WO2021190588A1 - Herba lycopi extract and preparation method therefor and use thereof - Google Patents

Abstract

The present invention belongs to the field of medicines, and relates to a herba lycopi extract and a preparation method therefor and use thereof. In particular, the present invention relates to a preparation method for herba lycopi extract. The preparation method comprises the following steps: (1) using 25%-95% ethanol solution to extract herba lycopi to obtain an extracting solution; and (2) concentrating the extracting solution to obtain a first concentrate. The present invention further relates to the herba lycopi extract prepared by means of the preparation method. The herba lycopi extract of the present invention has a good anti-osteoporosis effect.

Description

Eupatorium adenophorum extract, its preparation method and use Technical field

The invention belongs to the field of medicine, and relates to an extract of Eupatorium adenophorum, its preparation method and application. Specifically, the present invention relates to the medical use of Eupatorium adenophorum extract.

Background technique

Osteoporosis (OP) is a systemic bone metabolic disease that is caused by multiple factors, which is characterized by the decrease of bone mass per unit volume and the destruction of bone tissue microstructure, which leads to increased bone fragility and susceptibility to fractures. The U.S. National Institutes of Health defines osteoporosis as a bone disease with damage to bone strength, deterioration of bone tissue microstructure (cancellous bone trabecular thinning, fracture, and number reduction) and increased risk of fractures. Bone strength is A comprehensive reflection of bone density and bone quality. Osteoporosis can be divided into two categories: primary OP and secondary OP. Both postmenopausal OP and senile OP belong to primary OP. Osteoporosis patients generally have no special clinical manifestations, but severe hips and secondary OP often occur. Fractures of the vertebral body or the distal radius. Currently commonly used OP treatment drugs include estrogen, bone resorption inhibitors, bone formation promoters and bone mineral compounds.

For postmenopausal osteoporosis, hormone replacement therapy (hormone replacement therapy, HRT) is the traditional "gold standard" method for preventing and treating osteoporotic fractures in postmenopausal women. HRT can prevent bone loss and reduce the incidence of fractures. In addition, HRT can also improve menopausal symptoms and prevent colon cancer. However, long-term use of HRT can cause estrogen-like effects, which are mainly manifested as an increase in the risk of breast cancer, and the risk of endometrial cancer is increased without the addition of progesterone. Other side effects include fluid retention, breast tenderness, headache, and irregular vaginal bleeding. In animal experiments, the most direct evidence of estrogen-like effects is that after ovariectomy, the secretion of estrogen is greatly reduced, and the uterus of rats should shrink rapidly, and the uterine index will decrease. After estrogen supplementation, the uterine index will greatly increase. These adverse reactions limit the application of HRT. In general, the estrogen-like effect caused by traditional Chinese medicine is far lower than that of estrogen replacement therapy, and the treatment of osteoporosis by traditional Chinese medicine has many years of historical basis, so traditional Chinese medicine is playing an increasingly important role in the prevention and treatment of osteoporosis.

Eupatorium (Lycopi Herba) is the dry above-ground part (sometimes also referred to as dry whole grass) of Lycopus lucidus Turcz.vat.hirtus Regel, which is divided into wetlands and moist side of low-lying valleys in mountains and wilds. In bushes or tall grasses along the banks of streams. Eupatorium adenophorum has the main pharmacological effects of enhancing uterine contraction, anticoagulation, and improving blood stasis. Clinically, it is used for irregular menstruation, amenorrhea, dysmenorrhea, postpartum stasis, abdominal pain, carbuncle, swelling toxin, edema and ascites.

At present, there is still a need to develop new drugs or methods for preventing and treating osteoporosis.

Summary of the invention

After in-depth research and creative work, the inventor surprisingly found that Eupatorium or Eupatorium extract (for example, the ethanol extract of Eupatorium) has a good effect on preventing and treating osteoporosis, especially postmenopausal osteoporosis. . The inventors have further discovered that its use for preventing and treating osteoporosis does not cause estrogen-like effects.

This provides the following inventions:

One aspect of the present invention relates to a method for preparing Eupatorium adenophorum extract, which includes the following steps:

A method for preparing Eupatorium adenophorum extract includes the following steps:

(1) Use 25%-95% ethanol solution to soak and extract Eupatorium to obtain the extract;

(2) Concentrating the extract to obtain the first concentrate;

Preferably, it further includes steps (3), (4) and (5):

(3) The extract of step (1) or the first concentrate of step (2) is diluted with water and centrifuged to obtain the supernatant and precipitate. Take the supernatant and load the macroporous resin column (for example Macroporous resin D101, D101-Ⅰ, DA201, DM301, HPD100 or DM130),

(4) Use water or 5%-60% ethanol solution for elution to obtain an eluate;

(5) Concentrate the eluate to obtain a second concentrate.

Either the first concentrate or the second concentrate can be used as the adenophora extract of the present invention.

In one or more embodiments of the present invention, the preparation method, wherein step (1) satisfies any one, any two, any three, any four, any of the following I.-VII. 5 kinds, any 6 kinds or all 7 kinds:

I. The concentration of the ethanol solution is 45%-75%, 50%-75%, 50%-70%, 55%-70%, 55%-65%, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%; particularly preferably 60%;

II. The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times of Eupatorium;

III. Eupatorium is crushed or not crushed Eupatorium;

IV. The immersion time is at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours or at least 12 hours;

V. The extraction is reflux extraction;

VI. The number of extractions is one or more times, such as 1-4 times; preferably, the extracts obtained from each extraction are combined; and

VII. The time for each extraction is at least 0.2 hours, at least 0.5 hours, at least 1 hour, 1-24 hours, 1-10 hours or 1-5 hours.

Those skilled in the art know that during reflux extraction, the heating temperature is to keep the extraction system or the ethanol solution in a boiling state.

In one or more embodiments of the present invention, the preparation method, wherein, in step (2) and/or step (5),

The concentration is concentration under reduced pressure;

The temperature of concentration is 40°C-60°C, preferably 50°C;

Preferably, the first concentrate and/or the second concentrate is an extract.

In one or more embodiments of the present invention, the preparation method, wherein, in step (3),

Repeat loading at least once, 1-10 times, 1-6 times, 1-3 times or 1-2 times;

and / or

The dilution factor is 2-10 times or 4-5 times.

In one or more embodiments of the present invention, the preparation method, wherein, in step (3), centrifugation is performed at 2°C-30°C and 5°C-25°C. Preferably, the rotation speed of the centrifugation is greater than 1000 revolutions per minute, greater than 2000 revolutions per minute, greater than 3000 revolutions per minute, greater than 5000 revolutions per minute, or 2000-6000 revolutions per minute. Preferably, the centrifugation time is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, 1-30 minutes, 5-20 minutes, or 10-20 minutes.

In one or more embodiments of the present invention, the preparation method, wherein, in step (3), centrifugation is performed at a speed of 5000 revolutions per minute at 25° C. for 5 minutes.

In one or more embodiments of the present invention, the preparation method, wherein, in step (4),

The concentration of the ethanol solution is 5%-45%, 5%-35%, 5%-25% or 5%-15%;

In one or more embodiments of the present invention, the preparation method, wherein, in step (4),

The amount of water or ethanol solution used is greater than or equal to 1 column volume, greater than or equal to 2 column volume, greater than or equal to 3 column volume, 1-10 column volume; preferably 4-5 column volume.

Another aspect of the present invention is an Eupatorium adenophorum extract, which is prepared by the preparation method described in any one of the present invention.

In some embodiments of the present invention, the Eupatorium adenophorum extract is used to treat and/or prevent osteoporosis;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the treatment and/or prevention of osteoporosis does not cause estrogen-like effects;

Preferably, the dosage of Eupatorium adenophorum extract is 50mg-1000mg, 50mg-500mg, 100mg-1000mg, 100mg-500mg, 150mg-400mg, 100mg-300mg, 120mg-300mg, 130mg-300mg, 130mg-per kilogram of body weight per day. 280 mg, 150 mg-300 mg, 150 mg-250 mg, 200 mg or 250 mg; the body weight may be the body weight of a mammal (such as a human).

The inventors surprisingly found that the ethanol extract of Eupatorium adenophorum, for example, the extract of Eupatorium adenophorum prepared by the preparation method of the previous steps (1)-(2), has significant effects on the treatment and/or prevention of osteoporosis. The water extract of Eupatorium adenophorum does not have the effect of preventing and treating osteoporosis. As shown in the results of Experimental Example 3 of the present invention, the water extract of Eupatorium adenophorum (extract 7) has no effect on the bone mineral density of the femur of rats. This shows that the effective ingredients of Eupatorium adenophorum are mainly extracts obtained from ethanol solution.

The inventors have further discovered that the extract obtained after centrifugation and macroporous resin water elution of the Eupatorium adenophorum ethanol extract, for example, includes the Eupatorium adenophorum extract prepared by the preparation method of the previous steps (1)-(5) , Its effect of treating and/or preventing osteoporosis is more significant. As shown in the experimental results of Experimental Example 2, it is even better than the ethanol extract of Adenophora adenophora without centrifugation and macroporous resin water elution. It shows that the main active ingredient of Eupatorium adenophorum for preventing and treating osteoporosis is the extracting ingredient with greater polarity.

Another aspect of the present invention relates to a pharmaceutical composition comprising the adenophora adenophorum extract of the present invention;

Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;

Preferably, the pharmaceutical composition further comprises one or more selected from the following drugs:

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

Preferably, the pharmaceutical composition is used to treat and/or prevent osteoporosis.

In some embodiments of the present invention, the unit dose of the pharmaceutical composition, taking the weight of a mammal (such as a human) of about 70 kg as an example, and calculating according to the weight of the Eupatorium adenophorum extract of the present invention, is 3.5 g-70 g , 3.5g-35g, 7g-70g, 7g-35g, 10.5g-28g, 7g-21g, 8.4g-21g, 9.1g-21g, 9.1g-19.6g, 10.5g-21g, 10.5g-17.5g, 14g or 17.5g.

In some embodiments of the present invention, the pharmaceutical composition comprises an effective amount of Eupatorium adenophorum extract of the present invention.

Another aspect of the present invention relates to a combination drug product, which comprises a first product and a second product that are individually packaged,

in,

The first product comprises the Eupatorium adenophorum extract of the present invention;

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

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

Preferably, the first product and the second product also independently include one or more pharmaceutically acceptable excipients.

In some embodiments of the present invention, the first product contains an effective amount of Eupatorium adenophorum extract of the present invention.

In some embodiments of the present invention, the Eupatorium adenophorum extract of the present invention contained in the first product is 3.5g-70g, 3.5g-35g, 7g-70g, 7g-35g, 10.5g-28g, 7g-21g , 8.4g-21g, 9.1g-21g, 9.1g-19.6g, 10.5g-21g, 10.5g-17.5g, 14g or 17.5g.

Another aspect of the present invention relates to the use of Eupatorium adenophorum or Eupatorium adenophorum ethanol extract in the preparation of a medicament for the treatment and/or prevention of osteoporosis;

Preferably, the Eupatorium adenophorum ethanol extract is the Eupatorium adenophorum extract of the present invention;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.

In one or more embodiments of the present invention, the use, wherein the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.

In one or more embodiments of the present invention, the use, wherein the dosage of adenophora ethanol extract is 50mg-1000mg, 50mg-500mg, 100mg-1000mg, 100mg-500mg, 150mg per kilogram of body weight per day -400mg, 100mg-300mg, 120mg-300mg, 130mg-300mg, 130mg-280mg, 150mg-300mg, 150mg-250mg, 200mg or 250mg; the body weight may be the body weight of a mammal (such as a human).

Another aspect of the present invention relates to a method for treating and/or preventing osteoporosis, comprising the step of administering to a subject in need an effective amount of Eupatorium adenophorum ethanol extract, such as the Eupatorium adenophorum extract of the present invention;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the method of treating and/or preventing osteoporosis does not cause estrogen-like effects.

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

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

Generally, the pharmaceutical composition of the present invention contains 0.1%-90% by weight of the adenophora adenophorum extract of the present invention as the main drug. The pharmaceutical composition can be prepared according to methods known in the art. When used for this purpose, if necessary, the main drug can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare an appropriate administration form or dosage form for human use.

The term "adjuvant" in this application refers to the excipients or vehicles used to administer the main drug, including but not limited to diluents, disintegrants, precipitation inhibitors, surfactants, glidants, viscosities Mixtures, lubricants, coating materials, etc. Excipients are generally described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Examples of excipients include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethyl cellulose, sodium carboxymethyl cellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, Hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxy octadecyl hydroxystearate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, lactose monohydrate, magnesium stearate, mannitol , Microcrystalline cellulose, etc.

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

Depending on the disease to be treated and the patient and the route of administration, the pharmaceutical preparation of the present invention can be administered in different doses once or multiple times a day. The dosage depends on many factors, such as the severity of the osteoporosis to be treated or adjuvanted, the patient's gender, age, weight and individual response, the route of administration, and the number of administrations. The above-mentioned dosage can be administered in a single dosage form or divided into several, for example, two, three, or four dosage forms.

The actual dosage level of each main drug in the pharmaceutical composition of the present invention can be changed, so that the desired therapeutic response can be effectively obtained for the specific patient, composition and administration method. The dosage level must be selected according to the route of administration, the severity of the condition to be treated, and the condition and past medical history of the patient to be treated. However, the practice in the art is to start the dose lower than the level required to obtain the desired therapeutic effect, and gradually increase the dose until the desired effect is obtained.

In some embodiments of the present invention, the dosage of Eupatorium adenophorum extract is 50mg-1000mg, 50mg-500mg, 100mg-1000mg, 100mg-500mg, 150mg-400mg, per kilogram of body weight of mammals (such as humans) per day. 100mg-300mg, 120mg-300mg, 130mg-300mg, 130mg-280mg, 150mg-300mg, 150mg-250mg, 200mg or 250mg.

The term "effective amount" refers to a dose that can treat, prevent, alleviate and/or alleviate the disease or condition described in the present invention in a subject. In some embodiments of the present invention, the subject is a mammal, preferably a human.

The beneficial effects of the invention

The Eupatorium adenophorum extract prepared by the present invention has any one or more of the following technical effects:

(1) Improve bone density of bones such as femur and/or tibia;

(2) Increase bone calcium content of bones such as femur and/or tibia;

(3) Improve the bone tissue microstructure of bones such as femur and/or tibia;

(4) Does not cause estrogen-like effects;

(5) It has obvious effects of preventing and/or treating osteoporosis.

Description of the drawings

Figure 1A-Figure 1H: Femoral micro-CT images, where Figure 1A-Figure 1D are the longitudinal section of the distal end of the femur, and Figure 1E-Figure 1H are the transverse section of the distal end of the femur. in:

Figure 1A is a cross-sectional view of the femur in the sham operation group (SHAM);

Figure 1B is a cross-sectional view of the femur in the model group (OVX);

Figure 1C is a cross-sectional view of the femur in the estradiol group;

Figure 1D is a cross-sectional view of a group 1 femur with Eupatorium adenophorum extract;

Figure 1E is a longitudinal section view of the femur in the sham operation group (SHAM);

Figure 1F is a longitudinal section view of the femur in the model group (OVX);

Figure 1G is a longitudinal section view of the femur in the estradiol group;

Fig. 1H is a longitudinal section view of the femur in group 1 of Eupatorium adenophorum extract.

Figure 2A-2D: Paraffin section of the distal end of the femur. in:

Figure 2A is a paraffin section of the distal femur in the sham operation group (SHAM);

Figure 2B is a paraffin section of the distal femur in the model group (OVX);

Figure 2C is a paraffin section of the distal femur in the estradiol group;

Figure 2D is a paraffin section of the distal femur of group 1 with Eupatorium adenophorum extract.

Figure 3A-Figure 3H: Osteoclast differentiation map. in:

Figure 3A is a morphological diagram of the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 10 days;

Figure 3B shows the morphology of the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL) and RANKL (50ng/mL) for 7 days. Figure; The box in the figure shows osteoclasts with multinucleated fusion;

Figure 3C shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Eupatorium 20% ethanol The morphology of the extract (25μg/mL) after a total of 7 days of treatment; the box in the figure shows the osteoclasts with multinucleated fusion;

Figure 3D shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Eupatorium 40% ethanol The morphology of the extract (25μg/mL) after a total of 7 days of treatment; the box in the figure shows the osteoclasts with multinucleated fusion;

Figure 3E shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Eupatorium 60% ethanol The morphology of the extract (25μg/mL) after a total of 7 days of treatment; the box in the figure shows the osteoclasts with multinucleated fusion;

Figure 3F shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Eupatorium 80% ethanol The morphology of the extract (25μg/mL) after a total of 7 days of treatment; the box in the figure shows the osteoclasts with multinucleated fusion;

Figure 3G shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, and then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Eupatorium 95% ethanol The morphology of the extract (25μg/mL) after a total of 7 days of treatment; the box in the figure shows the osteoclasts with multinucleated fusion;

Figure 3H is a statistical analysis diagram of the number of osteoclasts in Figures 3A-3G.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific conditions are indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Preparation Example 1: Preparation of Eupatorium adenophorum extract 1

1. Raw materials, reagents and instruments

Decoction pieces of Eupatorium adenophorum, 60% (v/v) ethanol solution.

2. Eupatorium extract 1 preparation steps:

(1) Soak 4 kg of Eupatorium adenophorum in a 60% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 1.

Preparation example 2-6: Preparation of Adenophora adenophorum extract 2-6

1. Raw materials, reagents and instruments

Decoction pieces of Eupatorium adenophorum, 60% (v/v) ethanol solution, 25% (v/v) ethanol solution, 50% (v/v) ethanol solution, 75% (v/v) ethanol solution, 95% (v/v) v) Ethanol solution, macroporous resin D101 (particle size: 0.3-1.25mm, average pore size:

), macroporous resin column (column length×inner diameter: 150cm×15cm), high-speed centrifuge (Beckman).

2. 2-6 preparation steps of Eupatorium extract:

(1) Soak 20 kg of Eupatorium adenophorum decoction pieces in 10-12 times the weight of 60% (v/v) ethanol solution for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C. to 60° C. to obtain an extract, which is the total extract of Eupatorium adenophorum (weight about 4.1 kg).

(4) Dilute the total extract of Eupatorium adenophorum (weight: about 4.1kg) obtained in step (3) with 20L of water, centrifuge at 25°C for 5 minutes at 5000 rpm, and take the supernatant to obtain The aqueous supernatant of the total extract of Eupatorium adenophorum, leaving a precipitate;

(5) Use macroporous resin D101 for segmented separation. First, put 10kg of macroporous resin D101 into the macroporous resin column, and soak the macroporous resin D101 with 95% (v/v) ethanol solution for about 12 hours. Make it fully swell, and then rinse the macroporous resin column with water to replace the ethanol in it; then, the aqueous supernatant of the total extract of Eupatorium adenophorum obtained in step (4) is repeatedly loaded 6 times, the purpose is to make the macroporous resin D101 Fully adsorb the aqueous supernatant of the total extract of Eupatorium adenophorum;

(6) Use 100L of water to elute the macroporous resin D101, and concentrate the eluent under reduced pressure at 40°C-60°C to obtain Eupatorium adenophorum extract 2;

(7) Use 10L 25% (v/v) ethanol solution to dissolve the precipitate in step (4), use a high-speed centrifuge at 25°C at 5000 rpm for 5 minutes, take the supernatant, and leave the precipitate. Add the supernatant to the macroporous resin D101 that has been eluted with water in step (6) to continue adsorption, and then use 100L 25% (v/v) ethanol solution to elute the macroporous resin D101. The eluent is in Concentrate under reduced pressure at 40°C-60°C to obtain Eupatorium adenophorum extract 3;

(8) Use 10L 50% (v/v) ethanol solution to dissolve the precipitate in step (7), use a high-speed centrifuge at 25°C at 5000 rpm for 5 minutes, take the supernatant, and leave the precipitate. Add the supernatant to the macroporous resin D101 that has been eluted with 25% (v/v) ethanol solution in step (7) to continue adsorption, and then use 100L 50% (v/v) ethanol solution to treat the macroporous resin D101 Elution was performed, and the eluate was concentrated under reduced pressure at 40°C-60°C to obtain Eupatorium adenophorum extract 4;

(9) Use 10L 75% (v/v) ethanol solution to dissolve the precipitate in step (8), use a high-speed centrifuge at 25°C at 5000 rpm for 5 minutes, take the supernatant, and leave the precipitate. Add the supernatant to the macroporous resin D101 that has been eluted with 50% (v/v) ethanol solution in step (8) to continue adsorption, and then use 100L 75% (v/v) ethanol solution to treat the macroporous resin D101 Elution is performed, and the eluate is concentrated under reduced pressure at 40°C-60°C to obtain Eupatorium adenophorum extract 5;

(10) Use 10L 95% (v/v) ethanol solution to dissolve the precipitate in step (9), use a high-speed centrifuge at 25°C at 5000 rpm for 5 minutes, take the supernatant, and leave the precipitate. Add the supernatant to the macroporous resin D101 that has been eluted with 75% (v/v) ethanol solution in step (9) to continue adsorption, and then use 100L 95% (v/v) ethanol solution to treat the macroporous resin D101 Elution was performed, and the eluate was concentrated under reduced pressure at 40°C-60°C to obtain Eupatorium adenophorum extract 6;

The final ratio of Eupatorium adenophorum extract 2 to the total extract of Eupatorium adenophorum is 27.68%, the ratio of Eupatorium adenophorum extract 3 is 14.76%, the ratio of Eupatorium adenophorum extract 4 is 10.64%, and the ratio of Eupatorium adenophorum extract 5 is 3.49%, the proportion of Eupatorium adenophorum extract 6 is 4.71% (Note: The calculation of this proportion is mainly to determine the dose of animal gavage in the next step, in order to make the sample obtained from further macroporous resin separation and the total extract corresponding to the gavage dose The ingredients are given the same dose for intragastric administration).

The above-mentioned extracts 2-6 are all extracts.

Preparation Example 7: Preparation of Eupatorium adenophorum extract 7

1. Raw materials, reagents and instruments

Decoction pieces of Zelan Chinese medicine, Buchner funnel, filter bottle.

2. 7 preparation steps of Eupatorium extract:

(1) Soak 500g of Chinese herbal medicine pieces of Eupatorium adenophorum in 10-12 times the weight of water for about 12 hours, heat and reflux for extraction, reflux for 2 hours, and extract once;

(2) Combine the products obtained in step (1), filter with gauze, and filter with Buchner funnel to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 7.

Preparation Example 8-9: Preparation of Adenophora adenophorum extract 8-9

1. Raw materials, reagents and instruments

Decoction pieces of Eupatorium adenophorum, 60% (v/v) ethanol solution, 95% (v/v) ethanol solution, macroporous resin D101 (particle size: 0.3-1.25mm, average pore size:

), macroporous resin column (column length×inner diameter: 120cm×9.5cm), high-speed centrifuge (Beckman).

2. 8-9 preparation steps of Eupatorium extract:

(1) Soak 5kg of Eupatorium adenophorum in 60% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at 40°C-60°C to obtain an extract, which is Eupatorium adenophorum extract 8.

(4) Dilute 2/3 of the Eupatorium extract 8 obtained in step (3) with 4L of water, and centrifuge at 25°C for 5 minutes at a speed of 5000 rpm for 5 minutes, and take the supernatant to obtain the Eupatorium extract The supernatant of the aqueous solution of substance 8, leaving a precipitate;

(5) Use macroporous resin D101 for segmented separation. First, put 3kg of macroporous resin D101 into the macroporous resin column, soak the macroporous resin D101 with a 95% (v/v) ethanol solution, and soak for about 12 hours. Make it fully swell, and then rinse the macroporous resin column with water to replace the ethanol in it; then, the aqueous supernatant of Eupatorium adenophorum extract 8 obtained in step (4) is repeatedly loaded 4 times to make the macroporous resin D101 fully adsorb Aqueous supernatant of Eupatorium adenophorum extract 8;

(6) The macroporous resin D101 was eluted with 14L of water, and the eluate was concentrated under reduced pressure at 40°C-60°C to obtain an extract, which is Eupatorium adenophorum extract 9.

The extract 1, the total extract, and the extract 8 prepared above are substantially the same, and they are all extracts obtained after extraction and concentration with a 60% ethanol solution;

The extract 9 is substantially the same as the extract 2, and both are the extract obtained after the macroporous resin is eluted with water and concentrated.

Preparation Example 10: Preparation of Eupatorium adenophorum extract 10

1. Raw materials, reagents and instruments

Eupatorium adenophorum decoction pieces, 20% (v/v) ethanol solution.

2. 10 preparation steps of Eupatorium extract:

(1) Soak 4 kg of Eupatorium adenophorum decoction pieces in 20% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 10.

Preparation Example 11: Preparation of Eupatorium adenophorum extract 11

1. Raw materials, reagents and instruments

Decoction pieces of Eupatorium adenophorum, 40% (v/v) ethanol solution.

2. 11 preparation steps of Eupatorium adenophorum extract:

(1) Soak 4 kg of Eupatorium adenophorum in a 40% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 11.

Preparation Example 12: Preparation of Adenophora adenophorum extract 12

1. Raw materials, reagents and instruments

Eupatorium Chinese medicine decoction pieces, 80% (v/v) ethanol solution.

2. 12 preparation steps of Eupatorium adenophorum extract:

(1) Soak 4 kg of Eupatorium adenophorum in 80% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 12.

Preparation Example 13: Preparation of Eupatorium adenophorum extract 13

1. Raw materials, reagents and instruments

Decoction pieces of Eupatorium adenophorum, 95% (v/v) ethanol solution.

2. 13 preparation steps of Eupatorium adenophorum extract:

(1) Soak 4 kg of Eupatorium adenophorum in a 95% (v/v) ethanol solution of 10-12 times the weight for about 12 hours, heat and reflux for extraction, extract 4 times, and reflux for 1 hour each time;

(2) Combine the products obtained in step (1) and filter to obtain an extract;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 40° C.-60° C. to obtain an extract, which is Eupatorium adenophorum extract 13.

Experimental example 1: Detection of osteoporosis index of Eupatorium adenophorum extract 1

1) Experimental method

1.1 Animals, materials, equipment and drugs

SD clean rat, female, 3 months old, purchased from the Laboratory Animal Center of Xiamen University; chloral hydrate was purchased from Shanghai Shenggong; β-estradiol was purchased from Sigma-Aldlrich; Micro-CT was from Simense Inveon, Germany PET/CT; solid density measuring instrument is German OHAUS; electronic analytical balance is German OHAUS.

Eupatorium adenophorum extract 1 was prepared by Preparation Example 1.

1.2 Ovariectomized rat model of osteoporosis (OVX)

After the female rats have their ovaries removed, the bone metabolism is abnormally enhanced, the loss of cancellous bone is accelerated, and the bone strength decreases significantly. The rats are very similar to humans in the location of bone loss and the occurrence of osteoporotic fractures. One of the classic models for studying osteoporosis in women after menopause. The specific operation method is as follows:

Female SD rats were anesthetized by intraperitoneal injection of 30 mg/kg chloral hydrate solution. The surgical area was cut and disinfected, the dorsal midline was cut longitudinally, and bilateral ovarian extraction was performed. The control group underwent a sham operation, that is, the ovaries were not removed during the operation, and only a small amount of fat was removed. After the ovary or a small amount of fat is removed, the incision is sutured.

1.3 Grouping and administration

One month after the rats were ovary removed, the experimental group was set as the sham operation control group (SHAM), the model group (OVX), and the positive control estradiol group (E ₂ ), with 6 rats in each group. The _{dose of E 2} group was 1 mg/kg/d. Another adjuvant adenophora extract 1 administration group was set up, and the administration dose was 125 mg/kg/d and 500 mg/kg/d two groups, with 3 rats in each group. The dispensing solvent is 0.5% sodium carboxymethyl cellulose solution, the sham operation control group and the model control group are respectively given the same volume of 0.5% sodium carboxymethyl cellulose solution, and the present invention adopts intragastric administration. During the experiment, the rats' food, drinking, and activity conditions were observed, weighed once a week, and the dosage was adjusted according to changes in body weight. By the 90th day, the rats were sacrificed and the osteoporosis-related indexes were detected.

2) Testing indicators

2.1 Micro-CT detection of bone density: take the middle to the lower 1/3 of the left femur (telecentric end), and use X-ray computed tomography to calculate the bone density. The unit is HU.

2.2 Uterine index: The rats were weighed, and after the cervical vertebrae were sacrificed, the uterus was taken out, the adipose tissue near the uterus was stripped, the wet weight was weighed, and the uterine index (uterine wet weight/body weight) was calculated.

2.3 Bone tissue morphometric parameters: Take the right femur, fix it according to routine, decalcify, slice, HE stain, use an upright microscope to take pictures and record, multi-functional true color pathological image analysis software to count and analyze the pathological parameters of the femur. The meaning and calculation formula of each index are shown in the following table:

Table 1: Parameters and calculation formulas of bone tissue microstructure

parameter	symbol	unit	Explanation and formula
Bone tissue area	T.Ar	mm 3	Bone tissue area

Trabecular bone area	Tb.Ar	mm 2	Trabecular bone area
Trabecular bone circumference	Tb.Pm	mm	Trabecular bone circumference length
Trabecular bone thickness	Tb.Th	mm	(2000/1.199)/(Tb.Ar/Tb.Pm)
Trabecular bone area ratio	%Tb.Ar	%	Tb.Ar/T.Ar×％
Number of trabeculae	Tb.N	#/mm	(1.199/2)/(Tb.Pm/T.Ar)
Trabecular bone separation	Tb.Sp	mm	(2000/1.199)x(T.Ar-Tb.Ar)/Tb.Pm

3) result

3.1 The images obtained from Micro-CT scanning of the left femur of the rat are shown in Figure 1A-Figure 1H (the scale bar is 0.5mm in the figure). The measured femoral bone density is listed in Table 2.

Compared with the sham operation control group, the cancellous bone of the model group was significantly reduced, and the bone density was significantly reduced (P<0.001), indicating that the model of osteoporosis caused by ovariectomy was successful. Compared with the model group, the _{number of cancellous bone in the estradiol group (E 2} ) and the adenophorum adenophorum extract 1 administration group were significantly increased, and adenophorum adenophorum extract 1 could increase the bone density of rats in a dose-dependent manner (P<0.05 ), indicating that Eupatorium adenophorum extract 1 can effectively prevent the cancellous bone density of the left femur of osteoporotic rats from decreasing.

3.2 Uterine Index: The results are shown in Table 2 for Uterine Index.

Table 2: Effects of Eupatorium adenophorum extract 1 on bone mineral density and uterine index of rats

Group	Concentration (mg/kg)	Bone density (HU)	Uterine index (g/kg)
SHAM control group	/	1348.3±192.8	2.450±0.598
OVX model group	/	764.7±144.8 ###	0.400±0.068 ###
E 2	1	879.1±90.6 *	1.122±0.074 ***
Eupatorium extract 1	125	839±152.1 *	0.350±0.079
Eupatorium extract 1	500	853.4±76.8 *	0.415±0.078

Note: ^### P＜0.001 is the comparison between the model group and the sham operation control group; ^* P＜0.05 and ^*** P＜0.001 are the comparison between the administration group and the model group.

It can be seen from the data in Table 2 that the uterine index of the model control group decreased significantly (P<0.001), and the endothelial hyperplasia was significantly reduced, indicating that the model of osteoporosis in rats caused by ovariectomy was successful. Compared with the OVX group, the estradiol group (E ₂ ) significantly increased the uterine index (P<0.001), exerted an estrogen-like effect, and stimulated uterine endothelial hyperplasia. However, Eupatorium extract 1 (125mg/kg/d, 500mg/kg/d) has no significant difference in the effect of rat uterus weight, indicating that Eupatorium extract 1 does not have a direct estrogen-like effect. Compared with estradiol, Eupatorium extract 1 is not prone to endometrial hyperplasia, bleeding and other adverse reactions, so it reduces the risk of endometrial cancer and breast cancer.

3.3 Bone tissue morphometric detection: The paraffin sections of the right femur of the rat are shown in Figure 2A-2D (the scale bar in the figure is 200 μm), and the data analysis results are shown in Table 3.

Table 3: Effect of Eupatorium adenophorum extract 1 on the microstructure of rat femur bone tissue

Note: ^### P＜0.001 is the comparison between the model group and the sham operation control group; ^* P＜0.05 and ^*** P＜0.001 are the comparison between the administration group and the model group.

The results showed that the model group was compared with the sham operation control group, the trabecular bone area, trabecular bone thickness and number of the model group were significantly reduced, and the trabecular separation degree was significantly increased, indicating that the model was successful. Compared with the model group, both estradiol (E ₂ ) and Eupatorium adenophorum extract 1 can significantly increase the area of trabecular bone, the thickness of trabecular bone, the number of trabecular bone, and reduce the degree of trabecular bone separation (P<0.001 ), indicating that Eupatorium adenophorum extract 1 can effectively increase bone formation, reduce bone resorption, and prevent bone loss, thereby providing an effective way to prevent or treat osteoporosis.

Experimental Example 2: Effect of Eupatorium adenophorum 1-6 on rats with osteoporosis induced by ovariectomized

1) Test method

1.1 Animals, materials, equipment, modeling

SD clean-grade rat, female, 3 months old, purchased from the Experimental Animal Center of Xiamen University, chloral hydrate purchased from Shanghai Shenggong, Micro-CT is Siemense Inveon PET/CT, Germany, according to experimental example 1 The described method performs ovarian extraction.

Eupatorium adenophorum extracts 1-6 were prepared from Preparation Examples 1-6, respectively.

1.2 Grouping and administration

The rats were divided into sham operation control group (SHAM), model group (OVX), and administration group one month after operation. There are 6 groups in the administration group, which are Adenophora adenophorum extract 1-6, and the corresponding doses are given by gavage according to the extraction ratio in Table 4 (Adenophora adenophorum extract 1 is 500 mg/kg/d, 2 is 276.8mg/kg/d, Eupatorium extract 3 is 147.6mg/kg/d, Eupatorium extract 4 is 106.4mg/kg/d, Eupatorium extract 5 is 34.9mg/kg/d, Eupatorium The extract 6 was 47.1 mg/kg/d). The sham operation group and the model group were given the same volume of 0.5% sodium carboxymethyl cellulose solution. The rats were weighed once a week, and the dosage was adjusted according to changes in body weight. Rats were sacrificed 90 days after gavage, the left femur was taken, and the bone density was calculated using Micro-CT scanning imaging.

2) Results

As shown in Table 4.

Table 4: Effects of Eupatorium adenophorum 1-6 on bone mineral density in rats

Group	Extraction ratio (%)	Bone density (HU)
SHAM control group	/	1391.8±173.8
OVX model group	/	831.1±42.4 ###
Eupatorium extract 1	/	979.0±116.9 **
Eupatorium extract 2	27.68	1159.1±46.3 ***
Eupatorium extract 3	14.76	860.1±39.3
Eupatorium extract 4	10.64	911.1±42.1
Eupatorium extract 5	3.49	903.8±40.7
Eupatorium extract 6	4.71	897.1±87.7

Note: ^### P＜0.001 is the comparison between the model group and the sham operation control group; ^** P＜0.01 and ^*** P＜0.001 are the comparison between the administration group and the model group. There were 6 rats in the sham operation control group and the model group, and 3 rats in each administration group.

Micro-CT detection of bone mineral density: Table 4 shows that compared with the sham operation control group, the bone density of the model group was significantly reduced (P<0.001), indicating that the model of osteoporosis caused by ovariectomy was successful. Compared with the model group, Eupatorium extract 1 and Eupatorium extract 2 can greatly increase the bone density of rats (P<0.001), while the Eupatorium extract 3-6 group has no significant difference.

3) Conclusion

Eupatorium 60% ethanol extract (Euphorax extract 1) and its water-eluted fraction (Euphorax extract 2) can significantly increase the bone density of rats, and the effect of Eupatorium water eluted components is more significant It shows that the main active ingredient of Eupatorium adenophorum extract for preventing and treating osteoporosis is the extract with greater polarity.

Experimental example 3: Effects of Adenophora adenophorum extract 7 and Adenophorum adenophorum extract 8 on ovariectomized rats with osteoporosis

1) Test method

1.1 Animals, materials, equipment, modeling

SD clean-grade rat, female, 3 months old, purchased from the Experimental Animal Center of Xiamen University, chloral hydrate purchased from Shanghai Shenggong, Micro-CT is Siemense Inveon PET/CT, Germany, according to experimental example 1 The described method performs ovarian extraction.

Eupatorium adenophorum extracts 7-8 were prepared by preparation examples 7-8, respectively.

1.2 Grouping and administration

One month after the operation, the rats were divided into sham operation control group (SHAM), model group (OVX), and administration group (500mg/kg/d Eupatorium extract 7 and 500mg/kg/d Eupatorium extract 8) ,6 per group. The sham operation group and the model group were given the same dose of 0.5% sodium carboxymethyl cellulose solution. By 90 days, the rats were sacrificed, and the left femur was taken, and the bone density was calculated using Micro-CT scanning imaging.

2) Results

As shown in Table 5.

Table 5: Effect of Eupatorium extract 7 and Eupatorium extract 8 on bone mineral density in rats

Group	Bone density (HU)
SHAM control group	1501.8±229.8
OVX model group	765.4±28.2 ###
Eupatorium extract 7	762.9±63.1
Eupatorium extract 8	954.6±124.4 **

Note: ^### P＜0.001 is the comparison between the model group and the sham operation control group; ^** P＜0.01 is the comparison between the administration group and the model group.

It can be seen from Table 5 that Adenophora adenophorum extract 8 can significantly increase the bone mineral density of the rat femur, while the adenophorum adenophorum extract 7 has no effect on the bone mineral density of the rat femur. This shows that the anti-osteoporosis active ingredient of Eupatorium adenophorum is mainly the extract obtained from the ethanol solution, and that the ethanol extract of Eupatorium adenophorum rather than the water extract has a significant effect of treating and/or preventing osteoporosis.

Experimental example 4: The effect of different doses of Eupatorium adenophorum 9 on ovariectomized rats with osteoporosis

1) Test method

1.1 Animals, materials, equipment, modeling

SD clean-grade rat, female, 3 months old, purchased from the Experimental Animal Center of Xiamen University, chloral hydrate purchased from Shanghai Shenggong, Micro-CT is Siemense Inveon PET/CT, Germany, according to experimental example 1 The described method performs ovarian extraction.

Eupatorium adenophorum extract 9 was prepared by Preparation Example 9.

1.2 Grouping and administration

One month after the operation, the rats were divided into sham operation control group (SHAM), model group (OVX), and administration group (divided into low-dose group, middle-dose group, and high-dose group according to the dose), with 6 rats in each group. The administration group was administered intragastrically at three doses of 69.2 mg/kg/d, 138.4 mg/kg/d and 276.8 mg/kg/d. The sham operation group and the model group were given the same dose of 0.5% carboxymethyl cellulose. Sodium solution. By 90 days, the rats were sacrificed, and the left femur was taken, and the bone density was calculated using Micro-CT scanning imaging.

2) Results

As shown in Table 6.

Table 6: Effect of Eupatorium adenophorum 9 on bone mineral density of rat femur

Group	Concentration (mg/kg/d)	Bone density (HU)
SHAM control group	/	1501.8±229.8
OVX model group	/	765.4±28.2 ###
Low-dose group	69.2	768.8±47.7
Medium dose group	138.4	839.9±16.6 *
High dose group	276.8	1012.4±90.8 ***

Note: ^### P＜0.001 is the comparison between the model group and the sham operation control group; ^* P＜0.05 and ^*** P＜0.001 are the comparison between the administration group and the model group, N=6.

It can be seen from Table 6 that the medium and high doses of Adenophora adenophorum extract 9 can significantly increase the bone density of rats, indicating that Adenophorum adenophorum extract 9 can effectively increase bone formation and prevent bone loss, thereby providing a preventive or therapeutic bone An effective means of poor quality.

Experimental example 5: Detection of anti-osteoporosis activity of Eupatorium adenophorum extract 10-13 and Eupatorium adenophorum extract 1

1. Laboratory animals, reagents and instruments

As shown in Table 7 below.

Table 7

2. Experimental method

2.1. Construction of osteoclast differentiation model

Excessive activation of osteoclasts is one of the main mechanisms of the pathogenesis of osteoporosis. Most of the drugs currently used clinically are osteoclast inhibitors, so osteoclast inhibitors have the potential to prevent or treat osteoporosis. The effect of disease. In the body, osteoclasts are derived from blood mononuclear-macrophages, and bone marrow contains a large number of monocyte-macrophage precursor cells. The monocyte-macrophage precursor cells isolated in vitro can form monocyte-macrophages under the induction of M-CSF, and then can differentiate into osteoclasts under the co-induction of M-CSF and RANKL. The establishment of this osteoclast differentiation model in vitro can be used for in vitro drug screening and evaluation of osteoporosis.

2.2. Extraction of mouse monocyte-macrophage precursor cells

C57BL/6J mice aged 6-8 weeks were selected, and the mice were sacrificed by the method of neck breaking, and they were immersed in 75% ethanol for 5 minutes for disinfection and sterilization. The femurs of the mice were separated aseptically in a biological safety cabinet. And tibia. Prepare the α-MEM medium containing the 10% FBS and 1% penicillin mixture, fill the sterile syringe with the medium, use the syringe to flush out the whole bone marrow cells from the femur and tibia, and set a pressure of 5×10 ⁴ /cm ² Inoculate it in a 10cm petri dish, add M-CSF (50ng/mL) and culture overnight, and then collect the upper layer of suspended cells.

2.3. Differentiation of osteoclasts

The collected bone marrow cells are cultured for 3 days under the treatment of M-CSF (50ng/mL) and can become mouse monocyte-macrophages. Mouse monocyte macrophages are osteoclast precursor cells with multi-differentiation potential. . The above-mentioned mononuclear-macrophages were ^{planted in a 96-well cell culture plate at a density of 5×10 4} /cm ² , and M-CSF (50ng/mL) and RANKL (50ng/mL) were treated at the same time for 7 days to induce differentiation into Osteoclasts.

2.4. Extract processing

While the cells were processing M-CSF and RNAKL, the ethanol extracts (25μg/mL) of different concentrations of Adenophora adenophora were processed at the same time. After the cells were cultured for 7 days, they were observed under a microscope.

2.4. Indicator detection

Compared with mononuclear-macrophages, osteoclasts have a larger volume and obvious cytoplasmic structure. In addition, a mature osteoclast usually has 3-100 nuclei, which is a multinuclear fusion cell. At the same time, osteoclasts will express a large amount of tartrate-resistant acid phosphatase, which will be stained red by the TRAP staining kit. The cell culture plate was fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then stained with a tartrate-resistant acid phosphatase staining kit for 1 hour at 37 degrees Celsius, and photographed with an inverted microscope to observe and count the multinucleated fusion osteoclasts. Graph Prism 8.0 software was used to analyze the data, and the t test was used to analyze the difference. *** is P<0.001, which represents the significant difference between the model group and the extract treatment group.

3. Experimental results

As shown in Figures 3A-3H, the extracted monocytes (Figure 3A, blank control group) are small in size and cannot be stained red by the TRAP staining kit. In the cells treated with M-CSF and RANKL (Figure 3B, model group), a large number of multinucleated fusion osteoclasts appeared, and the cell volume was significantly increased, with obvious cytoplasmic structure (as shown in the box), and can be The TRAP staining kit is stained red. This indicates that the osteoclast differentiation model was successfully constructed. While the cells were induced to differentiate by M-CSF and RANKL, the number of osteoclasts was significantly reduced by processing Eupatorium esphaeroides (25μg/mL) extracted with different concentrations of ethanol (Figure 3C-3G). As shown in 3H, Eupatorium 20% ethanol extract (Euphoran extract 10), 40% ethanol extract (Euphoran extract 11), 60% ethanol extract (Euphoran extract 1), 80% ethanol extraction (Adenophora adenophorum extract 12) and 95% ethanol extract (Adenophora adenophorum extract 13) can significantly reduce the number of osteoclasts in a 96-well plate (***). Therefore, Eupatorium adenophorum 20% ethanol extract, 40% ethanol extract, 60% ethanol extract, 80% ethanol extract, 95% ethanol extract all have the effect of inhibiting the formation of osteoclasts and have potential anti-osteoporosis effect.

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

Claims (14)

1. A method for preparing Eupatorium adenophorum extract includes the following steps:

   (1) Use 25%-95% ethanol solution to soak and extract Eupatorium to obtain the extract;

   (2) Concentrating the extract to obtain the first concentrate;

   Preferably, it further includes steps (3), (4) and (5):

   (3) Dilute the extract of step (1) or the first concentrate of step (2) with water and centrifuge, take the supernatant, and load the macroporous resin column (such as macroporous resin D101, D101-I, DA201) , DM301, HPD100 or DM130),

   (4) Use water or 5%-60% ethanol solution for elution to obtain an eluate;

   (5) Concentrate the eluate to obtain a second concentrate.
2. The preparation method according to claim 1, wherein step (1) satisfies any one, any two, any three, any four, any five, any six, or all of the following I.-VII. 7 types:

   I. The concentration of the ethanol solution is 45%-75%, 50%-70%, 55%-65% or 60%;

   II. The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times of Eupatorium;

   III. Eupatorium is crushed or not crushed Eupatorium;

   IV. The soaking time is at least 1 hour, at least 5 hours or at least 10 hours;

   V. The extraction is reflux extraction;

   VI. The number of extractions is one or more times, such as 1-4 times; preferably, the extracts obtained from each extraction are combined; and

   VII. The time for each extraction is at least 0.2 hour, at least 0.5 hour or at least 1 hour.
3. The preparation method according to any one of claims 1 to 2, wherein, in step (2) and/or step (5),

   The concentration is concentration under reduced pressure;

   The temperature of concentration is 40°C-60°C, preferably 50°C;

   Preferably, the first concentrate and/or the second concentrate is an extract.
4. The preparation method according to any one of claims 1 to 3, wherein in step (3), the sample loading is repeated at least once, 1-10 times or 1-6 times;

   and / or

   The dilution factor is 2-10 times or 4-5 times.
5. The preparation method according to any one of claims 1 to 4, wherein in step (4), the concentration of the ethanol solution is 5%-45% or 5%-15%;

   The amount of water or ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
6. An extract of Eupatorium adenophorum, which is prepared by the preparation method according to any one of claims 1 to 5.
7. A pharmaceutical composition comprising the Eupatorium adenophorum extract according to claim 6;

   Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;

   Preferably, the pharmaceutical composition further comprises one or more selected from the following drugs:

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

   Preferably, the pharmaceutical composition is used to treat and/or prevent osteoporosis;

   Preferably, the unit dose of the pharmaceutical composition is 3.5g-70g, 3.5g-35g, 10.5g-21g, 10.5g-17.5g, 14g or 17.5 calculated according to the weight of the adenophora adenophorum extract of claim 6. g.
8. A combination drug product, which comprises a first product and a second product that are individually packaged,

   in,

   The first product comprises the Eupatorium adenophorum extract of claim 6;

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

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

   Preferably, the first product and the second product also independently contain one or more pharmaceutically acceptable excipients;

   Preferably, the first product contains the Eupatorium adenophorum extract of claim 6 in the range of 3.5 g to 70 g, 3.5 g to 35 g, 10.5 g to 21 g, 10.5 g to 17.5 g, 14 g or 17.5 g.
9. Use of Eupatorium or Eupatorium ethanol extract in the preparation of medicines for the treatment and/or prevention of osteoporosis;

   Preferably, the Eupatorium adenophorum ethanol extract is the Eupatorium adenophorum extract according to claim 6;

   Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

   Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

   Preferably, the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.
10. The use according to claim 9, wherein the administration dose of Eupatorium adenophorum ethanol extract is 50mg-1000mg, 50mg-500mg, 150mg-300mg, 150mg-250mg, 200mg or 250mg per kilogram of body weight per day.
11. The Eupatorium adenophorum extract according to claim 6, which is used to treat and/or prevent osteoporosis;

    Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

    Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

    Preferably, the treatment and/or prevention of osteoporosis does not cause estrogen-like effects;

    Preferably, the dosage of Eupatorium adenophorum extract is 50mg-1000mg, 50mg-500mg, 150mg-300mg, 150mg-250mg, 200mg or 250mg per kilogram of body weight per day.
12. A method for treating and/or preventing osteoporosis, comprising the step of administering to a subject in need an effective amount of Eupatorium adenophorum ethanol extract, such as the Eupatorium adenophorum extract according to claim 6;

    Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

    Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

    Preferably, the method of treating and/or preventing osteoporosis does not cause estrogen-like effects.
13. The method for treating and/or preventing osteoporosis according to claim 12, wherein:

    The dosage of Eupatorium adenophorum extract is 50mg-1000mg, 50mg-500mg, 150mg-300mg, 150mg-250mg, 200mg or 250mg per kilogram of body weight per day.
14. The method for treating and/or preventing osteoporosis according to any one of claims 12 to 13, wherein the subject is a mammal, preferably a human.

Images