WO2022237873A1 - Pharmaceutical composition for preventing and treating osteoporosis - Google Patents

Abstract

A pharmaceutical composition for treating and/or preventing osteoporosis, the pharmaceutical composition comprising 2-8 parts by weight of Ligustri lucidi fructus, 2-8 parts by weight of Ecliptae herba, 1-5 parts by weight of Spatholobi caulis, and 1-5 parts by weight of Achyranthis bidentatae radix. The pharmaceutical composition can effectively prevent and treat osteoporosis, perimenopausal syndrome, constipation or obesity and reduce blood fat, and has a better effect than that of the Erzhi formula.

Description

Pharmaceutical composition for preventing and treating osteoporosis technical field

The invention belongs to the field of traditional Chinese medicine and relates to a pharmaceutical composition, in particular to a pharmaceutical composition for treating and/or preventing osteoporosis.

Background technique

Osteoporosis (Osteoporosis, OP) is a systemic bone metabolic disease caused by multiple factors, characterized by decreased bone mass per unit volume and destruction of bone tissue microstructure, resulting in increased bone fragility and susceptibility to fracture. The National Institutes of Health defines osteoporosis as a skeletal disease characterized by impaired bone strength, microarchitectural degeneration of bone tissue (trabecular bone of cancellous bone becomes thinner, broken, and reduced in number) and increased risk of fracture. It is a comprehensive reflection of bone density and bone quality. Osteoporosis can be divided into two categories: primary OP and secondary OP. Both postmenopausal OP and senile OP belong to primary OP. Patients with osteoporosis generally have no special clinical manifestations, but severe hip, Fractures of the vertebral body or distal radius. Currently commonly used OP treatment drugs include estrogens, bone resorption inhibitors, bone formation accelerators, and bone mineralizers.

Perimenopause is a special stage that women must go through. During this period, women's ovarian function declines and estrogen levels drop, causing multi-system dysfunction such as the endocrine system, cardiovascular system, and nervous system, causing a series of symptoms of varying degrees collectively known as perimenopausal syndrome (Perimenopausal Syndrome, PS) . The specific manifestations of perimenopausal syndrome vary from person to person, and most symptoms can be improved through self-regulation and psychological counseling ^[1] . However, perimenopausal osteoporosis, as a very serious complication of perimenopausal syndrome, will not only bring great harm to women's psychology and physiology, but even endanger the lives of patients ^[2] .

According to statistics, the number of perimenopausal women suffering from osteoporosis accounts for about 25% of the total number of perimenopausal women in my country. Its onset is characterized by a long course of disease, and it is not easy to be detected in the early stage. It will not be paid attention until severe bone pain, vertebral degeneration or even fracture occurs, and the prognosis is poor, which is very likely to cause lifelong disability or even death of the patient ^[3,4] .

At present, estrogen replacement therapy (HRT) is commonly used clinically to treat perimenopausal osteoporosis. The curative effect is good but there are many adverse reactions. Research results show that long-term use of estrogen therapy will increase the incidence of breast cancer and endometrial cancer, and at the same time increase the risk of stroke, coronary arteriosclerosis, pulmonary embolism and deep vein thrombosis ^[5-7] . In addition, according to statistics, other types of drugs for the treatment of osteoporosis will also cause different types of adverse reactions, which may even endanger the lives of patients. Therefore, Chinese medicine has gradually entered people's sight because of its good therapeutic effect, small side effects and good compliance, and the research on Chinese medicine treatment of perimenopausal diseases has attracted increasing attention.

Erzhi Fang is composed of Ligustrum lucidum and Eclipta chinensis. The fruit of Ligustrum lucidum is used as medicine, which is harvested during the winter solstice, while the whole herb of Eclipta edulis is used as medicine, which is harvested during the summer solstice; the pills are called Erzhi pills. Among them, Ligustrum lucidum often uses wine Ligustrum lucidum, that is, the processed product of Ligustrum lucidum. Erzhi Fang has the functions of nourishing the liver and kidney, nourishing yin and stopping bleeding. Modern studies have shown that the pharmacological effects of Erzhi Fang mainly include immune regulation, anti-oxidation, anti-aging, liver protection, anti-osteoporosis, anti-inflammatory, anti-tumor, hormone-like effects, etc. ^[8] .

Ligustrum lucidum in the prescription is the fruit of privet privet of Osmanthus fragrans. It is sweet in taste and cool in nature. It returns to the liver and kidney meridian. The treatment of sore knees, premature graying of beard and hair, and blurred eyesight. Modern studies have shown that Ligustrum lucidum is rich in medicinal substances, mainly including triterpenoids, iridoids, phenethyl alcohols, flavonoids, Ligustrum lucidum polysaccharides, etc., and the 2015 edition of "Chinese Pharmacopoeia" will The iridoid glycoside compound privetin is used as the quality detection component of Ligustrum lucidum ^[9] .

In the recipe, Eclipta (commonly known as Eclipta) is the whole herb of the Compositae plant, which is sweet and sour, cool in nature, and returns to the liver and kidney meridians. It has the effects of nourishing liver and kidney, cooling blood and stopping bleeding, and is mainly used for the treatment of diseases such as cooling blood, stopping bleeding, nourishing kidney, and benefiting yin. Modern studies have shown that its medicinal substances mainly include flavonoids, triterpenes and their derivatives, vanillin compounds, thiophenes, steroids, and volatile oils. The 2015 edition of the "Chinese Pharmacopoeia" used the vanillin compound wedelolide as the quality detection component of Eclipta japonica ^[10] .

Spatholobus Spatholobus is a commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis. It belongs to the liver and kidney channels. "Compendium of Materia Medica" points out that "Spathiphyllum japonica, promotes blood circulation and relaxes tendons, treats dry blood labor of men and women, and all fatigue injuries...". Clinically, it is mainly used for irregular menstruation, blood deficiency and chlorosis, numbness and paralysis, and rheumatic arthralgia. Modern studies have found that the chemical composition of Spatholobus is complex, containing ten types of flavans (such as catechin), terpenoids (such as lupeol), sterols (β-sitosterol), anthraquinones (such as emodin), etc. other types of compounds.

Achyranthes bidentata is the root of Achyranthes bidentata plant of Amaranthaceae. Bitter, sour, flat in nature, it returns to the liver and kidney meridian, and has the effects of nourishing the liver and kidney, strengthening muscles and bones, promoting blood circulation and stimulating menstruation, leading qi and blood to descend, diuresis and treating stranguria. It was first recorded in "Shen Nong's Materia Medica": "It is mainly used for cold-damp impotence, spasms of limbs, knee pain that cannot be flexed and stretched, expels blood, hurts heat and burns, and long-term use makes light of body and endures old age."

At present, there is still a need to develop more effective drug means that can prevent and treat diseases such as osteoporosis.

Contents of the invention

After intensive research and creative work, the present inventor has obtained a pharmaceutical composition for preventing and treating diseases such as osteoporosis and an extract thereof. The inventors surprisingly found that the drug combination or its extract can effectively prevent and treat osteoporosis, especially perimenopausal osteoporosis, prevent and treat constipation and/or lower blood lipid. The effect of the pharmaceutical composition or extract of the invention is better than that of Erzhi Fang (Erzhi Pill), and the safety of long-term use is good. The following inventions are thus provided:

One aspect of the present invention relates to a pharmaceutical composition comprising:

Ligustrum 2-8 parts by weight

Eclipta 2-8 parts by weight

Spatholobus 1-5 parts by weight

Achyranthes knuckle 1-5 parts by weight.

In some embodiments of the present invention, the pharmaceutical composition includes:

Ligustrum 2-8 parts by weight

Eclipta 2-8 parts by weight

Spatholobus 2-4 parts by weight

Achyranthes knuckle 1-3 parts by weight;

Preferably, include:

Ligustrum 4-6 parts by weight

Eclipta 4-6 parts by weight

Spatholobus 2-4 parts by weight

Achyranthes knuckle 1-3 parts by weight;

More preferably, including:

Ligustrum 4.5-5.5 parts by weight

Eclipta 4.5-5.5 parts by weight

Spatholobus 2.5-3.5 parts by weight

Achyranthes knuckle 1.5-2.5 parts by weight;

Further preferably, including:

Ligustrum 4.8-5.2 parts by weight

Eclipta 4.8-5.2 parts by weight

Spatholobus 2.8-3.2 parts by weight

Achyranthes knuckle 1.8-2.2 parts by weight;

Particularly preferably, include:

Ligustrum 5 parts by weight

Eclipta 5 parts by weight

Spatholobus 3 parts by weight

Achyranthes knuckle 2 parts by weight.

In some embodiments of the present invention, the unit dose of the pharmaceutical composition is 10-60g, preferably 15-40g, 20-30g, 22-28g or 24-26g, more preferably 25g.

In the pharmaceutical composition of the present invention, various medicinal materials (for example, 2, 3 or 4 of them) may be mixed or relatively independent (not mixed).

In some embodiments of the present invention, the pharmaceutical composition is composed of the above four herbs.

In some embodiments of the present invention, the pharmaceutical composition is composed of the above four herbs and pharmaceutically acceptable excipients.

Another aspect of the present invention relates to an extract prepared from any one of the pharmaceutical compositions of the present invention.

In some embodiments of the present invention, the extract is prepared by a preparation method comprising the following steps:

(1) Extracting the Ligustrum lucidum, Eclipta chinensis, Millipeda and Achyranthes bidentata in any one of the parts by weight described above with water or ethanol solution, to obtain an extract;

(2) Concentrating the extract to obtain a concentrate, which is an extract.

In some embodiments of the present invention, the extract is characterized by any one or more of the following items ①-⑧:

1. In step (1), the extraction is one or more times, and when it is multiple times, the extracts of each time are combined;

② In step (1), extract the mixture of Ligustrum lucidum, Eclipta chinensis, Caulis Spatholobus and Achyranthes bidentata; or extract any 1 flavor and the mixture of the remaining 3 flavors to obtain the respective extracts, combined or not Each extract; or extract the mixture of any 2 flavors and the mixture of the remaining 2 flavors separately to obtain the extracts, combine or not combine the extracts; or extract any 1 flavor, the other 1 flavor and the mixture of the remaining 2 flavors extracting separately to obtain each extract, merging or not merging each extract; or extracting 4 medicinal materials separately to obtain each extract, merging or not merging each extract;

③ In step (1), the concentration of the ethanol is 10%-99%, 20%-90%, 30%-80%, 40%-80%, 50%-80%, 50%-70% or 60% %-80%;

④ In step (1), the extraction is reflux extraction or decoction;

5. In step (1), the extraction time is at least 0.5 hour, at least 1 hour, at least 2 hours or 1-5 hours;

6. In step (1), the consumption of water or ethanol solution is 5-20 times the amount (ml/g), preferably 8-16 times the amount;

7. In step (2), the concentration is concentration under reduced pressure;

8. In the step (2), the concentrate is an extract.

In some embodiments of the present invention, the extract, wherein, in step (2), the concentrate is one or more ( eg 2, 3 or 4). In the case of not combining extracts in item ②, if there are several extracts, several concentrates will be obtained; at this time, the several concentrates are extracts.

In some embodiments of the present invention, the extract, wherein Ligustrum lucidum and Eclipta chinensis are respectively extracted to obtain Ligustrum lucidum concentrate and Eclipta edulis extract; Extract with the mixture of Spatholobus Spatholobus and Achyranthes bidentata to obtain the concentrate of Ligustrum lucidum Spatholobus Spatholobus and Achyranthes bidentata; the three obtained concentrates are the extracts of the present invention.

In some embodiments of the present invention, the extract, wherein,

Step (1) comprises the following steps:

Extracting Ligustrum lucidum and Eclipta edulis separately to obtain Ligustrum lucidum extract and Eclipta edulis extract; extracting the mixture of Ligustrum lucidum extract and the mixture of Spatholobus Spatholobus and Achyranthes bidentata to obtain Ligustrum lucidum Spatholobus Spatholobus Achyranthes knuckle extract;

Preferably, step (2) comprises the steps of:

Concentrate the Ligustrum lucidum extract, the Eclipta chinensis extract and the Ligustrum lucidum Caulis Spatholobus Achyranthes extract respectively and then mix or combine the extracts and then concentrate to obtain the extract.

Another aspect of the present invention relates to a pharmaceutical preparation, which comprises the extract described in any one of the present invention, and one or more pharmaceutically acceptable excipients;

Preferably, the unit dose of the pharmaceutical preparation is 10-60 g, preferably 15-40 g or 20-30 g, more preferably 25 g, calculated according to the mass of the crude drug.

In some embodiments of the present invention, the pharmaceutical preparation, wherein the extract is the only active ingredient.

In some embodiments of the present invention, the pharmaceutical preparation consists of the extract and one or more pharmaceutically acceptable excipients.

In some embodiments of the present invention, the pharmaceutical preparation is a pill, tablet, granule, capsule, tincture or suspension.

In some embodiments of the present invention, the pharmaceutical preparation further comprises one or more selected from the following drugs:

Calcium, vitamin D, bisphosphonates, estrogens such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone, and osteoprotegerin.

According to the pharmaceutical composition according to any one of the present invention, the extract according to any one of the present invention or the pharmaceutical preparation according to any one of the present invention, it is used for treating and/or preventing osteoporosis , perimenopausal syndrome, constipation or obesity or hypolipidemia;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the osteoporosis is perimenopausal osteoporosis;

Preferably, the obesity is climacteric obesity.

Yet another aspect of the present invention relates to a combination pharmaceutical product comprising a first product and a second product in separate packages,

in,

The first product comprises the pharmaceutical composition according to any one of the present invention, the extract according to any one of the present invention or the pharmaceutical preparation according to any one of the present invention;

The second product contains one or more selected from the following drugs:

Calcium, vitamin D, bisphosphonates, estrogens such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone, and osteoprotegerin;

Preferably, the second product further comprises one or more pharmaceutically acceptable excipients;

Preferably, the unit dose of the first product is 10-60g, preferably 15-40g or 20-30g, more preferably 25g, calculated according to the mass of the crude drug.

The "first" or "second" in the "first product" or "second product" is only for distinguishing or clearly denoting, and has no order meaning.

Another aspect of the present invention relates to the pharmaceutical composition described in any one of the present invention, the extract described in any one of the present invention or the pharmaceutical preparation described in any one of the present invention in preparation treatment and/or prophylaxis Osteoporosis, perimenopausal syndrome, constipation or obesity drugs, or the use of blood lipid-lowering drugs;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the osteoporosis is perimenopausal osteoporosis;

Preferably, the obesity is climacteric obesity.

In some embodiments of the present invention, the use, wherein the dosage per kilogram of body weight per day is 0.1g-2g, 0.2g-1g or 0.4g-0.8g, preferably 0.5g.

Another aspect of the present invention relates to a method for preparing an extract, comprising the steps of:

(1) extract Ligustrum lucidum, Eclipta chinensis, Millipeda and Achyranthes bidentata in any one of the present invention by using water or ethanol solution to obtain an extract;

(2) Concentrate the extract to obtain a concentrate, which is the extract.

In some embodiments of the present invention, the preparation method is characterized in that any one or more of the following items ①-⑧:

1. In step (1), the extraction is one or more times, and when it is multiple times, the extracts of each time are combined;

② In step (1), extract the mixture of Ligustrum lucidum, Eclipta chinensis, Caulis Spatholobus and Achyranthes bidentata; or extract any 1 flavor and the mixture of the remaining 3 flavors to obtain the respective extracts, combined or not Each extract; or extract the mixture of any 2 flavors and the mixture of the remaining 2 flavors separately to obtain the extracts, combine or not combine the extracts; or extract any 1 flavor, the other 1 flavor and the mixture of the remaining 2 flavors extracting separately to obtain each extract, merging or not merging each extract; or extracting 4 medicinal materials separately to obtain each extract, merging or not merging each extract;

③ In step (1), the concentration of the ethanol is 10%-99%, 20%-90%, 30%-80%, 40%-80%, 50%-80%, 50%-70% or 60% %-80%;

④ In step (1), the extraction is reflux extraction or decoction;

⑤ In step (1), the extraction time is at least 0.5 hour, at least 1 hour, at least 2 hours or 1-5 hours;

6. In step (1), the consumption of water or ethanol solution is 5-20 times the amount (ml/g), preferably 8-16 times the amount;

7. In step (2), the concentration is concentration under reduced pressure;

8. In the step (2), the concentrate is an extract.

In some embodiments of the present invention, the preparation method, wherein,

Step (1) comprises the following steps:

Extracting Ligustrum lucidum and Eclipta edulis separately to obtain Ligustrum lucidum extract and Eclipta edulis extract; extracting the mixture of Ligustrum lucidum extract and the mixture of Spatholobus Spatholobus and Achyranthes bidentata to obtain Ligustrum lucidum Spatholobus Spatholobus Achyranthes knuckle extract;

Preferably, step (2) comprises the steps of:

Concentrate the Ligustrum lucidum extract, the Eclipta chinensis extract and the Ligustrum lucidum Caulis Spatholobus Achyranthes extract respectively and then mix or combine the extracts and then concentrate to obtain the extract.

The ethanol solution involved in the present invention refers to the aqueous solution of ethanol unless otherwise specified.

In the present invention, unless otherwise specified, the concentration of the ethanol or the concentration of the ethanol solution is the volume percent concentration (v/v)%.

In the present invention, unless otherwise specified, the term "Erzhi Fang" refers to a prescription composed of Ligustrum lucidum and Eclipta chinensis (commonly known as Eclipta), and the term "Erzhi Wan" refers to a preparation obtained through industrial production.

In the present invention, the pharmaceutical composition or extract of the present invention is also sometimes referred to as Jiawei Erzhifang.

Usually, the pharmaceutical preparation of the present invention contains 0.1% by weight to 90% by weight of the extract of the present invention as the main ingredient. Pharmaceutical formulations can be prepared according to methods known in the art. For this purpose, if necessary, the main drug can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare an appropriate administration form or dosage form for human use.

The term "adjuvant" in this application refers to the excipient or vehicle used to administer the main drug, which includes but not limited to diluents, disintegrating agents, precipitation inhibitors, surfactants, glidants, viscose Mixtures, lubricants, coating materials, etc. Excipients are generally described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Examples of excipients include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, Hydroxyethyl Cellulose, Hydroxy Methyl Cellulose, Hydroxy Octacyl Hydroxystearate, Hydroxy Propyl Cellulose, Hydroxy Propyl Methyl Cellulose, Lactose, Lactose Monohydrate, Magnesium Stearate, Mannitol , microcrystalline cellulose, etc.

The extracts of the present invention can be formulated into pharmaceutical preparations, including dosage forms suitable for oral administration, dosage forms suitable for parenteral injection (such as intravenous injection, subcutaneous injection) (such as as a solution), dosage forms suitable for topical administration (for example as an ointment, patch or cream), and formulations suitable for rectal administration (for example as a suppository) and the like.

Another aspect of the present invention relates to a method for treating and/or preventing osteoporosis, perimenopausal syndrome, constipation or obesity or lowering blood lipids, comprising administering an effective amount of any of the present invention to a subject in need Steps of one of the pharmaceutical compositions described in the present invention, the extracts described in any one of the present invention or the pharmaceutical preparations described in any one of the present invention;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the osteoporosis is perimenopausal osteoporosis;

Preferably, the obesity is climacteric obesity.

In some embodiments of the present invention, the method, wherein the dosage per kilogram of body weight per day is 0.1g-2g, 0.2g-1g or 0.4g-0.8g, preferably 0.5g.

Depending on the disease to be treated and the patient and the route of administration, the pharmaceutical preparations according to the invention can be administered once or several times a day in different doses. The dosage depends on many factors, such as the severity of the osteoporosis to be treated or prevented, the sex, age, body weight and individual response of the patient, the route of administration and the frequency of administration, etc. The above dose can be administered as a single dose or divided into several, eg two, three or four doses.

The actual dosage level of the principal ingredient (extract) in the pharmaceutical formulations of the present invention can be varied to effectively achieve the desired therapeutic response for a particular patient, composition and mode of administration. Dosage levels will be selected based on the route of administration, the severity of the condition being treated and the condition and previous medical history of the patient being treated. However, it is the practice in the art to start doses below that required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.

The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of the diseases or conditions described in the present invention in a subject.

Beneficial Effects of the Invention

The present invention achieves any one or more of the following technical effects (1)-(6):

(1) The pharmaceutical composition or extract of the present invention can effectively treat or prevent osteoporosis.

(2) The pharmaceutical composition or extract of the present invention can effectively treat or prevent constipation.

(3) The pharmaceutical composition or extract of the present invention can effectively lower blood fat, treat or prevent hyperlipidemia.

(4) The effect of the pharmaceutical composition or extract of the present invention in treating or preventing osteoporosis is better than that of Erzhi Fang (Erzhi Wan).

(5) The long-term use safety of the pharmaceutical composition or extract of the present invention is good.

(6) The preparation method of the present invention is superior to the traditional Erzhifang (Erzhiwan) preparation process, for example, under the same prescription, the extract prepared by the preparation method of the present invention has better anti-osteogeny Porous effects, such as better bone density.

Description of drawings

Figure 1: Curves of body weight of animals in each group over time.

Figure 2: Histogram of body weight of animals in each group before sampling (before sacrifice).

Figure 3: Histogram of liver mass index of animals in each group.

Figure 4: Histogram of spleen index of animals in each group.

Figure 5: Histogram of kidney indices of animals in each group.

Figure 6: Histogram of uterine index of animals in each group.

Figure 7: Representative hysterograms of animals in each group.

Figure 8: Histogram of animal fat index for each group.

Figure 9: Histogram of white fat index of animals in each group.

Figure 10: Histogram of serum LDL-C levels of animals in each group.

Figure 11: Histogram of bone density of animals in each group.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Preparation Example 1: Example of Preparation Process of Flavored Erzhifang Extract (1)

1. Preparation and analysis of Ligustrum lucidum extract

1.1 Preparation of Ligustrum lucidum extract

Experimental equipment: vacuum drying oven, heating mantle, rotary evaporator.

experiment procedure:

Take 300g of Ligustrum lucidum medicinal material, put it into a 20L round-bottomed flask, add 10L of 80% (v/v) ethanol, put it on a heating mantle for heating and reflux extraction, count from the slight boiling of the liquid, and turn off the heating mantle to cool after 2 hours , filter out the liquid in the flask with gauze, add 10L 80% ethanol again for extraction, combine the two extracts, concentrate under reduced pressure at 50°C to a thick extract, put it in a vacuum oven at 45°C for drying, and weigh.

Experimental results: A total of 72.0 g of dried samples were obtained, and the paste yield was 24%.

1.2 Detection method and single factor test results

1.2.1 HPLC conditions: instrument: waters2695, chromatographic column: Agilent 4.6×250mm, 5μm, temperature: 25°C, mobile phase: A: water, B: methanol, detection wavelength 224nm.

1.2.2 Liquid phase method:

As shown in Table 1 below.

Table 1

Preparation of the reference substance solution: Accurately weigh the privetin reference substance, and dissolve it in methanol to make a reference substance solution.

Inject 10 μl of reference substance solution and sample solution respectively, calculate the content of index components, and make statistics. Ligustrum lucidum extract contains privetin 19.82mg/g.

2. Preparation and Analysis of Eclipta Eclipta Extract

2.1 Eclipta 60% ethanol heating reflux extraction

Experimental equipment: vacuum drying oven, heating mantle, rotary evaporator.

experiment procedure:

Weigh 300g of Eclipta medicinal material, put it into a 20L round-bottomed flask, add 10L of 60% ethanol, put it on a heating mantle for heating and reflux extraction, count from the slight boiling of the liquid, turn off the heating mantle to cool after 2 hours, filter with gauze Take out the liquid in the flask, add 10L of 60% ethanol again for extraction, combine the two extracts, concentrate under reduced pressure at 55°C to a thick extract, dry in a vacuum oven at 45°C, and weigh.

Experimental results: A total of 53.25 g of dried samples were obtained, and the paste yield was 17.75%.

2.2 Detection method and single factor test results

2.2.1 HPLC conditions: instrument: waters2695, chromatographic column: Agilent 4.6×250mm, 5μm, temperature: 25°C, mobile phase: A: 0.5% acetic acid water, B: methanol, detection wavelength 351nm.

2.2.2 Liquid phase method:

As shown in Table 2 below.

Table 2

Preparation of the reference substance solution: Accurately weigh the wedelia lactone reference substance, dissolve it in methanol to prepare the reference substance solution.

Inject 10 μl of reference substance solution and sample solution respectively, calculate the content of index components, and make statistics. The content of wedelolide in the extract of Eclipta chinensis is 0.8mg/g.

3. Preparation and Analysis of Spatholobus Extract

3.1 Heat reflux with 60% ethanol from Spatholobus Spatholobus

Experimental equipment: vacuum drying oven, heating mantle, rotary evaporator.

experiment procedure:

Weigh 300g of Mildew Spatholobus medicinal material, put it into a 5L round-bottomed flask, add 3000ml of 60% ethanol, put it on a heating mantle for heating and reflux extraction, count from the slight boiling of the liquid, turn off the heating mantle to cool after 2 hours, filter with gauze Take out the liquid in the flask, add 3000ml of 60% ethanol again for extraction, combine the two extracts, concentrate under reduced pressure at 55°C to a thick extract, put it in a vacuum oven at 45°C for drying, and weigh.

Experimental results: 60.24 g of dry solid was obtained, and the paste yield was 20.08%.

4. Preparation and analysis of Achyranthes bidentata extract

4.1 Achyranthes bidentata 60% ethanol extraction

Achyranthes bidentata decoction pieces 250g, add 8 times the amount of 60% ethanol 2000ml to reflux for extraction for 2 hours, pour out the extract, then add 2000ml of 60% ethanol to reflux and extract for 2 hours, combine the two extracts, concentrate and dry to obtain 126.4g of Achyranthes bidentata extract , The cream yield is 50.6%.

Preparation example 2: Example of preparation process of flavored Erzhifang extract (2)

1. Take 200g of Ligustrum lucidum medicinal material, put it in a round bottom flask, add 2400ml of 60% ethanol, heat and reflux for extraction for 2 hours, pour out the filtrate to obtain the first extract of Ligustrum lucidum, then add 2400ml of 60% ethanol, heat Reflux extraction for 2 hours, pour out the filtrate to obtain the second extract of Ligustrum lucidum, and the medicinal residue of Ligustrum lucidum for later use, combine the two extracts, concentrate and dry to obtain 48.78 g of Ligustrum lucidum extract.

2. Get 200g Eclipta medicinal material, put in a round bottom flask, add 2000 milliliters of 70% ethanol, heat and reflux the extract for 3 hours, pour out the filtrate to obtain the first extract of Eclipta, then add 2000 milliliters of 70% ethanol, Heated and refluxed for 3 hours, poured out the filtrate to obtain the second extract of Eclipta chinensis, combined the two extracts, concentrated and dried to obtain 28.52 g of Eclipta eclipta extract.

3. Take the medicinal residue of Ligustrum lucidum obtained in the previous step 1, add 120g of Caulis Spatholobus and 80g of Achyranthes bidentata, put it in a round bottom flask, add 3200ml of water, heat and reflux for extraction for 2 hours, pour out the solution to obtain the first extraction solution, then add 3200 ml of water, heat and reflux for extraction for 2 hours, pour out the solution to obtain the second extract, combine the two extracts, concentrate and dry to obtain 67.56 g of Ligustrum lucidum Spatholobi Spatholobus Achyranthes bidentata extract.

Mix 48.78g of Ligustrum lucidum extract, 28.52g of Eclipta chinensis extract and 67.56g of Ligustrum lucidum Caulis Spatholobus Achyranthes extract to obtain the extract of the present invention, code-named XGY.

Without being bound by theory, the inventors found that the sample prepared by the preparation method of Preparation Example 2 is conducive to the formation of the preparation and the extraction of possible active ingredients with higher content (Ligustrum lucidum polysaccharide, verbascoside and/or ecclidene glycoside A, etc.) , Better efficacy.

In addition, any two or any three of the four medicinal materials may be mixed for extraction.

Preparation example 3: Example of preparation process of flavored Erzhifang extract (3)

Take 200g of Ligustrum lucidum medicinal material, 200g of Eclipta chinensis medicinal material, 120g of Millet Spatholobus and 80g of Achyranthes bidentata, put them in a round bottom flask, add 6000ml of 70% ethanol, heat and reflux for extraction, extract twice, each time for 2 hours, combine the two The secondary extract was concentrated and dried to obtain 140.58 g of flavored Erzhi Fang extract, code-named XF.

Experimental example 1: Anti-osteoporosis experiment

1. Materials and methods

1.1 Experimental animals

SPF grade female C57BL/6 mice, 8 weeks old, weighing 19-20g. Purchased from Speiford (Beijing) Biotechnology Co., Ltd., certificate number SCXK (Beijing) 2016-0002. They were raised in the Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Tianjin, with a temperature of 23-25°C, a humidity of 50%-60%, and a 12-hour cycle of light and dark.

1.2 Drugs

Flavored Erzhi Prescription Group: Ligustrum lucidum extract, Eclipta chinensis extract, Caulis Spatholobus extract, and Achyranthes bidentata extract were all prepared according to the method of Preparation Example 1.

Preparation method: Weigh 14.4g of Ligustrum lucidum extract, 10.65g of Eclipta officinalis extract, 7.23g of Caulis Spatholobus extract, and 12.14g of Achyranthes bidentata extract, dissolve in 0.2% CMCNa, and dilute to 100mL, as the flavored two to Prescription group administration solution. Store at 4°C.

1.3 Preparation of ovariectomized animal model

After one week of adaptive feeding, the mice were randomly divided into a sham operation group (sham) and an operation group (OVX) according to body weight. During the operation, the animals were anesthetized with tribromoethanol, the mice were fixed on the operating board, the middle and lower 1/3 of the back of the mice were depilated and skinned, disinfected with povidone iodine, and about 2 cm was cut longitudinally in the center of the bilateral backs. Make an incision, separate the skin layer and muscle layer, gently pull out the ovary with sterile ophthalmic forceps, ligate the end of the fallopian tube with surgical thread (to prevent postoperative bleeding), remove all the ovaries with ophthalmic scissors, and finally slowly bring the uterus into the abdominal cavity. The bilateral ovaries were removed in the same way, and the muscle layer and skin layer were sutured separately. The sham operation group was operated in the same way, but only the adipose tissue around the ovaries was removed, and the ovaries did not need to be removed. Continuous intraperitoneal injection of penicillin sodium (0.1 mL/cause, 200,000 units/mL) for three days after operation to prevent internal infection caused by operation.

1.4 Experimental group administration

As shown in Table 3 below.

table 3

The experimental animals were given 0.1mL/10g/d orally for 8 weeks. During the period of administration, the growth of the mice was observed, and the weight was recorded every week. The whole process was fed with standard feed, and they had free access to food and water.

2. Experimental results

2.1 Effects of Modified Erzhi Recipe on Body Weight and Viscera of Ovariectomized Mice

Gastrointestinal administration was carried out once a day for 8 consecutive weeks, and the changes in the body weight and organs of the mice in each group were observed. The results showed that the weight of the mice in the Model group increased significantly, and the modified Erzhifang group could significantly reduce the castration rate. The body weight of the mice increased (P<0.05). There were no significant changes in internal organs of mice in each group.

2.2 Effects of Modified Erzhi Recipe Administration on the Uterus of Ovariectomized Mice

After castration, the weight of the uterus of the mice was significantly reduced (P<0.01), and the uterus appeared to shrink, and the uterine atrophy could be improved after being given the modified Erzhi Formula.

2.3 Effects of Modified Erzhi Recipe on Fat Content in Castrated Mice

The results showed that compared with the sham operation group, there was no significant change in the fat content of the back of the mice after castration, but the modified Erzhi Recipe could significantly increase the fat content of the back of the mice (P<0.01). The results of the abdominal fat content showed that the abdominal fat content of the castrated mice increased significantly (P<0.01), and the modified Erzhi Fang could significantly reduce the abdominal fat content (P<0.01).

2.4 Effect of Modified Erzhi Prescription on Bone Density of Ovariectomized Mice

The bone mineral density (BMD) of the mouse tibia was detected by dual-energy X-ray absorptiometry, and the results showed that, compared with the sham operation group, the bone mineral density (BMD) of the mouse tibia in the model group decreased significantly (P<0.01). The administration of the modified Erzhi Fang can increase the bone density of the tibia.

Experimental example 2: Anti-osteoporosis experiment

1. Materials and methods

1.1 Experimental animals

SPF grade female C57BL/6 mice, 8 weeks old, weighing 19-20g. Purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., certificate number SCXK (Beijing) 2016-0006. They were raised in the Animal Center of Tianjin University of Traditional Chinese Medicine, with a temperature of 23-25°C, a humidity of 50%-60%, and a 12-hour cycle of light and dark.

1.2 Drugs

Modified Erzhi Formula Group (XGY): Ligustrum lucidum extract, Eclipta chinensis extract, and Ligustrum lucidum, Caulis Spatholobus and Achyranthes bidentata mixed extracts were all prepared according to the method of Preparation Example 2. Preparation method: weigh 14.63g of Ligustrum lucidum extract, 8.56g of Eclipta officinalis extract, and 20.27g of mixed extract of Ligustrum lucidum and Caulis Spatholobus Achyranthes bidentata, dissolve in 0.2% CMCNa, dilute to 100mL, and use it as flavored Erzhi recipe Group (XGY) was administered the solution. Store at 4°C.

Added flavored Erzhi formula group (XF): take the mixed medicinal materials of Ligustrum lucidum, Eclipta chinensis, Caulis Spatholobus and Achyranthes bidentata, and prepare according to the method of Preparation Example 3. Preparation method: Weigh 42.2 g of the above-mentioned XF extract, dissolve it with 0.2% CMCNa, and dilute to 100 mL, and use it as the dosing solution of the flavored Erzhifang group (XF). Store at 4°C.

Erzhifang Group (EZF): Ligustrum lucidum extract and Eclipta chinensis extract were both prepared according to the method of Preparation Example 1. Preparation method: Weigh 14.4 g of the above-mentioned Ligustrum lucidum extract and 10.65 g of the Eclipta chinensis extract, dissolve them in 0.2% CMCNa, and dilute to 100 mL, as the administration solution for the Erzhifang group (EZF). Store at 4°C.

1.3 Preparation of ovariectomized animal model

After one week of adaptive feeding, the mice were randomly divided into a sham operation group (sham) and an operation group (OVX) according to body weight. During the operation, the animals were anesthetized with tribromoethanol, the mice were fixed on the operating board, the middle and lower 1/3 of the back of the mice were depilated and skinned, disinfected with povidone iodine, and about 2 cm was cut longitudinally in the center of the bilateral backs. Make an incision, separate the skin layer and muscle layer, gently pull out the ovary with sterile ophthalmic forceps, ligate the end of the fallopian tube with surgical thread (to prevent postoperative bleeding), remove all the ovaries with ophthalmic scissors, and finally slowly bring the uterus into the abdominal cavity. The bilateral ovaries were removed in the same way, and the muscle layer and skin layer were sutured separately. The sham operation group was operated in the same way, but only the adipose tissue around the ovaries was removed, and the ovaries did not need to be removed. Continuous intraperitoneal injection of penicillin sodium (0.1 mL/cause, 200,000 units/mL) for three days after operation to prevent internal infection caused by operation.

1.4 Experimental group administration

As shown in Table 4 below.

Table 4

The experimental animals were given 0.1mL/10g/d orally for 8 weeks. During the period of administration, the growth of the mice was observed, and the weight was recorded every week. The whole process was fed with standard feed, and they had free access to food and water.

1.5 Detection of LDL-C content in serum

Before the test, the mouse serum samples were taken out from the -80°C refrigerator and equilibrated to room temperature, and the low-density lipoprotein cholesterol (LDL-C ) assay kit (batch number: 142020006) to detect LDL-C content in mouse serum.

2. Experimental results

2.1 Effects of Modified Erzhi Recipe on Body Weight and Viscera of Ovariectomized Mice

The intragastric administration was carried out once a day for 8 consecutive weeks, and the changes in the body weight and organs of the mice in each group were observed. The results are shown in Figures 1 to 5.

The results showed that the weight of the mice in the Model group increased significantly, and the Erzhifang group, the flavored Erzhifang group (XGY) and the flavored Erzhifang group (XF) could all significantly reduce the weight gain of castrated mice (Figure 1 and Figure 2, P<0.05). There were no significant changes in internal organs of the mice in each group (Fig. 3 to Fig. 5).

2.2 Effects of Modified Erzhi Recipe Administration on the Uterus of Ovariectomized Mice

The results are shown in Figure 6 and Figure 7.

The results showed that the weight of the uterus of the castrated mice was significantly reduced (P<0.01), and the uterus appeared atrophy, and the uterine atrophy could be improved after administration of Jiawei Erzhifang (XGY) and Jiawei Erzhifang (XF).

From the analysis of the results in Figure 6 to Figure 7, the uterine indicators of the Jiawei Erzhifang (XGY) group and the Jiawei Erzhifang (XF) group are more normal than those of the Erzhifang group (EZF), suggesting that the Jiawei Erzhifang (XGY ) group and modified Erzhifang (XF) group had more uterine nourishing effect than Erzhifang group, and showed stronger estrogen-like effect than Erzhifang group.

2.3 Effects of Erzhi Fang Compatible Administration on Fat Content in Castrated Mice

The results are shown in Figure 8 and Figure 9.

The results showed that, compared with the sham operation group, the abdominal fat content of castrated mice was significantly increased (P<0.01), among which Erzhifang, Jiawei Erzhifang (XGY) and Jiawei Erzhifang (XF) could significantly reduce Abdominal fat content (P<0.01). At the same time, the detection of body fat composition showed that the body fat composition of castrated mice increased significantly (P<0.01), and Erzhifang, Jiawei Erzhifang (XGY) and Jiawei Erzhifang (XF) could significantly reduce the weight of castrated mice. The content of body fat components in rats (P<0.01). In addition, the results in Figure 8 show that the flavored Erzhi Formula (XF) has a stronger effect on preventing fat gain in castrated mice than Erzhi Formula (EZF), suggesting that the flavored Erzhi Formula (XF) has a stronger effect on preventing and treating menopausal obesity. effect.

2.4 Effects of Erzhifang Compatible Administration on LDL-C Levels in Castrated Mice

Compared with the sham operation group, the serum LDL-C level in the model group increased, while Erzhifang, Jiawei Erzhifang (XGY) and Jiawei Erzhifang (XF) all had the effect of lowering LDL-C (Figure 10).

2.5 Effects of Erzhifang Compatible Administration on Bone Density of Ovariectomized Mice

The bone mineral density of the mouse femur was detected by dual-energy X-ray absorptiometry, and the results are shown in FIG. 11 .

The results showed that, compared with the sham operation group, the bone mineral density (BMD) of the mouse femur in the model group was significantly decreased (P<0.01). The administration of Erzhifang, Modified Erzhifang (XGY) and Modified Erzhifang (XF) could increase the BMD of femur (P<0.05 or P<0.01). The results also showed that, compared with the Erzhifang group (EZF), the modified Erzhifang group (XGY) had a more obvious effect on improving bone mineral density; the effect of the modified Erzhifang group (XGY) was even better than that of four traditional Chinese medicine combined Craft's flavored two-to-square set (XF).

references

[1] Liu Enling, Zhou Yuxiu. Efficacy of hormone replacement therapy with estradiol and drospirenone tablets for menopausal syndrome [J]. Chinese Journal of Gerontology, 2014, 26(19): 5566-5567.

[2] Chen Wei, Liu Dongling, Jiang Xingpeng. Observation on the clinical efficacy of Huiyang Acupuncture in the treatment of menopausal syndrome of spleen and kidney yang deficiency [J]. Chinese Journal of Basic Medicine of Traditional Chinese Medicine, 2016, 22(3): 401-403.

[3] Shen Hao, Xie Yanming. Research status of risk factors for osteoporotic fractures and risk assessment of TCM syndrome elements [J]. Chinese Orthopedics, 2014, 27(3): 261-265.

[4] Guo Huaping, Yu Yanyan, Chen Wenhua, Yu Bo, Qi Qi. Analysis of related risk factors and preventive measures for postmenopausal osteoporosis [J]. Chinese Journal of Rehabilitation Medicine, 2011, 26(5): 424-428 .

[5] Huang Jianli, Wang Gang, Li Lintao. Observation on the Effect of Chinese and Western Medicine in Treating Senile Osteoporosis [J]. Chinese Journal of Osteoporosis, 2016, 22(12): 1573-1575+1584.

[6] Zhang Jianxiang. Comparison of adverse reactions of Chinese and Western medicine in the treatment of depression [J]. Chinese Practical Medicine, 2010,5(19):130-131.

[7] Bai Junli. Clinical observation of hormone replacement therapy combined with alendronate sodium in the treatment of perimenopausal women with osteoporosis [J]. Clinical Medicine Practice, 2017,26(2):107-109.

[8] Zou Yong, Zuo Zhengyun, Zhao Haimei, et al. Research progress on pharmacological effects of Erzhi Pills [J]. Jiangxi Traditional Chinese Medicine. 2015, 46(03): 75-76+80.

[9] Liu Tingting, Wang Meng. Research progress on chemical constituents and pharmacological effects of Ligustrum lucidum [J]. Chinese Journal of Experimental Formulas. 2014,20(14):228-234.

[10] Fang Yue, Li Xichen, Zhang Chaofeng. Research progress on chemical constituents and pharmacological activities of Eclipta chinensis [J]. Strait Pharmacy. 2015,27(06):1-3.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (19)

1. A pharmaceutical composition comprising:

   
2. The pharmaceutical composition according to claim 1, comprising:

   

   Preferably, include:

   

   More preferably, including:

   

   Further preferably, including:

   

   Particularly preferably, include:

   

   
3. According to any one of claims 1 to 2, the pharmaceutical composition has a unit dose of 10-60 g, preferably 15-40 g or 20-30 g, more preferably 25 g.
4. An extract prepared from the pharmaceutical composition according to any one of claims 1 to 3.
5. Extract according to claim 4, it is made by the preparation method comprising the following steps:

   (1) Extracting Ligustrum lucidum, Eclipta chinensis, Gallis Spatholobus and Achyranthes bidentata of any one of claims 1 to 3 by weight with water or ethanol solution, to obtain an extract;

   (2) Concentrate the extract to obtain a concentrate, which is the extract.
6. The extract according to claim 5, characterized in that any one or more of the following ①-⑧ items:

   1. In step (1), the extraction is one or more times, and when it is multiple times, the extracts of each time are combined;

   ② In step (1), extract the mixture of Ligustrum lucidum, Eclipta chinensis, Caulis Spatholobus and Achyranthes bidentata; or extract any 1 flavor and the mixture of the remaining 3 flavors to obtain the respective extracts, combined or not Each extract; or extract the mixture of any 2 flavors and the mixture of the remaining 2 flavors separately to obtain the extracts, combine or not combine the extracts; or extract any 1 flavor, the other 1 flavor and the mixture of the remaining 2 flavors extracting separately to obtain each extract, merging or not merging each extract; or extracting 4 medicinal materials separately to obtain each extract, merging or not merging each extract;

   ③ In step (1), the concentration of the ethanol is 10%-99%, 20%-90%, 30%-80%, 40%-80%, 50%-80%, 50%-70% or 60% %-80%;

   ④ In step (1), the extraction is reflux extraction or decoction;

   ⑤ In step (1), the extraction time is at least 0.5 hour, at least 1 hour, at least 2 hours or 1-5 hours;

   6. In step (1), the consumption of water or ethanol solution is 5-20 times the amount (ml/g), preferably 8-16 times the amount;

   7. In step (2), the concentration is concentration under reduced pressure;

   8. In the step (2), the concentrate is an extract.
7. The extract according to any one of claims 5 to 6, wherein,

   Step (1) comprises the following steps:

   Extracting Ligustrum lucidum and Eclipta edulis separately to obtain Ligustrum lucidum extract and Eclipta edulis extract; extracting the mixture of Ligustrum lucidum extract and the mixture of Spatholobus Spatholobus and Achyranthes bidentata to obtain Ligustrum lucidum Spatholobus Spatholobus Achyranthes knuckle extract;

   Preferably, step (2) comprises the steps of:

   Concentrate the Ligustrum lucidum extract, the Eclipta chinensis extract and the Ligustrum lucidum Caulis Spatholobus Achyranthes extract respectively and then mix or combine the extracts and then concentrate to obtain the extract.
8. A pharmaceutical preparation comprising the extract according to any one of claims 4 to 7, and one or more pharmaceutically acceptable excipients;

   Preferably, the unit dose of the pharmaceutical preparation is 10-60 g, preferably 15-40 g or 20-30 g, more preferably 25 g, calculated according to the mass of the crude drug.
9. The pharmaceutical preparation according to claim 8, which is pill, tablet, granule, capsule, tincture or suspension.
10. The pharmaceutical preparation according to any one of claims 8 to 9, further comprising one or more selected from the following drugs:

    Calcium, vitamin D, bisphosphonates, estrogens such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone, and osteoprotegerin.
11. A combination pharmaceutical product comprising a first product and a second product in separate packages,

    in,

    The first product comprises the pharmaceutical composition according to any one of claims 1 to 3, the extract according to any one of claims 4 to 7, or the extract according to any one of claims 8 to 9. the above-mentioned pharmaceutical preparations;

    The second product contains one or more selected from the following drugs:

    Calcium, vitamin D, bisphosphonates, estrogens such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone, and osteoprotegerin;

    Preferably, the second product further comprises one or more pharmaceutically acceptable excipients;

    Preferably, the unit dose of the first product is 10-60g, preferably 15-40g or 20-30g, more preferably 25g, calculated according to the mass of the crude drug.
12. The pharmaceutical composition according to any one of claims 1 to 3, the extract according to any one of claims 4 to 7 or the pharmaceutical preparation according to any one of claims 8 to 9 are prepared Use in medicines for treating and/or preventing osteoporosis, perimenopausal syndrome, constipation or obesity, or medicines for lowering blood lipids;

    Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

    Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

    Preferably, the osteoporosis is perimenopausal osteoporosis;

    Preferably, the obesity is climacteric obesity.
13. The use according to claim 12, wherein the dosage per kilogram of body weight per day is 0.1g-2g, 0.2g-1g or 0.4g-0.8g, preferably 0.5g.
14. A method for preparing an extract, comprising the steps of:

    (1) Extracting Ligustrum lucidum, Eclipta chinensis, Gallis Spatholobus and Achyranthes bidentata of any one of claims 1 to 3 by weight with water or ethanol solution, to obtain an extract;

    (2) Concentrate the extract to obtain a concentrate, which is the extract.
15. The preparation method according to claim 14, characterized in that any one or more of the following ①-⑧ items:

    1. In step (1), the extraction is one or more times, and when it is multiple times, the extracts of each time are combined;

    ② In step (1), extract the mixture of Ligustrum lucidum, Eclipta chinensis, Caulis Spatholobus and Achyranthes bidentata; or extract any 1 flavor and the mixture of the remaining 3 flavors to obtain the respective extracts, combined or not Each extract; or extract the mixture of any 2 flavors and the mixture of the remaining 2 flavors separately to obtain the extracts, combine or not combine the extracts; or extract any 1 flavor, the other 1 flavor and the mixture of the remaining 2 flavors extracting separately to obtain each extract, merging or not merging each extract; or extracting 4 medicinal materials separately to obtain each extract, merging or not merging each extract;

    ③ In step (1), the concentration of the ethanol is 10%-99%, 20%-90%, 30%-80%, 40%-80%, 50%-80%, 50%-70% or 60% %-80%;

    ④ In step (1), the extraction is reflux extraction or decoction;

    ⑤ In step (1), the extraction time is at least 0.5 hour, at least 1 hour, at least 2 hours or 1-5 hours;

    6. In step (1), the consumption of water or ethanol solution is 5-20 times the amount (ml/g), preferably 8-16 times the amount;

    7. In step (2), the concentration is concentration under reduced pressure;

    8. In the step (2), the concentrate is an extract.
16. The preparation method according to any one of claims 14 to 15, wherein,

    Step (1) comprises the following steps:

    Extracting Ligustrum lucidum and Eclipta edulis separately to obtain Ligustrum lucidum extract and Eclipta edulis extract; extracting the mixture of Ligustrum lucidum extract and the mixture of Spatholobus Spatholobus and Achyranthes bidentata to obtain Ligustrum lucidum Spatholobus Spatholobus Achyranthes knuckle extract;

    Preferably, step (2) comprises the steps of:

    Concentrate the Ligustrum lucidum extract, the Eclipta chinensis extract and the Ligustrum lucidum Caulis Spatholobus Achyranthes extract respectively and then mix or combine the extracts and then concentrate to obtain the extract.
17. A method for treating and/or preventing osteoporosis, perimenopausal syndrome, constipation or obesity or lowering blood lipids, comprising administering to a subject in need an effective amount of any one of claims 1 to 3 The steps of the pharmaceutical composition, the extract according to any one of claims 4 to 7, or the pharmaceutical preparation according to any one of claims 8 to 9;

    Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

    Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

    Preferably, the osteoporosis is perimenopausal osteoporosis;

    Preferably, the obesity is climacteric obesity.
18. The method according to claim 17, wherein the dosage per kilogram of body weight per day is 0.1g-2g, 0.2g-1g or 0.4g-0.8g, preferably 0.5g.
19. According to the pharmaceutical composition according to any one of claims 1 to 3, the extract according to any one of claims 4 to 7 or the pharmaceutical preparation according to any one of claims 8 to 9, It is used for the treatment and/or prevention of osteoporosis, perimenopausal syndrome, constipation or obesity or lowering blood lipids;

    Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

    Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

    Preferably, the osteoporosis is perimenopausal osteoporosis;

    Preferably, the obesity is climacteric obesity.

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