CN114344361B

CN114344361B - Pharmaceutical composition for preventing and treating osteoporosis

Info

Publication number: CN114344361B
Application number: CN202210093858.1A
Authority: CN
Inventors: 高秀梅; 张伯礼; 刘二伟; 毛浩萍; 杨文志; 陈晓鹏; 韩立峰; 张晗; 陶蕊; 陈璐
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2021-05-14
Filing date: 2022-01-26
Publication date: 2023-12-15
Anticipated expiration: 2042-01-26
Also published as: WO2022237873A1; CN114344361A; CN117651563A

Abstract

The application belongs to the field of traditional Chinese medicines, relates to a pharmaceutical composition, and in particular relates to a pharmaceutical composition for treating and/or preventing osteoporosis. Specifically, a pharmaceutical composition comprising: 2-8 parts of glossy privet fruit, 2-8 parts of eclipta, 1-5 parts of suberect spatholobus stem and 1-5 parts of achyranthes root. The pharmaceutical composition can effectively prevent and treat osteoporosis, has better effect than two formulas, has good safety after long-term administration, and has good application prospect.

Description

Pharmaceutical composition for preventing and treating osteoporosis

Technical Field

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

Background

Osteoporosis (OP) is a systemic bone metabolic disease caused by multiple factors characterized by reduced bone mass per unit volume and destruction of bone tissue microstructure, resulting in increased bone fragility and susceptibility to fracture. Osteoporosis is a skeletal disease defined by the national institutes of health as a disease of bone that is impaired in bone strength, deterioration of bone tissue microstructure (small Liang Bianxi, broken, reduced number of cancellous bone) and increased risk of fracture, bone strength being a comprehensive reflection of bone density and bone mass. Osteoporosis can be divided into two major categories, namely primary OP and secondary OP, postmenopausal OP and senile OP both belong to primary OP, and patients with osteoporosis generally have no special clinical manifestations, but fracture at the far ends of hips, vertebral bodies or radius and the like is serious and frequent. The OP therapeutic drugs commonly used at present include estrogen, bone resorption inhibitor, bone formation promoter, bone mineralizer, etc.

Perimenopause is a special stage that women have to go through. During this period, women have reduced estrogen levels due to hypoovaries, resulting in dysfunction of the endocrine system, cardiovascular system, nervous system, etc., and a series of symptoms of varying degrees are collectively known as perimenopausal syndrome (Perimenopausal Syndrome, PS). Perimenopausal syndrome has its specific manifestations varied from person to person, and most of the symptoms can be improved by self-regulation and psychological dispersion ^[1] . However, perimenopausal osteoporosis, which is a very serious complication in perimenopausal syndrome, can cause serious injury to the psychology and physiology of females and even endanger the lives of patients ^[2] 。

According to statistics, the number of women suffering from osteoporosis in perimenopause in China accounts for about 25% of the total number of women in perimenopause. The disease is characterized by long disease course, and is not easy to be detected in early stage, and the disease is important until serious bone pain, vertebral degeneration, fracture and the like occur, and the disease is bad after healing, thus being very easy to cause the life-long disability and even death of patients ^[3,4] 。

At present, estrogen replacement therapy (HRT) is commonly used for treating perimenopausal osteoporosis in clinic, and the curative effect is good but the adverse reaction is quite large. The research results show that the long-term intake of estrogen therapy can increase the incidence rate of breast cancer and endometrial cancer and aggravate the risks of cerebral apoplexy, coronary arteriosclerosis, pulmonary embolism and deep vein thrombosis ^[5-7] . In addition, it is also counted that other types of drugs for osteoporosis can cause different types of adverse reactions, and serious and even life-threatening patients. Therefore, the traditional Chinese medicine gradually enters the sight of people due to good treatment effect, small toxic and side effects and good compliance, and the research on treating perimenopausal symptoms by the traditional Chinese medicine is increasingly paid attention to.

The second formula consists of glossy privet fruit, eclipta and other mass compatibility, the glossy privet fruit is used as a medicine, the eclipta is harvested in winter, the eclipta is used as a medicine in whole herb, and the eclipta is harvested in summer; the pill is called two-to-one pill, wherein fructus Ligustri Lucidi is usually processed with wine fructus Ligustri Lucidi. The formula II has the effects of tonifying liver and kidney, nourishing yin and stopping bleeding. Modern researches have shown that the pharmacological actions of the two formulas mainly comprise immunoregulatory action, antioxidant action, antiaging action, liver protecting action, osteoporosis resisting action, antiinflammatory action, antitumor action, hormone-like action, etc ^[8] 。

In the formula, the glossy privet fruit is the fruit of the glossy privet of the luteolinaceae, has sweet taste, cool nature, and liver and kidney meridian return, has the functions of nourishing liver and kidney, improving eyesight and blackening hair, and is mainly used for treating symptoms such as deficiency of liver-yin and kidney-yin, dizziness and tinnitus, soreness and weakness of waist and knees, premature graying of beard and hair, dim eyesight and the like. Modern research formThe glossy privet fruit contains abundant medicinal substances, mainly including triterpenes, iridoids, phenethyl alcohol, flavonoids, glossy privet fruit polysaccharide, etc., and the 2015 edition of Chinese pharmacopoeia takes the iridoid glycoside compound terglossy privet glycoside as the quality detection component of the glossy privet fruit ^[9] 。

In the recipe, eclipta (commonly called eclipta alba) is the whole herb of eclipta prostrata of Compositae, and is sweet and sour in taste, cool in nature, enters liver and kidney meridians. Has effects of nourishing liver and kidney, cooling blood and stopping bleeding, and can be used for treating diseases such as blood cooling, hemostasis, kidney invigorating, yin nourishing, etc. Modern researches have shown that the effective substances mainly comprise flavonoids, triterpenes and derivatives thereof, vanilloid, thiophen, steroid and volatile oil. The 2015 edition of Chinese pharmacopoeia takes the vanilla ether compound wedelolactone as the quality detection component of eclipta ^[10] 。

Caulis Spatholobi is a common traditional Chinese medicine for promoting blood circulation to remove blood stasis, and has the effects of liver and kidney channels, relaxing tendons, activating collaterals, promoting blood circulation, tonifying blood, regulating menstruation and relieving pain. The "Ben Cao Bei Yao" indicates "caulis Spatholobi, promoting blood circulation and relaxing tendons, and treating the dry blood fatigue of men and women and all deficiency and strain … …". Clinically, the traditional Chinese medicine composition is mainly used for irregular menstruation, sallow complexion due to blood deficiency, numbness and paralysis and rheumatalgia. Modern researches have found that suberect spatholobus stem has complex chemical composition and contains more than ten types of compounds such as flavans (such as catechin), terpenes (such as lupeol), sterols (beta-sitosterol), anthraquinones (such as emodin) and the like.

The radix Achyranthis bidentatae is root of radix Achyranthis bidentatae of achyranthes of Amaranthaceae. Bitter and sour nature, calm, enter liver and kidney meridians, and has the effects of tonifying liver and kidney, strengthening tendons and bones, promoting blood circulation, dredging meridians, guiding qi and blood downwards, inducing diuresis, treating stranguria and the like. Starting from Shennong Ben Cao Jing: the main cold-dampness flaccidity arthralgia, spasm of limbs, pain of knee, difficulty in flexing and extending, blood and qi expelling, impairment of heat and fire, and long-term taking of the medicine is light and durable. "

At present, more effective drug means capable of preventing and treating osteoporosis and other diseases are required to be developed.

Disclosure of Invention

The inventor of the present application has conducted intensive research and creative work to obtain a pharmaceutical composition for preventing and treating osteoporosis and other diseases and an extract thereof. The inventors have surprisingly found that the pharmaceutical combination or extract thereof is effective in preventing osteoporosis, in particular perimenopausal osteoporosis, in preventing constipation and/or in reducing blood lipid. The effect of the pharmaceutical composition or the extract of the application is better than that of the two-to-one pill, and the safety of long-term use is good. The following application is thus provided:

one aspect of the application relates to a pharmaceutical composition comprising:

in some embodiments of the application, the pharmaceutical composition comprises:

preferably, it comprises:

more preferably, it comprises:

further preferably, the method comprises:

particularly preferably, it comprises:

in some embodiments of the application, the pharmaceutical composition has a unit dose of 10-60g, preferably 15-40g, 20-30g, 22-28g or 24-26g, more preferably 25g.

In the pharmaceutical composition of the present application, the individual medicinal materials (e.g., 2, 3 or 4 of them) may be mixed or may be relatively independent (not mixed).

In some embodiments of the application, the pharmaceutical composition consists of the above 4 medicinal materials.

In some embodiments of the application, the pharmaceutical composition comprises the above 4 medicinal materials and pharmaceutically acceptable auxiliary materials.

Another aspect of the application relates to an extract made from the pharmaceutical composition of any of the application.

In some embodiments of the application, the extract is prepared by a preparation process comprising the steps of:

(1) Extracting any one of the above fructus Ligustri Lucidi, ecliptae herba, caulis Spatholobi and Achyranthis radix with water or ethanol solution to obtain extractive solution;

(2) Concentrating the extractive solution to obtain concentrate, which is extract.

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

(1) in the step (1), the extraction is carried out once or more times, and when the extraction is carried out more times, the extracting solutions of the times are combined;

(2) in the step (1), the mixture of glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root is extracted; or extracting the mixture of 1 or 3 of the above materials to obtain extractive solutions, and mixing or not mixing the extractive solutions; or extracting the mixture of any 2 kinds of materials and the mixture of the rest 2 kinds of materials respectively to obtain all extracting solutions, and combining or not combining all extracting solutions; or extracting the mixture of 1, one other and the other 2 to obtain each extract, and mixing or not mixing each extract; or extracting 4 medicinal materials respectively to obtain extractive solutions, and mixing or not mixing the extractive solutions;

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

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

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

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

(7) in the step (2), the concentration is reduced pressure concentration;

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

In some embodiments of the application, the extract, wherein in step (2), the concentrate is one or more (e.g., 2, 3, or 4). In the case where the extracts are not combined in the item (2), there are several kinds of extracts, and several kinds of concentrates are obtained; the several concentrates are now extracts.

In some embodiments of the application, the extract is prepared by separately extracting fructus Ligustri Lucidi and Ecliptae herba to obtain fructus Ligustri Lucidi concentrate and Ecliptae herba concentrate; extracting the residue after the extraction of the glossy privet fruit with the mixture of the suberect spatholobus stem and the achyranthes root to obtain the glossy privet fruit suberect spatholobus stem achyranthes root concentrate; the three concentrates obtained are the extracts of the present application.

In some embodiments of the application, the extract, wherein,

the step (1) comprises the following steps:

extracting fructus Ligustri Lucidi and Ecliptae herba respectively to obtain fructus Ligustri Lucidi extractive solution and Ecliptae herba extractive solution; extracting the residue after the extraction of the glossy privet fruit with the mixture of the suberect spatholobus stem and the achyranthes root to obtain glossy privet fruit suberect spatholobus stem achyranthes root extract;

preferably, step (2) comprises the steps of:

concentrating fructus Ligustri Lucidi extract, ecliptae herba extract and radix Achyranthis bidentatae extract respectively, mixing or mixing the extracts, and concentrating to obtain extract.

A further aspect of the application relates to a pharmaceutical formulation comprising an extract according to any one of the application, together with one or more pharmaceutically acceptable excipients;

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

In some embodiments of the application, the pharmaceutical formulation, wherein the extract is the only active ingredient.

In some embodiments of the application, the pharmaceutical formulation consists of the extract and one or more pharmaceutically acceptable excipients.

In some embodiments of the application, the pharmaceutical formulation is a pill, tablet, granule, capsule, tincture, or suspension.

In some embodiments of the application, the pharmaceutical formulation further comprises one or more of the following drugs selected from the group consisting of:

calcium agents, vitamin D, bisphosphates, estrogens such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone and osteoprotegerin.

Yet another aspect of the application relates to a combination pharmaceutical product comprising a first product and a second product which are individually packaged,

wherein,

the first product comprises the pharmaceutical composition of any one of the present application, the extract of any one of the present application or the pharmaceutical formulation of any one of the present application;

the second product comprises one or more of the following drugs:

calcium agents, vitamin D, bisphosphates, 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 on the mass of the crude drug.

The "first" or "second" in the "first product" or "second product" is merely for distinction or reference, and does not have an ordinal meaning.

A further aspect of the application relates to the use of a pharmaceutical composition according to any one of the application, an extract according to any one of the application or a pharmaceutical formulation according to any one of the application for the manufacture of a medicament for the treatment and/or prophylaxis of osteoporosis, perimenopausal syndrome, constipation or obesity, or a medicament for reducing blood lipid;

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 application, the use, wherein the dosage administered per kilogram of body weight per day is 0.1g-2g, 0.2g-1g or 0.4g-0.8g, preferably 0.5g.

Yet another aspect of the application relates to a method of preparing an extract comprising the steps of:

(1) Extracting glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root which are required by any one of the application in parts by weight with water or ethanol solution to obtain an extracting solution;

In some embodiments of the application, the method of preparation is characterized by any one or more of the following items (1) - (8):

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

(7) in the step (2), the concentration is reduced pressure concentration;

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

In some embodiments of the application, the method of making, wherein,

the step (1) comprises the following steps:

preferably, step (2) comprises the steps of:

The ethanol solutions according to the present application refer to aqueous ethanol solutions unless otherwise specified.

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

In the present application, unless otherwise specified, the term "two-to-one" means a prescription composed of glossy privet fruit and eclipta (commonly known as eclipta alba), and the term "two-to-one" means a preparation obtained by industrial production.

In the present application, the pharmaceutical composition or extract of the present application is sometimes referred to as a modified second ingredient.

Typically, the pharmaceutical formulation of the present application contains from 0.1% to 90% by weight of the extract of the present application as a main drug. Pharmaceutical formulations may be prepared according to methods known in the art. For this purpose, the primary drug may be combined, if desired, with one or more solid or liquid pharmaceutical excipients and/or adjuvants, to form a suitable administration form or dosage form for human use.

The term "excipient" in the present application refers to an excipient or vehicle used to administer the primary drug, including but not limited to diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, coating materials, and the like. Adjuvants are generally described in "Remington's Pharmaceutical Sciences" of e.w. martin. Examples of excipients include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethyl cellulose, sodium carboxymethyl cellulose, crospovidone, glycerol isostearate, glycerol monostearate, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyoctacosanol stearate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, and the like.

The extracts of the present application may be formulated into pharmaceutical preparations, including dosage forms suitable for oral administration, dosage forms suitable for parenteral injection (e.g., intravenous injection, subcutaneous injection) (e.g., as solutions), dosage forms suitable for topical administration (e.g., as ointments, patches or creams), and dosage forms suitable for rectal administration (e.g., as suppositories), and the like.

Depending on the disease to be treated and the patient and the route of administration, the pharmaceutical formulation of the application may be administered at different doses one or more times daily. The dosage to be administered depends on many factors such as the severity of osteoporosis to be treated or prevented, the sex, age, weight and individual response of the patient, the route and frequency of administration, etc. The above-mentioned doses may be administered in a single dosage form or divided into several, e.g. two, three, four dosage forms.

The actual dosage level of the primary drug (extract) in the pharmaceutical formulation of the present application may be varied so as to be effective in achieving the desired therapeutic response for a particular patient, composition and mode of administration. The dosage level will be selected based on the route of administration, the severity of the condition being treated, and the condition and past history of the patient to be treated. However, it is practiced in the art to administer doses that begin at levels below that required to achieve the desired therapeutic effect and gradually increase until the desired effect is achieved.

The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present application in a subject.

Advantageous effects of the application

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

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

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

(3) The pharmaceutical composition or the extract of the application can effectively reduce blood lipid and treat or prevent hyperlipidemia.

(4) The effect of the pharmaceutical composition or the extract of the application for treating or preventing osteoporosis is better than that of two-to-one (two-to-one pill).

(5) The pharmaceutical composition or the extract of the application has good safety in long-term use.

(6) The preparation method of the application is superior to the traditional two-to-one (two-to-one) preparation process, for example, under the condition of the same composition, the extract prepared by the preparation method of the application has better anti-osteoporosis effect, for example, the bone density can be better improved.

Drawings

Fig. 1: graph of body weight change over time for each group of animals.

Fig. 2: bar graph of body weight before drawing (before death of animals) for each group of animals.

Fig. 3: bar graph of liver weight index for each group of animals.

Fig. 4: bar graph of spleen index for each group of animals.

Fig. 5: bar graph of kidney index for each group of animals.

Fig. 6: bar graph of uterine index for each group of animals.

Fig. 7: representative uterine map for each group of animals.

Fig. 8: bar graphs of animal fat index for each group.

Fig. 9: bar graph of white fat index for each group of animals.

Fig. 10: bar graph of serum LDL-C levels for each group of animals.

Fig. 11: bar graph of bone density for each group of animals.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Preparation example 1: preparation process example of Jiawei two-to-one extract (1)

1. Preparation and analysis of glossy privet fruit extract

1.1 preparation of glossy privet fruit extract

Experimental instrument: vacuum drying oven, heating jacket, rotary evaporator.

The experimental process comprises the following steps:

300g of glossy privet fruit medicinal material is taken, placed into a 20L round bottom flask, 80% (v/v) ethanol is added into the flask for 10L, the flask is placed on a heating sleeve for heating reflux extraction, the heating sleeve is closed for cooling after 2 hours from the time of liquid micro-boiling, the liquid in the flask is filtered out by using gauze, 10L of 80% ethanol is added again for extraction, the two extracts are combined, the mixture is decompressed and concentrated to thick extract at 50 ℃, and the thick extract is placed into a 45 ℃ vacuum drying box for drying and weighing.

Experimental results: 72.0g of dry sample was obtained, and the paste yield was 24%.

1.2 detection method and Single factor test results

1.2.1HPLC condition: instrument: waters2695, column chromatography: agilent 4.6X250 mm,5 μm, temperature: 25 ℃, 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 a control solution: precisely weighing the terprivet glycoside reference substance, and dissolving with methanol to obtain reference substance solution.

And (5) respectively injecting 10 μl of the reference substance solution and the sample solution, calculating the content of the index component, and counting. The fructus Ligustri Lucidi extract contains terligustrum glycoside 19.82mg/g.

2. Preparation and analysis of eclipta extract

2.1 thermal reflux extraction of Ecliptae herba with 60% ethanol

Experimental instrument: vacuum drying oven, heating jacket, rotary evaporator.

The experimental process comprises the following steps:

weighing 300g of eclipta medicinal material, putting into a 20L round bottom flask, adding 60% ethanol 10L, placing on a heating sleeve for heating reflux extraction, performing micro-boiling time on liquid, closing the heating sleeve for cooling after 2 hours, filtering out liquid in the flask by using gauze, adding 10L 60% ethanol again for extraction, combining the two extraction solutions, concentrating under reduced pressure at 55 ℃ to thick extract, putting into a 45 ℃ vacuum drying oven for drying and weighing.

Experimental results: 53.25g of dry sample was obtained, and the paste yield was 17.75%.

2.2 detection method and Single factor test results

2.2.1HPLC condition: instrument: waters2695, column chromatography: agilent 4.6X250 mm,5 μm, temperature: 25 ℃, 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 a control solution: precisely weighing wedelolactone reference substance, dissolving with methanol, and making into reference substance solution.

And (5) respectively injecting 10 μl of the reference substance solution and the sample solution, calculating the content of the index component, and counting. The eclipta extract has wedelolactone content of 0.8mg/g.

3. Preparation and analysis of caulis Spatholobi extract

3.1 caulis Spatholobi 60% ethanol under reflux by heating

Experimental instrument: vacuum drying oven, heating jacket, rotary evaporator.

The experimental process comprises the following steps:

300g of suberect spatholobus stem medicinal material is weighed, placed into a 5L round bottom flask, added with 3000ml of 60% ethanol, placed on a heating sleeve for heating reflux extraction, counted by micro boiling of liquid, cooled by closing the heating sleeve after 2 hours, liquid in the flask is filtered out by gauze, added with 3000ml of 60% ethanol again for extraction, the two extracts are combined, concentrated to thick extract under reduced pressure at 55 ℃, placed into a vacuum drying oven at 45 ℃ for drying and weighing.

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

4. Preparation and analysis of achyranthes extract

4.1 60% ethanol extraction of achyranthes

250g of achyranthes root decoction pieces, 2000ml of 60% ethanol with the amount of 8 times is added for reflux extraction for 2 hours, 2000ml of 60% ethanol is added into the decoction dregs for reflux extraction for 2 hours after the extract is poured out, the two extracts are combined, 126.4g of achyranthes root extract is obtained by concentrating and drying, and the ointment yield is 50.6%.

Preparation example 2: preparation process example of Jiawei two-to-one extract (2)

1. 200g of glossy privet fruit medicinal material is taken, placed in a round-bottom flask, 2400 ml of 60% ethanol is added, heating reflux extraction is carried out for 2 hours, filtrate is poured out to obtain glossy privet fruit first extract, 2400 ml of 60% ethanol is added, heating reflux extraction is carried out for 2 hours, filtrate is poured out to obtain glossy privet fruit second extract, glossy privet fruit residues are reserved, the two extracts are combined, and 48.78g of glossy privet fruit extract is obtained through concentration and drying.

2. 200g of eclipta medicinal material is taken, placed in a round bottom flask, 2000ml of 70% ethanol is added, the extract is heated and refluxed for 3 hours, filtrate is poured out to obtain first extract of eclipta, 2000ml of 70% ethanol is added, the heating and refluxing extraction are carried out for 3 hours, filtrate is poured out to obtain second extract of eclipta, the two extracts are combined, and 28.52g of eclipta extract is obtained by concentrating and drying.

3. Adding 120g of suberect spatholobus stem and 80g of achyranthes root into the glossy privet fruit dregs prepared in the step 1, placing the glossy privet fruit dregs into a round bottom flask, adding 3200 ml of water, heating and reflux-extracting for 2 hours, pouring out the solution to obtain a first extract, adding 3200 ml of water, heating and reflux-extracting for 2 hours, pouring out the solution to obtain a second extract, combining the two extracts, concentrating and drying to obtain 67.56g of glossy privet fruit suberect spatholobus root extract.

The extract of the application is obtained by mixing 48.78g of the glossy privet fruit extract, 28.52g of the eclipta extract and 67.56g of the glossy privet fruit and suberect spatholobus stem extract, and the code number is XGY.

Without being bound by theory, the inventors found that the sample prepared by the preparation method of preparation example 2 is beneficial to the formation of the preparation, has higher content of possible effective components (ligustrum lucidum polysaccharide, acteoside and/or ecliptin A, etc.) and better drug effect.

In addition, any two or any three of the 4 medicinal materials can be mixed for extraction.

Preparation example 3: preparation process example of modified Erzhi square extract (3)

200g of glossy privet fruit medicinal material, 200g of eclipta medicinal material, 120g of suberect spatholobus stem and 80g of twotooth achyranthes root are taken, put into a round bottom flask, added with 6000 ml of 70% ethanol, heated and reflux extracted for 2 times, each time for 2 hours, the two extracts are combined, concentrated and dried to obtain 140.58g of the flavored second-to-first extract with the code of XF.

Experimental example 1: anti-osteoporosis test

1. Materials and methods

1.1 laboratory animals

SPF-grade female C57BL/6 mice, 8 weeks old, weighing 19-20g. Purchased from si Bei Fu (beijing) biotechnology limited, certification number SCXK (jing) 2016-0002. Raising in the institute of radiology of Chinese medical sciences of Tianjin, the raising temperature is 23-25 deg.c, the humidity is 50-60% and the light is 12 hr.

1.2 medicaments

Adding flavor two to formula group: fructus Ligustri Lucidi extract, ecliptae herba extract, caulis Spatholobi extract, and Achyranthis radix extract are prepared according to the method of preparation example 1.

The preparation method comprises the following steps: weighing fructus Ligustri Lucidi extract 14.4g, ecliptae herba extract 10.65g, caulis Spatholobi extract 7.23 and Achyranthis radix extract 12.14g, dissolving with 0.2% CMCna, and fixing volume to 100mL to obtain the final product. Preserving at 4 ℃.

1.3 preparation of an ovariectomized animal model

After one week of adaptive feeding, mice were randomly divided into sham surgery groups (sham) and surgery groups (OVX) by body weight. During operation, the animals are anesthetized by tribromoethanol, the mice are fixed on an operation plate, mao Beipi is removed from the 1/3 position of the lower middle part of the back of the mice, iodophor is disinfected, an incision of about 2cm is longitudinally cut at the center of the back of each side, a skin layer and a muscle layer are separated, the ovaries are gently pulled out by sterile ophthalmic forceps, ligation is carried out at the tail end of a fallopian tube by using an operation wire (post-operation bleeding is prevented), the ovaries are completely resected by ophthalmic scissors, the uterus is slowly taken into the abdominal cavity, the ovaries on each side are removed in the same way, and the muscle layer and the skin layer are respectively sutured. The sham operation group is operated by adopting the same method, but only the adipose tissues around the ovary are removed, and the ovary is not required to be removed. Sodium penicillin (0.1 mL/piece, 20 ten thousand units/mL) was continuously injected intraperitoneally for three days after the operation to prevent in vivo infection caused by the operation.

1.4 experimental group administration

As shown in table 3 below.

TABLE 3 Table 3

The experimental animals are administrated by lavage at 0.1mL/10g/d for 8 weeks, the growth condition of the mice is observed during the administration period, the mice are weighed and recorded every week, and the whole course standard feed is fed, and the mice are fed with free food and water.

2. Experimental results

2.1 Effect of two-to-one dosing on weight and organs of castrated mice

The mice are subjected to 1-time intragastric administration every day, the continuous intragastric administration is carried out for 8 weeks, the change condition of the weights and organs of each group of mice is observed, and the results show that the weight of the mice in the Model group is obviously increased, and the weight increase phenomenon (P is less than 0.05) of the castrated mice can be obviously reduced by adding the flavor to the formula II. There was no significant change in viscera in each group of mice.

2.2 Effect of modified two-to-one dosing on the uterus of castrated mice

The weight of the uterus of the mice is obviously reduced (P is less than 0.01) after castration, the uterus has atrophy phenomenon, and the mice have the phenomenon of improving uterine atrophy after the mice are flavored for two times.

2.3 Effect of modified two-to-one dosing on fat content in castrated mice

The results showed that there was no significant change in the back fat content of the mice after castration compared to sham surgery groups, whereas the addition of taste two to four could significantly increase the back fat content (P < 0.01). The abdominal fat content results show that the abdominal fat content of the mice is obviously increased (P < 0.01) after castration, wherein the abdominal fat content can be obviously reduced (P < 0.01) by adding flavor two to two.

2.4 Effect of modified two-to-one dosing on bone Density in castrated mice

The bone mineral density of the tibia of the mice was examined by a dual-energy X-ray bone densitometer, which showed that the Bone Mineral Density (BMD) of the tibia of the mice in the model group was significantly reduced (P < 0.01) compared to the sham surgery group. The second-to-fourth dosing can raise the bone density of the tibia.

Experimental example 2: anti-osteoporosis test

1. Materials and methods

1.1 laboratory animals

SPF-grade female C57BL/6 mice, 8 weeks old, weighing 19-20g. Purchased from beijing velarihua laboratory animal technologies limited, underwriter's SCXK (jing) 2016-0006. Raising in animal center of Tianjin university of Chinese medicine at 23-25 deg.c and humidity of 50-60% in 12 hr for light and shade circulation.

1.2 medicaments

Flavor two to square group (XGY): the glossy privet fruit extract, the eclipta extract and the glossy privet fruit and suberect spatholobus stem mixed extract are prepared according to the method of preparation example 2. The preparation method comprises the following steps: weighing 14.63g of glossy privet fruit extract, 8.56g of eclipta extract and 20.27g of glossy privet fruit and suberect spatholobus stem and achyranthes root mixed extract, dissolving the mixed extract with 0.2% of CMCNa, and fixing the volume to 100mL to obtain an additive second-formula (XGY) administration solution. Preserving at 4 ℃.

Odorizing two-to-one (XF): the preparation method of preparation example 3 is carried out by taking glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root mixed medicinal materials. The preparation method comprises the following steps: 42.2g of the XF extract was weighed out, dissolved in 0.2% CMCNA, and fixed to 100mL as an flavored two-to-one (XF) dosing solution. Preserving at 4 ℃.

Two-to-square group (EZF): both the glossy privet fruit extract and the eclipta extract were prepared according to the method of preparation example 1. The preparation method comprises the following steps: 14.4g of the glossy privet fruit extract and 10.65g of the eclipta extract are weighed, dissolved by 0.2% CMCNA, and fixed to 100mL to be taken as a two-to-one group (EZF) administration solution. Preserving at 4 ℃.

1.3 preparation of an ovariectomized animal model

1.4 experimental group administration

As shown in table 4 below.

TABLE 4 Table 4

1.5 detection of LDL-C content in serum

Before detection, the serum sample of the mouse is taken out from a refrigerator at-80 ℃ and equilibrated to room temperature, and the LDL-C content in the serum of the mouse is detected by using a full-automatic biochemical analyzer (model: BS-240VET, shenzhen Michael biomedical electronics Co., ltd.) and a low-density lipoprotein cholesterol (LDL-C) detection kit (batch number: 142020006).

2. Experimental results

2.1 Effect of two-to-one dosing on weight and organs of castrated mice

The administration was carried out by 1-day, and the mice were continuously fed with stomach for 8 weeks, and the body weight and organs of each group were observed, and the results are shown in fig. 1 to 5.

The results showed that Model mice significantly increased in weight, and that both the two-to-square, flavored two-to-square (XGY) and flavored two-to-square (XF) mice significantly reduced weight gain in castrated mice (FIGS. 1 and 2, P < 0.05). There was no significant change in internal organs of each group of mice (fig. 3 to 5).

2.2 Effect of modified two-to-one dosing on the uterus of castrated mice

The results are shown in fig. 6 and 7.

The results show that the mice after castration have significantly reduced uterine weight (P < 0.01), the uterus has atrophy, and the mice after administration of the flavor two to one (XGY) and flavor two to one (XF) have the effect of improving uterine atrophy.

From the analysis of the results of fig. 6-7, the uterus index of the flavored di-to-square (XGY) group and the flavored di-to-square (XF) group tended to be more normal than the two-to-square (EZF), suggesting that the flavored di-to-square (XGY) group and the flavored di-to-square (XF) group had a better uterine nourishing effect than the two-to-square group, and exhibited a stronger estrogenic effect than the two-to-square group.

2.3 Effect of two-to-one compatible administration on fat content in castrated mice

The results are shown in fig. 8 and 9.

The results show that the abdominal fat content of mice after castration is significantly increased (P < 0.01) compared to sham surgery groups, with two-to-square and flavored two-to-square (XGY) and flavored two-to-square (XF) being able to significantly reduce the abdominal fat content (P < 0.01). Meanwhile, body fat component content detection shows that the body fat component of the castrated mice is obviously increased (P is less than 0.01), and the body fat component of the castrated mice can be obviously reduced (P is less than 0.01) by a second-to-square method, a second-to-square method (XGY) and a second-to-square method (XF). In addition, the results of fig. 8 show that the flavored di-to-square (XF) has a stronger effect on preventing and treating climacteric obesity than the di-to-square (EZF) in resisting the increased fat in castrated mice.

2.4 Effect of two-to-formulation dosing on LDL-C levels in castrated mice

The serum LDL-C levels were elevated in the model group compared to the sham group, while both the two-to-party, the flavored two-to-party (XGY) and the flavored two-to-party (XF) had an LDL-C lowering effect (fig. 10).

2.5 Effect of two-to-one compatible administration on bone Density in castrated mice

Bone mineral density of the femur of the mice was measured using a dual energy X-ray bone densitometer, and the results are shown in fig. 11.

The results showed a significant reduction in Bone Mineral Density (BMD) of the mouse femur in the model group (P < 0.01) compared to the sham group. Both the two-to-side, the two-to-side with flavor (XGY) and the two-to-side with flavor (XF) administration can raise bone density of femur (P < 0.05 or P < 0.01). The results also show that the effect of the flavored two-to-one group (XGY) on increasing bone density is more pronounced than the two-to-one group (EZF); the effect of the flavor two-to-one group (XGY) is even better than that of the flavor two-to-one group (XF) adopting the combined extraction process of 4 traditional Chinese medicines.

Reference to the literature

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[3] Shen Hao and Xie Yanming the risk factors of osteoporotic fracture and the risk assessment of the Chinese syndrome factor are [ J ]. Chinese bone injury, 2014,27 (3): 261-265.

[4] Guo Huaping, yu Yanyan, chen Wenhua, residual wave, qi Ji. Analysis of risk factors related to the onset of postmenopausal osteoporosis and preventive measures discussion [ J ]. J.J.Chinese journal of rehabilitation, 2011,26 (5): 424-428.

[5] Huang Jianli, wang Gang, li Lintao. Observations of the effect of Chinese and Western medicines on senile osteoporosis [ J ]. J.Chinese osteoporosis, 2016,22 (12): 1573-1575+1584.

[6] Zhang Jianxiang adverse reactions of the Chinese and western medicines for treating depression are compared with [ J ]. Chinese practical medicine 2010,5 (19): 130-131.

[7] Bai Junli clinical observations of the treatment of perimenopausal women with the hormone replacement therapy in combination with alendronate sodium [ J ]. Clinical medical practice 2017,26 (2): 107-109.

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[9] Liu Tingting and Wang Meng the chemical components of fructus Ligustri Lucidi and the pharmaceutical effect are developed [ J ]. J.Chinese experimental prescription journal 2014,20 (14): 228-234.

[10] Fang Yue, li Xichen, zhang Chaofeng. Research on chemical components and pharmacological activities of eclipta [ J ]. Pharma pharmaceutical 2015,27 (06): 1-3.

Although specific embodiments of the application have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present application. The full scope of the application is given by the appended claims and any equivalents thereof.

Claims

1. An extract prepared from a pharmaceutical composition comprising:

4-6 parts of glossy privet fruit,

4-6 parts by weight of eclipta,

caulis Spatholobi 2-4 weight parts, and

1-3 parts of achyranthes root;

and the extract is prepared by a preparation method comprising the steps of:

(1) Respectively extracting the glossy privet fruit and the eclipta by using an ethanol solution to obtain glossy privet fruit extract and eclipta extract; extracting the residue after the extraction of the glossy privet fruit, the mixture of the caulis spatholobi and the achyranthes root with water to obtain glossy privet fruit and caulis spatholobi achyranthes root extract; the concentration of the ethanol solution is 50% -80%; the extraction time is at least 1 hour;

(2) Concentrating fructus Ligustri Lucidi extract, ecliptae herba extract and radix Achyranthis bidentatae extract respectively, mixing or mixing the extracts, and concentrating to obtain extract.

2. The extract of claim 1, wherein the pharmaceutical composition comprises the following:

4.5 to 5.5 weight portions of glossy privet fruit,

4.5 to 5.5 parts by weight of eclipta,

caulis Spatholobi 2.5-3.5 weight parts, and

1.5 to 2.5 parts by weight of achyranthes root.

3. The extract of claim 1, wherein the pharmaceutical composition comprises the following:

4.8 to 5.2 weight portions of glossy privet fruit,

4.8 to 5.2 parts by weight of eclipta,

caulis Spatholobi 2.8-3.2 weight parts, and

1.8 to 2.2 parts by weight of achyranthes root.

4. The extract of claim 1, wherein the pharmaceutical composition comprises the following:

5 parts by weight of glossy privet fruit,

5 parts by weight of eclipta,

3 parts by weight of spatholobus stem, and

2 parts of achyranthes root.

5. The extract according to any one of claims 1 to 4, wherein the pharmaceutical composition has a unit dose of 10-60g.

6. The extract according to any one of claims 1 to 4, wherein the pharmaceutical composition has a unit dose of 15-40g.

7. The extract according to any one of claims 1 to 4, wherein the pharmaceutical composition has a unit dose of 20-30g.

8. The extract of any one of claims 1 to 4, wherein the unit dose of the pharmaceutical composition is 25g.

9. The extract according to claim 1, characterized in that any one or more of the following (1) - (5):

(2) in the step (1), the extraction is reflux extraction or decoction;

(3) in the step (1), the dosage of the water or ethanol solution is 5-20 times of the dosage (mL/g) calculated according to the mL/g;

(4) in the step (2), the concentration is reduced pressure concentration;

(5) in the step (2), the concentrate is an extract.

10. The extract according to claim 1, wherein in step (1), the concentration of the ethanol solution is 50% -70%.

11. The extract according to claim 1, wherein in step (1), the concentration of the ethanol solution is 60% -80%.

12. The extract according to claim 1, wherein in step (1), the extraction time is at least 2 hours.

13. The extract according to claim 1, wherein in the step (1), the extraction time is 1 to 5 hours.

14. The extract according to claim 1, wherein in the step (1), the amount of water or ethanol solution is 8-16 times in terms of mL/g.

15. A pharmaceutical formulation comprising the extract of any one of claims 1 to 14, and one or more pharmaceutically acceptable excipients.

16. The pharmaceutical formulation according to claim 15, wherein the unit dose of the pharmaceutical formulation is 10-60g calculated on the mass of crude drug.

17. The pharmaceutical formulation according to claim 15, wherein the unit dose of the pharmaceutical formulation is 15-40g calculated on the mass of crude drug.

18. The pharmaceutical formulation according to claim 15, wherein the unit dose of the pharmaceutical formulation is 20-30g calculated on the mass of crude drug.

19. The pharmaceutical formulation of claim 15, wherein the pharmaceutical formulation has a unit dose of 25g calculated on a mass basis of the crude drug.

20. The pharmaceutical formulation of any one of claims 15 to 19, which is a pill, tablet, granule, capsule, tincture or suspension.

21. Use of an extract according to any one of claims 1 to 14 or a pharmaceutical formulation according to any one of claims 15 to 20 in the manufacture of a medicament for the treatment and/or prophylaxis of osteoporosis.

22. The use of claim 21, wherein the osteoporosis is primary or secondary osteoporosis.

23. The use of claim 21, wherein the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.

24. The use of claim 21, wherein the osteoporosis is perimenopausal osteoporosis.

25. A process for preparing the extract of any one of claims 1 to 14, comprising the steps of:

(1) Respectively extracting the following glossy privet fruits and eclipta by using an ethanol solution to obtain glossy privet fruit extract and eclipta extract; extracting the residue after the extraction of the glossy privet fruit, the mixture of the caulis spatholobi and the achyranthes root with water to obtain glossy privet fruit and caulis spatholobi achyranthes root extract; the concentration of the ethanol solution is 50% -80%; the extraction time is at least 1 hour;

4-6 parts of glossy privet fruit,

4-6 parts by weight of eclipta,

caulis Spatholobi 2-4 weight parts, and

1-3 parts of achyranthes root;

26. The preparation method of claim 25, wherein in the step (1), glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root with the following weight parts are extracted:

4.5 to 5.5 weight portions of glossy privet fruit,

4.5 to 5.5 parts by weight of eclipta,

caulis Spatholobi 2.5-3.5 weight parts, and

1.5 to 2.5 parts by weight of achyranthes root.

27. The preparation method of claim 25, wherein in the step (1), glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root with the following weight parts are extracted:

4.8 to 5.2 weight portions of glossy privet fruit,

4.8 to 5.2 parts by weight of eclipta,

caulis Spatholobi 2.8-3.2 weight parts, and

1.8 to 2.2 parts by weight of achyranthes root.

28. The preparation method of claim 25, wherein in the step (1), glossy privet fruit, eclipta, suberect spatholobus stem and achyranthes root with the following weight parts are extracted:

5 parts by weight of glossy privet fruit,

5 parts by weight of eclipta,

3 parts by weight of spatholobus stem, and

2 parts of achyranthes root.

29. The preparation method according to any one of claims 25 to 28, wherein the total unit dose of fructus Ligustri Lucidi, ecliptae herba, caulis Spatholobi and Achyranthis radix is 10-60g.

30. The preparation method according to any one of claims 25 to 28, wherein the total unit dose of fructus Ligustri Lucidi, ecliptae herba, caulis Spatholobi and Achyranthis radix is 15-40g.

31. The preparation method according to any one of claims 25 to 28, wherein the total unit dose of fructus Ligustri Lucidi, ecliptae herba, caulis Spatholobi and Achyranthis radix is 20-30g.

32. The preparation method according to any one of claims 25 to 28, wherein the total unit dose of fructus ligustri lucidi, eclipta, caulis spatholobi and achyranthes is 25g.

33. The production method according to claim 25, characterized in that any one or more of the following (1) to (5):

(2) in the step (1), the extraction is reflux extraction or decoction;

(3) in the step (1), the dosage of the water or ethanol solution is 5-20 times of the dosage of the water or ethanol solution according to the mL/g;

(4) in the step (2), the concentration is reduced pressure concentration;

(5) in the step (2), the concentrate is an extract.

34. The method according to claim 33, wherein in the step (1), the concentration of the ethanol solution is 50% to 70%.

35. The method according to claim 33, wherein in the step (1), the concentration of the ethanol solution is 60% to 80%.

36. The method according to claim 33, wherein in the step (1), the extraction time is at least 2 hours.

37. The process according to claim 33, wherein in the step (1), the extraction time is 1 to 5 hours.

38. The production process according to claim 33, wherein in the step (1), the amount of the water or ethanol solution is 8 to 16 times in terms of mL/g.