. 36 (4): 589-600] and [Lelas, S .1999. Behav Pharmacol 10 (l): 39-50] IC ₅₀ and.

The measurement results are shown in Figure 4:

Baicalin IC ₅ . 137. 07 ± 8. 50uM

Ki is 77. 10 soil 4. 79uM

The data and conclusions about the 50% competitive binding inhibitory concentration and competitive inhibitory constant of baicalin on the BDZ site of GABA receptor are blank points in the previous research in this field. The research conclusion of 50% competitive inhibition concentration and inhibition constant of GABA on GABA receptors provided the latest experimental theoretical basis for the active ingredients of the drug as a treatment for various types of mental anxiety.

2. Anti-anxiety effects of baicalin in mice at different doses. Experimental observation of animals using secondary Kunming mice; male; weight 16-18g. Provided by the Experimental Animal Center of the Academy of Medical Sciences. Certificate number: Jingdong Guanzi 200201, regular word raising in secondary animal room. Animal room temperature: 24 soil 2 ° C _; humidity: 60%; light time: 12 hours.

Test drug:

1) Baicalin (^ ic ^ iW dark brown powder; slightly bitter taste, poor solubility in water solvents, dry storage at room temperature; purity> 98. 6%.

2) Nitroazepam: As a positive control, white tablets; Specification: 5mg / tablet, commercially available. The drug has poor solubility in aqueous solvents.

The experimental animals were randomly divided into a high-dose group (30 mg / kg) _{, a} medium-dose group (15 mg / kg) and a low-dose group (7.5 mg / kg). A known positive drug, nitro-dazepine control group (lmg / kg), was also set up. ; And a normal blank control group.

Each type of test drug is prepared by dissolving in double distilled water according to the unit weight dose, and is administered by gastric lavage. The dosing volume was 0.5 ml. Each pointer measurement was started 60 minutes after gavage.

Laboratory equipment:

1) Elevated Cross Labyrinth: The cross labyrinth is made by a wooden board with a diameter of 0.5cm. The bottom of the labyrinth is cross-shaped. It is divided into four areas, that is, two symmetrical open areas with a bottom area of 25x5cm _; two symmetrical The closed area has a bottom area of 25x25cm, and the outer periphery of the closed area is enclosed by a 10cm high wooden board. The central area of the junction of the four zones is 5x5cm. The device is placed on a stand 40 cm above the ground. During the test, the test mice were placed in the central area, and the time and number of times the test animals entered the open or closed area within 300 seconds were recorded.

Experimental recording method: When the body of the experimental mouse enters the open or closed area, record the animal in The number of entries and the length of stay in the area. [Pellow, S. 1986. Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacology, Bioc enistry and Behavior 24 (3): 525- 529). The number of experimental mice selected to enter the open zone and the length of time they stay in the open zone indicate the degree of anti-anxiety effect of the drug.

2) Four-hole box: The four-hole box is made by a wood plate with a thickness of 0.5cm. Its cabinet size is 60x60x30cm. There are four round holes with a diameter of 4cm at the bottom of the box 5cm from the four corners. The box is placed 50cm from the ground. At the beginning of the test, the test animal is placed in the central area of the four-hole box, allowing the test animal to move freely in the box. Simultaneously record the number of probes and standing times of the test mice in a four-well box within 300 seconds.

Experimental recording method: When the nose of the test mouse touched the edge of the round hole from the lower edge of the orbit, record the probe of the test mouse once; when the forefoot of the test mouse leaves the ground, record the mouse standing up once. [File, SE. 1985. The effects of trizolobenzodiapines in two animal tests of anxiety and in the holeboard. British Journal of Pharmacology. 86 (3): 729- 735]. Compared with the animals in the normal blank control group, the reduction in the number of probes and standing times in the medication group reflected the level of sedation.

3) Horizontal suspension device: Fix a 1mm diameter and 20cm long rope at a position 20cm higher than the desktop. In the test, the front paws of the test mice were grasped by the cord, and then the hands were released, and the animal was suspended from the cord. At the same time, the number of times the test mice fell from the ropes within 60 seconds was recorded. [Bonetti, EP. 1982 Psychopharmacology (Berl.)-78 (l): 8-18], the number of drops of the mice from the suspension rope reflects the strength of the experimental mice.

The above experimental equipment is fixed in the laboratory. Try to be quiet after the start of the experiment, and try to avoid noise and other interference factors affecting the experimental results.

The relevant literature on mental anxiety and changes in animal testing behavior is explained as follows: In the elevated cross-maze test model, normal mice prefer to enter the closed area of the maze rather than the open area because they are afraid of height, and they are not willing to be on a relatively narrow elevated platform The reason for the activities in the open area is that it is not easy to maintain balance in the open space, and it is easy to worry about falling. Therefore, it is believed that the number of times the mouse moves in the open area and the length of time it stays in the open area reflect the degree of worry and anxiety of the experimental mice. The number of times that the experimental mice kept their heads upright with the probe and forelimbs off the ground in the four-hole box reflects the strength of the anxiety and dryness behavior of the experimental mice. Sedated mice had significantly fewer probes and uprights in a four-well box than anxious and arousal mice.

All experimental data are expressed as mean soil standard deviation (X ± S).釆 T test and χ ² test Statistical comparisons between groups were made.

(I) Observation of general pharmacological changes of mice in various dose groups after oral baicalin

The general conditions of the animals in each dose group were not significantly changed compared with the animals in the normal control group, and the activity and muscle strength were not changed. No abnormalities were found in the psychiatric nervous system and respiratory and circulatory systems, and no death occurred in all the animals.

Table 1.Experimental mice enter the open area and closed area of the cross table labyrinth within 300 seconds

Frequency and percentage comparison

Total number of open zones and closed zones (times / 300 seconds) Times% Times% High-dose group 22.42 ± 2.78 10.33 ± 2.27 * 46.21 ± 6.17 ** 12.00 ± 1.35 53.79 ± 6.17 Middle-dose group 25.83 ± 2.59 12.33 ± 1.88 ** 47.66 ± 4.21 ** 13.67 + 1.50 52.09 ± 4.52 Low dose group 23.00 ± 2.89 9.92 ± 1.49 43.08 ± 2.68 ** 13.08 ± 1.68 56.93 ± 2.68 stability group 30.33 ± 3.34 * 15.50 ± 3.07 ** 51.06 + 3.03 ** 14.83 ± 1.75 48.12 ± 3.12 Normal control group 25.67 ± 4.89 8.33 ± 1.76 33 · 64 ± 5 · 80 17.33 ± 3.77 66.36 ± 5.80 Compared with the normal control group: * ρ <0.05; ** ρ <0.01

(3) Comparison of the time of mice entering the open area and the closed area in each dose group after oral administration of baicalin

Time and percentage comparison

Open area closed area

Group time (second)% Time (second)% High dose group 78.25 ± 12.20 ** 26.09 ± 4.07 ** 213.00 ± 12.14 70.82 ± 3.77 Middle dose group 84.92 ± 12.15 ** 28.31 ± 4.05 ** 214.92 ± 14.16 71 · 64 ± 4.71 Low-dose group 72.08 ± 11.29 ** 24.05 ± 3.71 * 216.42 ± 17.86 72.13 ± 5.94 Stability group 91.67 ± 13.37 ** 30.55 ± 4.44 ** 199.08 ± 11.63 66.33 ± 3.89 Normal control group 53 · 25 ± 9.57 17 · 75 ± 3.19 238.42 + 11.91 79.47 ± 3.98 Compared with the normal control group: * ρ <0.05; ** ρ <0.01

(IV) Probes of mice in various dose groups after oral administration of baicalin; comparison of standing times and falling times Table 3. Experimental mice enter the probe within 300 seconds; comparison of standing up and falling times

The number of probes in the group The number of standing times The number of baicalin drops The high-dose group 9.00 ± 3.41 ** 16 · 00 ± 3 · 84 * 0.58 ± 0.99 The middle-dose group 17.58 ± 3.45 * 19.17 ± 4.45 * 0.42 + 0.67 The low-dose group 18.67 ± 4.80 17.67 ± 4.79 * 0.58 ± 0 · 79 Anding group 10 · 67 ± 3.70 10.75 ± 2.56 * 0.42 ± 0.67 Normal control group 19 · 42 ± 4 · 93 23.66 ± 4.48 0.25 ± 0.45 Compared with the normal control group:: * ρ <0. 05; It can be seen from the experimental results in Table 1-3:

1. Oral baicalin mice in the various dose groups in the elevated cross labyrinth entered the open area and the percentage of the open area were significantly increased compared with the normal blank control group, of which the increase was the most significant in the middle dose group (15mg / kg). There were no significant differences in the number and percentage of patients entering the closed side after taking the medicine in the three dose groups compared with the normal blank control group.

2. Oral baicalin mice in each dose group in the open cross labyrinth entered the open area and the percentage of time to enter the open area were significantly increased compared with the control group of the normal blank. The increase was most pronounced in the dose group (15mg / kg), p <0.01. The time and percentage of entry to the closed side after taking the three dose groups were not significantly different from those of the normal blank control group, P> 0.05. *

3. Oral baicalin each dose group in the four-hole box test results show all dose groups (7. 5- 30m _g / kg) were significantly different in mice and the vertical frequency and the probe body and the normal control group, p <0 . 05.

4. There was no drug-induced muscle relaxation in mice in each dose group of baicalin.

The above experimental results show that: Oral baicalin can significantly change the behavioral characteristics of the mice on the test device. The main manifestations are that the number of times to enter the open cross maze open area and the residence time and percentage are significantly increased compared with the normal control group. Reduced number of middle probes and uprights. The above experimental results confirm that baicalin has sedative and anxiolytic effects, and that the drug has no significant effect on the nervous system and muscle strength of experimental animals. Industrial applicability

According to the present invention, the baicalin and baicalin compounds have low toxicity and low drug side effects, and are an anti-anxiety medicine that can effectively treat and reduce various anxiety disorders.

Claims

Rights request

1. A new use of baicalin Baicalin as a main drug to treat various types of mental anxiety.

2. The new use of baicalin for treating various types of mental anxiety according to claim 1, characterized in that the effective oral dose per unit weight of the human body is 0.8-3.5 mg / kg.

3. The new use of baicalin for treating various types of mental anxiety according to claim 1, characterized in that the dosage forms of scopolamine as an anti-anxiety drug are: tablets, capsules, drip pills, oral liquids and injections Wait.