# US 2006/0068045 A1 · Method for the preparation of phytoprogestogenic extracts from rhizoma ligusticum chuanxiong and uses thereof

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- Number: US 2006/0068045 A1
- Document ID: US20060068045A1
- Status: Published application
- Kind: A1
- Application number: 10/525,447
- Filed: 2003-08-20
- Published: 2006-03-30
- Earliest claimed: 2002-08-20
- Inventors: Eu Yong; Lis Sa Elissa Lim; Mark Stuart Butler
- Web page: https://patentgrep.com/patent/US20060068045A1

## CPC

- A61K 36/236: Ligusticum (licorice-root)
- A61P 5/24: of the sex hormones
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 15/02: for disorders of the vagina
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- G01N 33/743: {Steroid hormones}
- G01N 2500/10: involving cells
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method of extracting phytoprogestogenic compounds from herbs and their use in preparing a medicament for treating humans requiring progesterone replacement or supplement. Assay systems to quantify progesterone and progestogenic receptor activity and a method for assembling a kit for the assays are also provided.

## Claims

### Claim at position 1

1-53. (canceled)

### Claim 54

An extract of Rhizoma Chuanxiong, characterized in that the extract is capable of eliciting progestogenic activity in a progestogen receptor assay.

### Claim 55 (depends on 54)

An extract as claimed in claim 54, wherein the extract is capable of eliciting an increase in progesterone reporter gene activity in the range 50-fold to 350-foid relative to a negative control.

### Claim 56 (depends on 55)

An extract according to claim 55, wherein the increase in progesterone reporter gene activity is 150 fold to 200 fold relative to a negative control.

### Claim 57 (depends on 55)

An extract according to claim 55, wherein the progesterone reporter gene comprises a luciferase reporter gene driven by two copies of progesterone response element from aminotransferase.

### Claim 58 (depends on 54)

An extract according to claim 54, wherein the extract is at a concentration in the range of 6.25 μg/ml to 100 μg/ml in the progestogen receptor assay.

### Claim 59 (depends on 58)

An extract according to claim 58, wherein the progestogen receptor assay comprises RPMI 1640 medium.

### Claim 60 (depends on 59)

An extract according to claim 59, wherein progestogen receptor assay comprises 10% charcoal-stripped fetal calf serum, 2 mM L-glutamine, 0.1 mM non essential amino acids and 1 mM sodium pyruvate, and wherein the extract is capable of eliciting the 50-fold to 350-fold increase in progesterone reporter gene activity after exposure for 42-48 hours at 37° C. in a 5% carbon dioxide incubator.

### Claim 61 (depends on 54)

An extract according to claim 54, comprising 3-butyl-4,5-dihydrophthalide and 3-butyl-phthalide.

### Claim 62 (depends on 61)

An extract according to claim 61, wherein the 3-butyl-4,5-dihydrophthalide is present in the extract in an amount of about 95wt % and the 3-butyl-phthalide is present in the extract in an amount of about 2wt %.

### Claim 63 (depends on 54)

A pharmaceutical composition comprising a therapeutically effective amount of the extract of claim 54, in a pharmaceutically acceptable vehicle.

### Claim 64 (depends on 63)

A pharmaceutical composition as claimed in claim 63, wherein the pharmaceutical composition is capable of being administered orally or subcutaneously.

### Claim 65 (depends on 63)

A pharmaceutical composition as claimed in claim 63, wherein the therapeutically effective amount is within the range 31.25 mg to 500 mg per 5000 ml of total blood volume.

### Claim 66 (depends on 1)

Use of the extract of claim 1, for the manufacture of a medicament for treating a progesterone condition in a human or animal.

### Claim 67 (depends on 66)

The use of claim 66, wherein the progesterone condition is selected from the group consisting of progesterone replacement, progesterone supplementation, menstrual disorders, amenorrhoea, menorrhagia, polycystic ovarian syndrome, pregnancy complications, endometriosis, contraception, menopause, and endometrial hyperplasia.

### Claim 68 (depends on 54)

Use of the extract of claim 54, for the manufacture of a medicament for treating stroke or brain injuries in a human or animal.

### Claim 69

A method of preparing an extract, the method comprising the step of subjecting Rhizoma Chuanxiong herb to an alcoholic solvent selected from the group consisting of methanol, ethanol, methanol water, ethanol water, and mixtures thereof, at a temperature and for a time to produce the extract characterized by being capable of eliciting progestogenic activity in a progestogen receptor assay.

### Claim 70 (depends on 69)

A method as claimed in claim 69, wherein the extract capable of eliciting an increase in progesterone reporter gene activity in the range 50-fold to 350-fold relative to a negative control.

### Claim 71 (depends on 69)

A method as claimed in claim 69, comprising at least partially removing one or more tannins from the extract to produce purified extract.

### Claim 72 (depends on 71)

A method as claimed in claim 71, wherein the removing comprises:
fractionating the extract by solid phase fractionation using a polar solvent, a non polar solvent and a mixed polarity solvent that has a polarity intermediate the polar and non polar solvents; and
extracting purified extract from the intermediate polar solvent.

### Claim 73 (depends on 22)

A method as claimed in claim 22, comprising using, as the mixed polarity solvent, a mixture comprising polar solvent and dichloromethane (DCM).

### Claim 74 (depends on 71)

A method as claimed in claim 71, wherein the removing comprises subjecting the extract to solvent-solvent extraction using mixtures of polar and non polar solvents.

### Claim 75 (depends on 74)

A method as claimed in claim 74, wherein the polar solvents are selected from the group consisting of water, ethanol, butanol, and mixtures thereof, and the non polar solvents are selected from the group consisting of hexane, dichloromethane (DCM), and mixtures thereof.

### Claim 76 (depends on 75)

A method as claimed in claim 75, comprising obtaining purified extract from the non polar solvent.

### Claim 77 (depends on 69)

A method as claimed in claim 69, further comprising, separating the extract from the Rhizoma Chuanxiong herb.

### Claim 78 (depends on 77)

A method as claimed in claim 77, further comprising drying the separated extract.

### Claim 79 (depends on 78)

A method as claimed in claim 78, comprising suspending the extract in an alcoholic medium.

### Claim 80 (depends on 69)

A method as claimed in claim 69, comprising suspending the extract at a concentration in the range 6.25 μg/ml to 10 Oμg/ml of an assay.

### Claim 81 (depends on 79)

A method as claimed in claim 79, comprising suspending the extract at a concentration of 50 μg/ml.

### Claim 82 (depends on 69)

A method as claimed in claim 69, wherein the subjecting is carried out for 5-7 days at 37° C.

### Claim 83 (depends on 69)

A method as claimed in claim 69, wherein the ethanol-water solvent comprises 50% or 70% ethanol in water by weight and wherein the methanol is 70% methanol in water by weight.

### Claim 84 (depends on 71)

A method as claimed in claim 71, wherein the removing comprises subjecting the extract to High Performance Liquid Chromatography.

### Claim 85

An extract of Rhizoma Chuanxiong, characterized in that the extract has a progestogenic activity to progesterone, in a range selected from the group consisting of: 23.39% to 79.86%; 23.39% to 48.17%; and 48.17% to 79.86%, and mixtures thereof.

### Claim 86

A method for the preparation of an alcoholic extract from Rhizoma Chuanxiong useful as a progestogen, said method comprising subjecting powdered Rhizoma Chuanxiong to a first extraction with alsoholic solvent selected from the group consisting of methanol, ethanol, methanol-water, ethanol-water, and mixtures thereof, separating a first Rhizoma Chuanxiong extract obtained thereby from a supernatant, subjecting the filtered first extract to a second extraction with an alcoholic solvent to obtain a second extract, wherein the first and second extractions are at a temperature and for a time to produce the extract, wherein the extract is capable of eliciting progestogenic activity in a progestogen receptor assay.

### Claim 87 (depends on 69)

An extract of Rhizoma Chuanxiong obtained from a process according to claim 69.

### Claim 88 (depends on 86)

An extract of Rhizoma Chuanxiong obtained from a process according to claim 86.