CN115844941A

CN115844941A - Composition of eucommia ulmoides extract and application of composition in preparation of anti-osteoporosis medicine

Info

Publication number: CN115844941A
Application number: CN202211672532.0A
Authority: CN
Inventors: 肖军平; 涂水才; 刘厚权; 龙军; 程志红
Original assignee: JIANGXI PUZHENG PHARMACEUTICAL CO Ltd
Current assignee: JIANGXI PUZHENG PHARMACEUTICAL CO Ltd
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-03-28
Anticipated expiration: 2042-12-26
Also published as: CN115844941B

Abstract

The invention provides a composition of eucommia ulmoides extracts and application thereof in preparation of anti-osteoporosis medicines, and belongs to the technical field of traditional Chinese medicine extraction, wherein the composition comprises eucommia ulmoides ethanol extracts, eucommia ulmoides water extracts and eucommia ulmoides powder; the eucommia ethanol extract comprises the following components: water extract of eucommia ulmoides: the mass ratio of the eucommia bark powder is (10-11): (6-7): (9-11). The composition of the eucommia ulmoides extract has a remarkable treatment effect on osteoporosis models caused by bilateral ovariectomy of female mice, and has a good osteoporosis resisting effect on osteoporosis models of aged male rats.

Description

Composition of eucommia ulmoides extract and application of composition in preparation of anti-osteoporosis medicine

Technical Field

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a composition of an eucommia ulmoides extract and application of the composition in preparation of an anti-osteoporosis medicine.

Background

Osteoporosis (OP) is a systemic disease characterized by decreased bone mass and damaged microstructure of bone tissue, resulting in increased bone fragility and fracture. Osteoporosis is defined by the world health organization as a progressive systemic bone disease with low bone mass and deterioration of bone tissue microstructure, which in turn increases bone fragility and susceptibility to fracture. The main pathological features are the damage of the bone microfeatures, while the thinning, widening and decreasing number of the trabecular bone structures are the main manifestations. Osteoporosis and fracture are one of the main causes of disability and death of old patients, and the quality and level of life of residents are sharply reduced. Meanwhile, osteoporosis brings a heavy economic burden to the society and families.

At present, clinical anti-osteoporosis medicines mainly comprise estrogens, calcitonin, bisphosphates, macromolecular monoclonal antibodies and the like, wherein the long-term use of the estrogens can cause diseases such as breast cancer, endometrial hyperplasia and the like; the biphosphate is a first-line anti-osteoporosis drug, can be specifically combined with hydroxyapatite in bone, and can inhibit osteoclast activity, thereby inhibiting bone absorption. Is used for treating osteoporosis, especially osteoporosis characterized by osteopenia and destruction of bone structure resulting in increased bone fragility and fracture rate.

Eucommia ulmoides is a common traditional Chinese medicine, and modern pharmacological research shows that the ingredients in the eucommia ulmoides extract have various pharmacological activities, and the main ingredients are chlorogenic acid and flavonoids. The active ingredient for resisting osteoporosis is mainly lignans, including compounds such as olivine, syringaresinol diglucoside, pinoresinol diglucoside, etc.

Clinical application shows that the effective components have anti-osteoporosis effect and are still difficult to meet the requirement of clinical application.

Chinese patent CN111686147A discloses an eucommia ulmoides extract and application thereof in treating osteoporosis, and the patent discloses an eucommia ulmoides extract which comprises the following components in parts by weight: 20-50 parts of eucommia petroleum ether extract and 10-30 parts of eucommia ethanol extract. The method is simple and easy to implement, but has poor effect of treating osteoporosis.

Chinese patent CN114949079a discloses a pharmaceutical composition for treating osteoporosis, which comprises the following components: eucommia bark, prepared rehmannia root, safflower, glossy privet fruit, tuber fleeceflower root, medlar, bee pollen, largehead atractylodes rhizome, psoralen, hemlock parsley lactone, saikosaponin A, dioscin, oleanolic acid, loganin and verbascoside isoflavone. The pharmaceutical composition can improve the bone density and the bone quality of bone tissues of mice with bilateral ovariectomy caused osteoporosis, but the pharmaceutical composition has complex components, and is difficult to control the mass ratio of eucommia bark, prepared rehmannia root, glossy privet fruit and tuber fleeceflower root to 1.7-1.4.

Therefore, the problem to be solved by the prior art is to provide a pharmaceutical composition for treating osteoporosis, which is simple in operation, good in safety, suitable for long-term administration, controllable in quality and good in effect.

Disclosure of Invention

Eucommia ulmoides (Eucommia ulmoides Oliver) is a plant of genus Eucommia of family Eucommiaceae, is slightly pungent in taste, warm in nature, and enters liver and kidney meridians. The main effects are indicated for deficiency of liver and kidney, soreness and pain of waist and knees, weakness of bones and muscles, dizziness, pregnancy leakage and threatened abortion.

The technical scheme of the invention comprises the following steps:

in one aspect, the invention provides a composition of an eucommia ulmoides extract, which comprises an ethanol eucommia ulmoides extract, a water eucommia ulmoides extract and eucommia ulmoides powder;

the eucommia ethanol extract comprises the following components: water extract of eucommia ulmoides: the mass ratio of Eucommiae cortex powder is (10-11)

(6-7)︰(9-11)；

The preparation method of the composition comprises the following steps:

(1) Extracting Eucommiae cortex with ethanol under reflux, mixing extractive solutions, and recovering solvent under reduced pressure to obtain extract and residue, wherein the extract is Eucommiae cortex ethanol extract;

(2) Extracting the dregs obtained in the step (1) by adding water, mixing the extracting solutions, and recovering the solvent to obtain an extract which is a eucommia ulmoides water extract;

(3) Pulverizing Eucommiae cortex into fine powder to obtain Eucommiae cortex powder;

(4) Mixing the above ethanol extract, water extract and powder of Eucommiae cortex.

Preferably, the ethanol extract of eucommia ulmoides: water extract of eucommia ulmoides: the mass ratio of Eucommiae cortex powder is (10.2-10.8): 6.5-7): 9.5-10.5.

Preferably, the eucommia ulmoides in the step (1) is dried eucommia ulmoides bark.

Further preferably, the volume concentration of the ethanol in the step (1) is 80-90%;

still further preferably, the amount of ethanol used in the step (1) is 80-120L;

preferably, the reflux extraction in the step (1) is performed for 1 to 2 times, and each time is 0.5 to 1.5 hours.

Preferably, the solvent recovery in step (1) is to recover the solvent from the extract under reduced pressure.

Preferably, the color of the extract in the step (1) is dark brown.

Preferably, the water adding amount in the step (2) is 90-110L.

Further preferably, the extraction method in the step (2) is heating reflux extraction.

Preferably, the reflux extraction in the step (2) is performed for 1 to 2 times, and each time is 0.5 to 1.5 hours.

Preferably, the solvent recovery in step (2) is to recover the solvent from the extract under reduced pressure.

Preferably, the color of the extract in the step (2) is brown.

Preferably, in the step (3), the eucommia ulmoides is eucommia ulmoides bark.

Further preferably, the step (3) further comprises removing coarse bark from the bark of eucommia ulmoides and washing the bark of eucommia ulmoides.

Further preferably, the step (3) further comprises a sterilization and drying step.

Still more preferably, the sterilization method in the step (3) is steam sterilization.

Preferably, the mesh number of the fine powder in the step (3) is 80-100 meshes.

In yet another aspect, the present invention provides a pharmaceutical composition.

The medicinal composition comprises the composition of the eucommia ulmoides extract with effective treatment amount or the composition prepared by the preparation method.

The pharmaceutical dosage forms include, but are not limited to, tablets, pills, injections, hard capsules, soft capsules, dripping pills, lozenges, dragees, granules, powders, syrups, emulsions, suspensions, creams, ointments, sprays, suppositories.

The pharmaceutical composition also comprises a medically acceptable carrier.

Preferably, the medically acceptable carrier includes, but is not limited to, disintegrants, fillers, excipients.

Further preferably, the disintegrant includes, but is not limited to, sodium carboxymethyl starch, low substituted hydroxypropyl cellulose, modified starch.

Further preferably, the filler includes, but is not limited to, starch, microcrystalline cellulose, powdered sugar.

Further preferably, the excipients include, but are not limited to, mannitol, lactose, calcium sulfate.

Preferably, the content of the eucommia ulmoides extract in the pharmaceutical composition is 50-100% by weight.

Further preferably, the content of the composition of the eucommia ulmoides extract in the pharmaceutical composition is 90-100% by weight.

Preferably, the pharmaceutical composition can be administered orally in solid dosage forms such as capsules, tablets, troches, lozenges, granules and powders, or in liquid dosage forms such as elixirs, syrups, emulsions, dispersions and suspensions. Other dosage forms which may be used to administer the pharmaceutical composition are ointments, creams, drops, transdermal patches or powders for topical administration. Both tablets and capsules can be prepared as sustained release products to provide sustained release of the drug over a period of hours. Liquid dosage forms for oral administration may contain coloring and flavoring agents to increase patient acceptance. Solutions for parenteral administration preferably contain water-soluble salts of the active ingredient, suitable stabilizers and, if desired, buffer substances. In addition, the parenteral solution may also contain a preservative.

Preferably, the pharmaceutical composition can be administered to a patient in need of treatment by oral route, at a dosage of 600-3000 mg/day per person, which can be adjusted according to the condition, age, etc. of the patient.

In another aspect, the invention provides an application of the pharmaceutical composition in preparing a medicament for treating osteoporosis.

Preferably, the osteoporosis includes, but is not limited to, bilateral ovariectomy-induced osteoporosis, aging-induced osteoporosis.

The beneficial effects of the invention include:

(1) The composition of the eucommia ulmoides extract has an obvious treatment effect on an osteoporosis model caused by bilateral ovariectomy of female mice, and has a good osteoporosis resistance effect on an osteoporosis model of aged male rats.

(2) The composition of the eucommia ulmoides extract has the obvious effect of improving the bone mineral density, and shows that the composition has an obvious effect of resisting osteoporosis. Has good safety and is suitable for long-term administration.

(3) The invention provides a medicine for treating osteoporosis, which has the advantages of definite curative effect, definite components and controllable quality, and can meet the requirement of clinical application.

(4) The composition of the eucommia ulmoides extract can promote the increase of bone mineral density, enhance the number and thickness of trabeculae and reduce the separation degree, and has an obvious effect of treating osteoporosis.

Detailed Description

The instrument comprises the following steps:

Micro-CT, model: skyscan 1174X-Ray Micromograph, bruker, belgium. Detection conditions are as follows: sample scan parameters were as follows: the rat femur was scanned at a voltage of 50kV and a current of 800 μ A at a scan resolution of 12 μm and a field size of 1304X 1024.

A dual energy X-ray bone densitometer model iNSiGHT VET DXA, available from Korea OsteoSys.

Preparation of ethanol extract of eucommia ulmoides in extract 1 group:

taking 10 kg of eucommia bark dry skin, preparing decoction pieces, performing reflux extraction for 1 time by using 100L of ethanol with the volume concentration of 85%, performing extraction for 2.0 hours when boiling starts, combining extracting solutions, and recovering a solvent from the extracting solutions under reduced pressure to obtain a dark brown extract and medicine residues, wherein the dark brown extract is the eucommia bark ethanol extract.

Preparation of ethanol extract of eucommia ulmoides in extract 2 group:

adding 100L pure water into the above residue, heating and reflux-extracting for 2 times, each for 1 hr, mixing extractive solutions, and recovering solvent under reduced pressure to obtain brown extract, which is water extract of Eucommiae cortex.

Preparation of ethanol extract of eucommia ulmoides in extract 3 groups:

taking cortex Eucommiae, removing coarse bark, cleaning, steam sterilizing, drying, and pulverizing into 100 mesh fine powder to obtain cortex Eucommiae powder.

Preparation of ethanol extract composition of eucommia ulmoides in composition group 4:

(1) Taking 10 kg of eucommia bark dry skin, preparing decoction pieces, performing reflux extraction for 1 time by 100L of ethanol with volume concentration of 85%, timing by boiling, extracting for 2.0 hours, combining extracting solutions, and recovering a solvent from the extracting solutions under reduced pressure to obtain a dark brown extract and dregs, wherein the dark brown extract is the eucommia bark ethanol extract.

(2) Adding 100L of pure water into the residue obtained in step (1), heating and reflux-extracting for 2 times, each time for 1 hr, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain brown extract which is water extract of Eucommiae cortex.

(3) Mixing the above ethanol extract and water extract at a mass ratio of 10.5: 6.5.

Composition 5 preparation of eucommia ulmoides extract composition:

(1) Taking 10 kg of dried bark of eucommia ulmoides, preparing decoction pieces, performing reflux extraction for 1 time by using 120L of ethanol with the volume concentration of 80%, performing extraction for 2.5 hours when boiling starts, combining extracting solutions, and recovering a solvent from the extracting solutions under reduced pressure to obtain brown extractum and dregs, wherein the brown extractum is the eucommia ulmoides ethanol extract.

(2) Adding 110L of pure water into the residue obtained in the step (1), heating and refluxing for extraction for 2 times, wherein each time lasts for 1.0 hour, and recovering the solvent under reduced pressure to obtain brown extract which is the water extract of eucommia ulmoides.

(3) Taking cortex Eucommiae, removing coarse bark, cleaning, steam sterilizing, drying, pulverizing into fine powder, and sieving with 100 mesh sieve to obtain cortex Eucommiae powder.

(4) Taking the prepared eucommia ethanol extract, eucommia water extract and eucommia powder according to the mass ratio of 10:6:9, mixing to obtain the product.

Preparation of composition of eucommia ulmoides extract in composition 6, 8 and 9 groups:

(1) Taking 10 kg of eucommia bark dry skin, preparing decoction pieces, performing reflux extraction for 1 time by using 100L of ethanol with the volume concentration of 85%, performing extraction for 2.0 hours when boiling starts, combining extracting solutions, and recovering a solvent from the extracting solutions under reduced pressure to obtain a dark brown extract and medicine residues, wherein the dark brown extract is the eucommia bark ethanol extract.

(2) Adding 100L of pure water into the residue obtained in step (1), heating and reflux-extracting for 2 times, each time for 0.5-1.5 hr, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain brown extract as water extract of Eucommiae cortex.

(3) Removing coarse bark from cortex Eucommiae, cleaning, steam sterilizing, drying, and pulverizing into 80-100 mesh fine powder to obtain cortex Eucommiae powder.

(4) Taking the eucommia ulmoides ethanol extract, the eucommia ulmoides water extract and the eucommia ulmoides powder which are prepared according to the mass ratio of 10.5:6.5:10, and mixing to obtain the final product.

Composition 7 preparation of composition of eucommia ulmoides extract:

(1) Taking 10 kg of dried bark of eucommia ulmoides, preparing decoction pieces, performing reflux extraction for 2 times by using 80L of ethanol with the volume concentration of 90%, wherein each time lasts for 1.5 hours, combining extracting solutions, and recovering a solvent from the extracting solution under reduced pressure to obtain dark brown extractum and medicine residues, wherein the dark brown extractum is eucommia ulmoides ethanol extract.

(2) Adding 90L of pure water into the residue obtained in the step (1), heating and refluxing for extraction for 2 times, wherein each time lasts for 1.5 hours, combining the extracting solutions, and recovering the solvent under reduced pressure to obtain brown extract which is the eucommia water extract.

(3) Taking cortex Eucommiae, removing coarse bark, cleaning, steam sterilizing, drying, and pulverizing into 100 mesh fine powder to obtain cortex Eucommiae powder.

(4) Taking the eucommia ethanol extract, the eucommia water extract and the eucommia powder which are prepared, and mixing the materials according to the mass ratio of 11:7:11, and mixing to obtain the product.

Composition 10 preparation of composition of eucommia ulmoides extract:

the eucommia ulmoides extract composition was prepared according to the technical scheme disclosed in patent CN111686167a, example 8.

Experimental example 1 pharmacodynamic study of bilateral ovariectomy-induced osteoporosis

Experimental animals:

170 female SD rats aged 7-8 weeks and weighing (200 + -20) g.

Construction of a model of osteoporosis due to bilateral ovariectomy:

selecting female SD rats aged 7-8 weeks, randomly selecting 10 rats as a sham operation group, carrying out intraperitoneal injection of 4% chloral hydrate for anesthesia, and carrying out bilateral ovariectomy on 170 rats except for the sham operation group in which only the abdominal opening is not used for ovariectomy. The postoperative intraperitoneal injection is carried out for 80 ten thousand IU/kg of penicillin potassium, and the administration is continuously carried out for 7 days at 1 time/day, so that the postoperative infection is prevented. Bone Mineral Density (BMD) of lumbar vertebrae and femurs of each rat was measured with a small animal dual-energy X-ray bone mineral density volume fraction analyzer (iNSiGHT VET DX) at week 5, 40 rats whose bone mineral density did not significantly decrease were excluded, and the remaining 120 SD rats (10 rats in the sham operation group were not counted) were randomly grouped.

Grouping experimental animals:

sham group: normal diet and survival were given, n =10;

model control group: normal diet and survival, n =10;

positive control group: alendronate, administered once weekly by oral gavage at a dose of 6mg/kg, n =10;

extract 1 group: the eucommia ulmoides ethanol extract for intragastric administration has the administration dosage of 100mg/kg, and n =10;

extract 2 group: the administration dosage of the eucommia ulmoides water extract for intragastric administration is 100mg/kg, and n =10;

extract 3 groups: mixing the eucommia ulmoides decoction piece fine powder (sieved by a 100-mesh sieve) into a feed, wherein the administration dose is 640mg/kg, the administration dose is calculated according to that the rat eats 5g of the feed per 100g of body weight per day (64 mg of the eucommia ulmoides decoction piece fine powder is added into about 5g of complete feed, mixed evenly and compacted), and n =10.

Composition 4 group: mixing the obtained ethanol extract and water extract of Eucommiae cortex at a mass ratio of 10.5:6.5, with administration dosage of 100mg/kg and n =10;

composition 5 group: the water solution of the eucommia ulmoides extract composition for gastric lavage comprises the mass ratio of an eucommia ulmoides ethanol extract, an eucommia ulmoides water extract and eucommia ulmoides powder in the eucommia ulmoides extract is 10:6:9, the administration dose is: 100mg/kg, n =10;

composition 6 group: the water solution of the eucommia ulmoides extract composition for intragastric administration is characterized in that the mass ratio of eucommia ulmoides ethanol extract, eucommia ulmoides water extract and eucommia ulmoides powder in the eucommia ulmoides extract is 10.5:6.5:10, the administration dose is: 100mg/kg, n =10;

composition 7 group: the water solution of the eucommia ulmoides extract composition for intragastric administration is characterized in that the mass ratio of an eucommia ulmoides ethanol extract to an eucommia ulmoides water extract to eucommia ulmoides powder in the eucommia ulmoides extract is 11:7:11, the dose administered was: 100mg/kg, n =10;

composition 8 group: the water solution of the eucommia ulmoides extract composition for intragastric administration is characterized in that the mass ratio of eucommia ulmoides ethanol extract, eucommia ulmoides water extract and eucommia ulmoides powder in the eucommia ulmoides extract is 10.5:6.5:10, the administration dose is: 25mg/kg, n =10;

composition 9 group: the water solution of the eucommia ulmoides extract composition for intragastric administration is characterized in that the mass ratio of an eucommia ulmoides ethanol extract to an eucommia ulmoides water extract to eucommia ulmoides powder in the eucommia ulmoides extract is 10.5:6.5:10, the administration dose is: 50mg/kg, n =10;

composition 10 group: preparing eucommia ulmoides extract according to the technical scheme disclosed in embodiment 8 of patent CN111686167A, and feeding the eucommia ulmoides extract into an aqueous solution of the eucommia ulmoides extract for intragastric administration at the following dosage: 100mg/kg, n =10.

The administration mode comprises the following steps:

the drugs in each group were orally administered for 16 weeks once a day (except for alendronate sodium 1 time per week).

Detection indexes are as follows:

(1) Bone density of rat lumbar vertebrae and femur

The experimental process comprises the following steps:

after the administration period of 16 weeks is finished, measuring the density of the femur and the lumbar vertebra of the rat in an anesthesia state; the rats were sacrificed and the femurs were removed and the trabecular bone structure was determined using Micro-CT. The anti-osteoporosis efficacy of the eucommia ulmoides extract composition is evaluated through the indexes.

The experimental results are as follows:

the results of bone density measurements of lumbar vertebrae and femurs of the rats in each group after 16 weeks of administration are shown in table 1 below.

Table 1 bone density measurement results (X ± SD, n = 10) of lumbar vertebrae and femurs of rats in each group 16 weeks after administration

Note: comparing with model control group<0.05, represents P<0.01; compared with the group of the pseudo-operation, ^## is represented by P<0.01; compared with the group of extract 1, ^▲ represents P<0.05， ^▲▲ Represents P<0.01; in comparison with the extract 2, the extract was,

represents P<0.05，/>

Represents P<0.01; in comparison with the group of composition 3, ^· represents P<0.05， ^·· Represents P<0.01; based on the comparison with combination 4>

Represents P<0.05，/>

Represents P<0.01。

The experimental conclusion is that:

as can be seen from Table 1, after ovariectomy is performed on the rat, the lumbar vertebrae bone density and the femoral bone density of the rat are both significantly reduced, and compared with a sham operation group, the difference has statistical significance (P < 0.01), which indicates that the rat osteoporosis model is successfully established. After 16 weeks of administration, the lumbar vertebrae density and the femoral bones density of the positive control group rats subjected to alendronate sodium intervention are respectively improved to 0.189 +/-0.014 and 0.262 +/-0.018, and are remarkably improved compared with the lumbar vertebrae density and the femoral bones density of the model control group rats, the difference is statistically significant (P <0.05, P < -0.01), and the fact that the administration of alendronate sodium for 16 weeks has a treatment effect on the reduction of the bone density of the rats is shown.

The medicines given to the extract 1 group and the extract 2 group are eucommia ethanol extracts and eucommia water extracts respectively, the bone densities of lumbar vertebrae and thighbones of rats are obviously higher than the corresponding bone densities of a model control group, the fact that the ethanol extracts or the water extracts of the eucommia have certain anti-osteoporosis effects is shown, but compared with middle-dose and high-dose groups of the eucommia extract composition, the anti-osteoporosis activity is weak, particularly the difference of the density of the lumbar vertebrae is statistically significant compared with the high-dose group, the fact that the anti-osteoporosis effect of the eucommia extract composition is stronger than that of the water extracts or the ethanol extracts singly under the same administration dose is shown, and the eucommia extract composition provided by the invention has unexpected anti-osteoporosis curative effect advantages. The test further evaluated that the fine powder of decoction pieces of eucommia ulmoides (group 3 extract) alone had a weaker anti-osteoporosis effect than the other groups administered 16 weeks after administration. The eucommia ulmoides ethanol extract and the eucommia ulmoides water extract are administered in the composition 4 group, the lumbar vertebrae density and the femoral bone density of a rat are obviously higher than those of a model control group, and the fact that the composition of the eucommia ulmoides ethanol extract and the eucommia ulmoides water extract has a certain osteoporosis resisting effect is shown, but the osteoporosis resisting effect is weaker compared with that of the composition of the eucommia ulmoides extract with the same dosage.

The mass ratio of the eucommia ulmoides ethanol extract, the eucommia ulmoides water extract and the eucommia ulmoides powder in the eucommia ulmoides extracts in the composition 5 group, the composition 6 group and the composition 7 group is respectively 10:6: 9. 10.5, 6.5, 10, 11, and 11, compared with a model control group, the rat has a certain degree of improvement in lumbar vertebra and femoral bone density, wherein the curative effect of the composition 6 group is better than that of the composition 5 and 7 groups, and after the low, medium and high doses of the eucommia ulmoides extract composition (composition 8 group, composition 9 group and composition 6 group) are respectively administered for 16 weeks, the rat has a certain degree of improvement in lumbar vertebra and femoral bone density compared with the model control group, wherein the low dose administration group has a curative effect equivalent to that of the positive control drug alendronate sodium, and as the dose is increased, the lumbar vertebra density of the medium and high dose group rats is higher than that of the low dose group, even the lumbar vertebra density of the high dose group is statistically different from that of the low dose group (P < 0.05), and although the femoral bone density of the medium and high dose group rats is not statistically different from that of the low dose group, the difference has biological significance, therefore, the eucommia ulmoides extract composition has dosage dependence on the anti-osteoporosis curative effect.

The technical scheme disclosed in patent CN111686167A example 8 of administration in the composition 10 group is used for preparing the eucommia ulmoides extract, compared with a model control group, the density of lumbar vertebrae and the density of femoral bones in the composition 10 group are not obviously different, and the effect of the composition of the eucommia ulmoides extract prepared by the invention on the anti-osteoporosis effect is better than that of the eucommia ulmoides extract prepared in patent CN111686167A example 8.

(2) Evaluation of the Structure of trabeculae

The experimental results are as follows:

changes in trabecular bone structure of the femurs of rats in each group after 16 weeks of administration are shown in table 2.

Table 2 change in trabecular bone structure of femur in each group of rats after 16 weeks of administration (X ± SD, n = 10)

Group of	BV/TV(％)	Tb.N(1/mm)	Tb.Sp(mm)
				Artificial operation group	37.8±6.8	4.12±0.58	0.25±0.06
Model control group	21.4±3.4 ^##	1.34±0.34 ^##	0.76±0.17 ^##
				Positive control group	28.0±5.9*	2.76±0.36*	0.50±0.12*
Extract 1 group	26.3±7.1	2.93±0.18**	0.59±0.17*
				Extract 2 group	24.6±6.6	2.80±0.32**	0.61±0.13
Extract 3 groups	22.2±6.0	1.73±0.49	0.71±0.19
				Composition 4 groups	24.3±3.8	2.69±0.32*	0.69±0.14
Composition No. 5	31.2±8.8**	3.09±0.44**	0.40±0.08**
				Composition 6 group	31.5±9.2**	3.19±0.59**	0.37±0.10** ^·
Composition 7 groups	30.1±7.4**	2.94±0.52**	0.45±0.10**
				Composition 8 groups	27.2±7.2*	2.47±0.46*	0.58±0.08*
Composition 9 groups	29.4±8.7**	3.02±0.40**	0.42±0.12**
				Composition 10 groups	24.8±6.8	2.81±0.52*	0.73±0.15

Note: p <0.05, as compared to the model group;

compared with the sham-operated group, the operation, ^## represents P < 0.01; compared with the positive control group, the test results show that, ^· is represented by P<0.05；

BV/TV: bone volume fraction, tb.n: trabecular number of bones, tb.sp: trabecular bone separation.

And (4) experimental conclusion:

through the modeling of the operation, the structure of the femoral trabecular bone of the rat is damaged, the number of the femoral trabecular bone is obviously reduced, the separation degree of the trabecular bone is increased, the number of the trabecular bone is obviously reduced, as shown in the table 2, the number of the trabecular bone BV/TV (%) of the model control group is reduced to 21.4 +/-3.4 and is obviously lower than that of the sham operation group, the number of the trabecular bone Tb.N (1/mm) of the model group is reduced to 1.34 +/-0.34 and is obviously lower than that of the sham operation group by 4.12 +/-0.58 (1/mm), and the separation degree of the trabecular bone is obviously increased (0.76 +/-0.17 &0.25 +/-0.06 and P < -0.01), which indicates that the establishment of the osteoporosis model of the rat in the experiment is successful. After 16 weeks of administration, the positive control group rats are administered alendronate sodium, the bone volume fraction BV/TV and the trabecular bone number Tb.N are respectively increased to 28.0 +/-5.9% and 2.76 +/-0.36 (1/mm), and compared with the model control group, the statistical difference (P is less than 0.05) shows that the alendronate sodium can significantly increase the number of the trabecular bone in unit size; the trabecular bone separation degree is an important parameter for measuring the bone microstructure health, the Tb.Sp of the positive control group is reduced to 0.50 +/-0.12 from 0.76 +/-0.17 of the model control group by administering alendronate for 16 weeks, and the difference has statistical significance (P is less than 0.05).

The number of trabeculae per unit length of femur of rats in the extract 1 group and the extract 2 group was significantly increased compared to the model control group, but the therapeutic effect was weaker than that of the latter two groups compared to the medium-dose and high-dose administration groups of the eucommia ulmoides extract composition. The eucommia ulmoides decoction piece fine powder (extract 3 group) is singly used, and after 16 weeks of administration, the volume fraction of the femoral bone and the number of the trabeculae of the rat are not obviously changed. The results show that 16-week administration of the eucommia ulmoides extract composition can obviously improve the trabecular bone volume fraction and the trabecular bone number per unit length of femurs of rats with osteoporosis, reduce the trabecular bone separation degree, and show good dose dependence, wherein the action strength is related to the administration dose. The effect of the dose group in the eucommia ulmoides extract composition is equivalent to that of the positive drug alendronate sodium, while the separation degree of the trabecular bone in the high dose group is lower than that of the positive control group, and the difference has statistical significance (P is less than 0.05). The results show that compared with the marketed positive medicines of alendronate sodium, eucommia water extract, eucommia ethanol extract and eucommia decoction piece powder, the eucommia extract composition has obvious advantages of improving the bone volume fraction and the number of bone trabeculae and reducing the separation degree of the bone trabeculae. Compared with the model control group, the eucommia ulmoides ethanol extract and the eucommia ulmoides water extract are administered in the composition 4 group, the trabecular bone volume fraction and the trabecular bone separation degree of the femur of the osteoporosis rat are not obviously changed, the number of the trabeculae in unit length is obviously increased, but the composition has weaker action advantage compared with the eucommia ulmoides extract composition (composition 6 group) with the same dosage.

After the low, medium and high dose groups (composition 8 group, composition 9 group and composition 6 group) of the eucommia ulmoides extract composition are administrated for 16 weeks, the volume fraction of the femoral bones and the number of the trabeculae of the rats are obviously higher than those of a model control group, especially the medium dose group and the high dose group, and compared with the model control group, P is less than 0.01, and the difference has statistical significance and biological significance; the bone trabecula separation degree of the rats in the high and medium dose groups of the eucommia ulmoides extract composition is obviously lower than that of the rats in the model control group, and the difference has statistical significance. The ratio of the composition 5 to the composition 7 to the composition 6 is changed to a small extent, and the small-scale change does not cause difference of drug effect in the aspect of anti-osteoporosis drug effect. In the composition 10 groups, the application patent CN111686167a in example 8 discloses a technical scheme for preparing an eucommia ulmoides extract, and compared with a model control group, the volume fraction and the separation degree of trabeculae of thighbones of osteoporosis rats in the composition 10 groups have no significant change, the number of trabeculae in unit length is significantly increased, but the effect is poorer than that of an eucommia ulmoides extract composition (composition 6 group) with the same dose. The composition of the eucommia ulmoides extract prepared by the present invention is superior to the eucommia ulmoides extract prepared in example 8 of patent CN111686167a in the effect of the amount of trabeculae per unit length.

As can be seen from the above, the eucommia ulmoides extract composition has a remarkable effect of improving the bone density of castrated female rats, and shows that the composition has a remarkable anti-osteoporosis effect. Increase the number and thickness of trabeculae and reduce the separation degree. Shows that the medicine has obvious effect of treating osteoporosis.

Experimental example 2 drug effect experiment for senile osteoporosis

Experimental animals:

SPF grade male SD rats 160, body weight (200. + -. 20) g.

Establishing an senile osteoporosis model:

male SD rats, 7-8 weeks old, 160 were selected and fed with normal diet for 18 months. A small animal dual-energy X-ray bone mineral density somatic component analyzer (iNSiGHT VET DX) is adopted to measure the Bone Mineral Density (BMD) of the lumbar vertebra and the thighbone of the rat, 30 rats with the bone mineral density not obviously reduced are removed, the remaining 120 SD rats are randomly grouped, 10 rats are fed with common complete feed each group. Another 10 male SD rats, 8 weeks old, were purchased before the end of the experiment and served as normal controls.

The groups of experimental animals, the dose and the frequency of administration were the same as those in Experimental example 1.

Administration to experimental animals:

the medicines in each group are orally taken for 12 months.

(1) Evaluation of bone Density

The experimental process comprises the following steps:

after 12 months of administration, the density of femoral and lumbar vertebrae of rats was measured under anesthesia, and the anti-osteoporosis efficacy of the eucommia ulmoides extract composition was evaluated.

The experimental results are as follows:

the results of bone density measurements of the femoral and lumbar vertebrae of each group of rats after 12 months of administration are shown in table 3 below.

TABLE 3 bone Density measurements of femoral and lumbar vertebrae in various groups of rats after 12 months of administration

Group of	Density of lumbar vertebrae (g/cm) ² )	Femoral bone density (g/cm) ² )
			Normal control group	0.224±0.010	0.288±0.016
Model control group	0.192±0.008 ^##	0.246±0.014 ^##
			Positive control group	0.216±0.015**	0.275±0.012**
Extract 1 group	0.208±0.014*	0.270±0.010*
			Extract 2 groups	0.203±0.017*	0.266±0.011*
Extract 3 groups	0.194±0.017	0.252±0.012
			Composition 4 groups	0.207±0.013*	0.262±0.014*
Composition 5 groups	0.224±0.010**	0.287±0.009**
			Composition 6 group	0.226±0.014**	0.292±0.015**
Composition 7 groups	0.220±0.011**	0.290±0.013**
			Composition 8 groups	0.212±0.012*	0.264±0.011*
Composition 9 groups	0.218±0.010**	0.279±0.017**
			Composition 10 groups	0.195±0.012	0.251±0.010

Note: compared with the control group of the model, ^* is represented by P<0.05， ^** Represents P<0.01；

Compared with the normal control group, ^# represents P<0.05。

The experimental conclusion is that:

as can be seen from Table 3, the lumbar vertebrae density and the femoral bone density of the aged rats are significantly lower than those of the normal control group (P < 0.01), indicating that the osteoporosis model of the aged rats is successfully established. After 12 months of continuous administration, the positive control group rats are subjected to alendronate sodium intervention, the density of lumbar vertebrae and the bone density of thighbone are respectively improved to 0.227 +/-0.014 and 0.287 +/-0.012 which are respectively higher than the corresponding bone density values of the model control group, the difference has statistical significance (P < 0.01), and the alendronate sodium can reverse the reduction of the bone density of the aged rats.

When the ethanol extract (extract 1 group) and the water extract (extract 2 group) of eucommia ulmoides are singly used, after 12 months of administration, the bone density of lumbar vertebrae and thighbone of rats is improved to a certain degree compared with the corresponding bone density value of a model control group, which indicates that the water extract or the ethanol extract of eucommia ulmoides has a certain osteoporosis resisting effect. The bone density of lumbar vertebrae and femur of rats was not significantly changed from the corresponding bone density values of the model control group by administration of only the eucommia ulmoides powder. When the composition of the ethanol extract and the water extract of eucommia ulmoides in the composition 4 group was administered, the lumbar vertebrae density and the femoral bone density were significantly increased as compared with the model control group, but the lumbar vertebrae density values of rats in the composition 4 group were lower than those in the compositions 5 to 9 group as compared with the compositions 5 to 9 group, suggesting that the anti-osteoporosis effect of the composition 4 was inferior to that of the compositions 5 to 9 group.

The composition is prepared from eucommia ulmoides water extract, eucommia ulmoides ethanol extract and eucommia ulmoides decoction piece powder according to a mass ratio of 10.5:6.5:10, the bone densities of lumbar vertebrae and thighbone of rats in low, medium and high dose groups (8 groups of composition, 9 groups of composition and 6 groups of composition) are obviously improved, especially the bone densities of lumbar vertebrae and thighbone of rats in the high dose group are numerically higher than those of corresponding bone densities of a positive control group (although no statistical difference exists, the biological significance is obvious), and the composition has an unexpected effect of resisting senile rat osteoporosis. The composition 5 group and the composition 7 group differed from the composition 6 in that the ratio of the compositions differed to a small extent, but the effect of the difference on the effect against osteoporosis was not significant.

The technical scheme disclosed in embodiment 8 of patent CN111686167A is used for administration in the composition 10 groups to prepare the eucommia ulmoides extract, and compared with a model control group, the density of lumbar vertebrae and the density of femoral bones in the composition 10 groups have no obvious change, which shows that the composition of the eucommia ulmoides extract prepared by the invention has better effect on resisting osteoporosis than the eucommia ulmoides extract prepared in embodiment 8 of patent CN 111686167A.

(2) Evaluation of trabecular bone Structure

The experimental results are as follows:

changes in the trabecular bone structure of the femurs of the rats in each group after 12 months of administration are shown in table 4.

Table 4 change in trabecular bone structure of femur of rats in each group 12 months after administration (X ± SD, n = 10)

Note: compared with the model control group, ^* represents P<0.05， ^** Represents P<0.01；

Compared with the normal control group, ^# represents P<0.05。

And (4) experimental conclusion:

as can be seen from Table 4, with the increase of the age in months, the femoral trabecular structures of aged rats were destroyed, the number was significantly reduced, the trabecular resolution was increased, the number of the trabecular scores was significantly decreased, the number of the trabecular scores BV/TV (%) of the model control group was decreased to 20.6. + -. 2.9, which was significantly lower than that of the orthodox control group, the number of the trabecular scores Tb.N (1/mm) of the model group was decreased to 1.57. + -. 0.23, which was significantly lower than that of the orthodox control group to 3.98. + -. 0.26 (1/mm), and the trabecular resolution was significantly increased (0.82. + -. 0.15 &0.33. + -. 0.08, P < -0.01), indicating that the establishment of the osteoporosis model of aged rats was successful in this experiment. After 12 months of continuous administration, the positive control group rats are administered alendronate sodium, the bone volume number BV/TV and the number Tb.N of trabeculae are respectively increased to 27.3 +/-3.2% and 2.58 +/-0.34 (1/mm), and compared with the model control group, the statistical difference is respectively realized (P <0.05 or P < 0.01), which shows that the alendronate sodium can obviously increase the number of trabeculae in unit size; the bone trabecular resolution is an important parameter for measuring the bone microstructure health, and Tb.Sp in the positive control group is reduced from 0.82 +/-0.15 in the model control group to 0.56 +/-0.11 in the positive control group after the administration of alendronate for 12 months, and the difference is statistically significant (P is less than 0.01).

The number of trabeculae per unit length of femur of rats in the extract 1 group and the extract 2 group was significantly increased compared to the model control group, but the treatment effect was weaker than that of the latter groups compared to the compositions 5, 6, 7, and 9. After the eucommia ulmoides decoction piece fine powder (the extract 3 groups) is singly used and is administered for 12 months, the volume fraction of the femoral bones and the number of the trabeculae of the rats are compared with the corresponding indexes of a model control group, and the difference has no statistical significance. The results show that the eucommia ulmoides extract composition can remarkably improve the trabecular bone volume fraction and the trabecular bone number per unit length of the femoral bone of an osteoporosis rat after being administrated for 12 months, reduce the trabecular bone separation degree, and show good dose dependence, wherein the action strength is related to the administrated dose. The composition 9 (the dosage group in the eucommia ulmoides extract composition) has the equivalent drug effect with the positive drug alendronate sodium, and the separation degree of the trabecular bone of the high-dosage group is lower than that of the positive control group, so that the difference has biological significance. The results show that compared with the marketed positive medicines of alendronate sodium, eucommia water extract, eucommia ethanol extract and eucommia decoction piece powder, the eucommia extract composition provided by the invention has obvious advantages in the effects of improving the bone volume fraction and the number of bone trabeculae and reducing the separation degree of the bone trabeculae.

After the low, medium and high dose groups (composition 8 group, composition 9 group and composition 6 group) of the eucommia ulmoides extract composition are administrated for 12 months, the volume fraction of the femoral bones and the number of trabeculae of a rat are obviously higher than those of a model control group, particularly the medium dose group and the high dose group, and compared with the model control group, P is less than 0.01, and the difference has statistical significance and biological significance; the bone trabecula separation degree of the rats in the high and medium dose groups of the eucommia ulmoides extract composition is obviously lower than that of the rats in the model control group, and the difference has statistical significance.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A composition of eucommia ulmoides extract is characterized by comprising an ethanol extract, a water extract and powder of eucommia ulmoides;

the eucommia ethanol extract comprises the following components: water extract of eucommia ulmoides: the mass ratio of the eucommia bark powder is (10-11): (6-7): (9-11);

the preparation method of the composition comprises the following steps:

2. The composition as claimed in claim 1, wherein the ethanol extract of eucommia ulmoides oliv: water extract of eucommia ulmoides: the mass ratio of the eucommia bark powder is (10.2-10.8): (6.5-7): (9.5-10.5).

3. The composition of claim 1, wherein the volume concentration of ethanol in step (1) of the preparation method is 80-90%.

4. The composition according to claim 1, wherein the number of reflux extractions in step (1) of the preparation method is 1 to 2, each for 1.5 to 2.5 hours; the extraction times in the step (2) are 1-2 times, and each time is 0.5-1.5h.

5. The composition according to any one of claims 1 to 4, wherein the eucommia ulmoides is eucommia ulmoides bark.

6. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of claims 1-5.

7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable carrier;

the medically acceptable carrier includes, but is not limited to, disintegrants, fillers, excipients.

8. The pharmaceutical composition according to any one of claims 6 to 7, wherein the composition is present in the pharmaceutical composition in an amount of 50 to 100% by weight.

9. Use of a pharmaceutical composition according to any one of claims 6 to 8 for the preparation of a medicament for the treatment of osteoporosis.

10. The use of claim 9, wherein the osteoporosis comprises bilateral ovariectomy-induced osteoporosis and aging-induced osteoporosis.