WO2021160104A1

WO2021160104A1 - Folium apocyni veneti extract, and preparation method therefor and use thereof

Info

Publication number: WO2021160104A1
Application number: PCT/CN2021/076120
Authority: WO
Inventors: 周虎; 陈杰波; 陈永炎; 高硕�; 曾志平; 刘聪巍; 王镇武
Original assignee: 厦门大学
Priority date: 2020-02-11
Filing date: 2021-02-09
Publication date: 2021-08-19
Also published as: CN112957380A; CN112957380B

Abstract

Provided is a Folium apocyni veneti extract for resisting osteoporosis. The method for preparing the Folium apocyni veneti extract comprises the following steps: (1) extracting Folium apocyni veneti with an ethanol solution at a concentration of 20% to 95% to obtain an extraction solution; and (2) concentrating the extraction solution in order to obtain a concentrate.

Description

Apocynum venetum leaf extract, its preparation method and application

Technical field

The invention belongs to the field of medicine and relates to apocynum venetum leaf extract, its preparation method and application. Specifically, the present invention relates to the medicinal use of Apocynum venetum leaf 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 skeletal disease in which the strength of bone is damaged, the microstructure of bone tissue is degraded (the cancellous bone trabecula becomes thinner, fractures, and the number decreases) and the risk of fracture increases. Bone strength It is a comprehensive reflection of bone density and bone quality. Osteoporosis can be divided into two categories: primary OP and secondary OP. Both postmenopausal OP and senile OP belong to primary OP. 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. Under normal circumstances, the estrogen-like effect caused by traditional Chinese medicine is far lower than that of estrogen replacement therapy, and the treatment of osteoporosis with traditional Chinese medicine has many years of historical basis, so traditional Chinese medicine plays an increasingly important role in the prevention and treatment of osteoporosis.

Apocynum venetum leaf is the leaf of Apocynum (Latin name: Apocynum.Venetum L.), a plant of Apocynaceae, and is one of the traditional Chinese medicines in my country. Modern pharmacological activity shows that Apocynum venetum leaves mainly protect the liver, antidepressant, lower blood pressure, lower blood sugar, and regulate blood lipids. Traditional Chinese medicine believes that Apocynum venetum leaves have a bitter taste and cool in nature. It has the effect of calming and suppressing liver yang and clearing liver heat. In clinical practice, Apocynum venetum leaves are mainly used to treat diseases such as hypertension, edema, and heart failure.

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 inventors were surprised to find that Apocynum venetum leaf or Apocynum venetum leaf extract (such as the ethanol extract of apocynum venetum leaf) has good prevention and treatment of osteoporosis, especially postmenopausal osteoporosis. The effect of disease. 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 apocynum venetum leaf extract, including the following steps:

(1) Extracting Apocynum venetum leaves with a 20%-95% ethanol solution to obtain an extract;

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

Preferably, it further includes steps (3), (4-1) and (5), steps (3), (4-2) and (5), or steps (3), (4-2), (4- 3) and (5):

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

(4-1) Perform elution with a 25%-95% ethanol solution to obtain an eluate;

(4-2) After eluting with water and 20%-30% ethanol solution (preferably 25% ethanol solution),

Elution is carried out with 35%-60% ethanol solution (preferably 50% ethanol solution) to obtain the eluent,

(4-3) Perform elution with 60%-80% ethanol solution (preferably 75% ethanol solution) to obtain an eluate;

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

The first concentrate or the second concentrate can be used as the Apocynum venetum leaf extract of the present invention.

The eluent in step (5) is the eluent obtained in step (4-1), the eluent obtained in step (4-2), the eluent obtained in step (4-3), or the eluent obtained in step ( 4-2) The eluent obtained by combining the eluent obtained in step (4-3).

In one or more embodiments of the present invention, the preparation method, wherein, in step (1),

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

The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times the amount of Apocynum venetum leaves;

Apocynum venetum leaf is crushed or not crushed apocynum venetum leaf;

The extraction is reflux extraction;

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

and / or

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 (4-1),

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

The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.

In one or more embodiments of the present invention, the preparation method, wherein, in step (4-2),

The amount of the ethanol solution is independently 1-10 times the column volume, preferably 4-5 times the column volume.

In one or more embodiments of the present invention, the preparation method, wherein, in step (4-3),

Another aspect of the present invention is apocynum venetum leaf extract, which is prepared by the preparation method described in any one of the present invention.

In one or more embodiments of the present invention, the apocynum venetum leaf extract is used to prepare a medicine for treating and/or preventing osteoporosis;

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 Apocynum venetum leaf extract, wherein the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.

In one or more embodiments of the present invention, the Apocynum venetum leaf extract, wherein the drug for treating and/or preventing osteoporosis does not cause estrogen-like effects.

In one or more embodiments of the present invention, the apocynum venetum leaf extract is used to treat and/or prevent osteoporosis without causing estrogen-like effects.

Another aspect of the present invention relates to a pharmaceutical composition comprising the Apocynum venetum leaf 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.

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 contains the Apocynum venetum leaf extract of the present invention;

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

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

Another aspect of the present invention relates to the use of apocynum venetum leaf or the ethanol extract of apocynum venetum leaf in the preparation of a medicine for treating and/or preventing osteoporosis;

Preferably, the apocynum venetum leaf ethanol extract is the apocynum venetum leaf extract of the present invention;

Preferably, the osteoporosis is primary osteoporosis or secondary 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 drug for treating and/or preventing osteoporosis does not cause estrogen-like effects.

Another aspect of the present invention relates to a method for treating and/or preventing osteoporosis, including the step of administering an effective amount of the Apocynum venetum leaf extract of the present invention to a subject (such as a human) in need;

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 Apocynum venetum leaf 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 Apocynum venetum leaf 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 of the medicine (for example, as an ointment, patch, or cream), and a dosage form suitable for rectal administration (for example, as a suppository), and the like.

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.

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.

The beneficial effects of the invention

The Apocynum venetum leaf 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;

(6) Compared with estradiol, it can increase the thickness of trabecular bone more effectively;

(7) Effectively inhibit the formation of osteoclasts.

Description of the drawings

Figure 1: The change curve of rat body weight.

Figure 2A-Figure 2H: Femur micro-CT images, where Figure 2A-Figure 2D are the longitudinal section of the distal end of the femur, and Figure 2E-Figure 2H are the transverse section of the distal end of the femur. in:

Figure 2A is a cross-sectional view of the femur in the sham operation group;

Figure 2B is a cross-sectional view of the femur in the model group;

Figure 2C is a cross-sectional view of the femur in the estradiol group;

Figure 2D is a cross-sectional view of a group 1 femur of Apocynum venetum leaf extract;

Figure 2E is a longitudinal section view of the femur in the sham operation group;

Figure 2F is a longitudinal section view of the femur in the model group;

Figure 2G is a longitudinal section view of the femur in the estradiol group;

Fig. 2H is a longitudinal section view of a group 1 femur of Apocynum venetum leaf extract.

Figure 3A-Figure 3F: Paraffin section of the distal end of the femur. in:

Figure 3A is a paraffin section of the femur in the sham operation group;

Figure 3B is a paraffin section of the femur in the OVX group;

Figure 3C is a paraffin section of the femur of the first group of Apocynum venetum leaf extract;

Figure 3D is a 4 times enlarged view of the paraffin section of the femur in the sham operation group;

Figure 3E is a 4 times enlarged view of the paraffin section of the femur in the OVX group;

Fig. 3F is a 4 times enlarged view of the paraffin section of the femur of group 1 of Apocynum venetum leaf extract.

Figure 4A-4F: Paraffin section of the proximal tibia. in:

Figure 4A is a paraffin section of the tibia in the sham operation group;

Figure 4B is a paraffin section of the tibia in the OVX group;

Fig. 4C is a paraffin section of tibia in group 1 of Apocynum venetum leaf extract;

Figure 4D is a 4 times enlarged view of the paraffin section of the tibia in the sham operation group;

Figure 4E is a 4 times enlarged view of the paraffin section of the tibia in the OVX group;

Fig. 4F is a 4 times enlarged view of the paraffin section of tibia in group 1 of Apocynum venetum leaf extract.

Figure 5A-Figure 5H: Osteoclast differentiation map. in:

Figure 5A is a morphological diagram of the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 10 days;

Figure 5B 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. picture;

Figure 5C 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 Apocynum venetum leaves 20% Morphology of ethanol extract (25μg/mL) treated for 7 days;

Figure 5D shows the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 3 days, then treated with M-CSF (50ng/mL), RANKL (50ng/mL) and Apocynum venetum 40% Morphology of ethanol extract (25μg/mL) treated for 7 days;

Figure 5E 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 Apocynum venetum 60% Morphology of ethanol extract (25μg/mL) treated for 7 days;

Figure 5F 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 apocynum venetum 80% Morphology of ethanol extract (25μg/mL) treated for 7 days;

Figure 5G 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 Apocynum venetum 95% Morphology of ethanol extract (25μg/mL) treated for 7 days;

Figure 5H is a statistical analysis diagram of the number of osteoclasts in Figures 5A-5G.

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 Apocynum venetum leaf extract 1

1. Raw materials, reagents and instruments

Apocynum venetum leaf herbal medicine, 60% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.

2. Apocynum venetum leaf extract 1 preparation steps:

(1) Crush 500g of apocynum venetum leaf herbal medicine, use 10 times the amount (weight/volume, unit is mL/g, the same below) 60% (v/v) ethanol solution soaked for 30 minutes, heated to boiling (about (81℃), reflux extraction, extraction 2 times, reflux for 1 hour each time;

(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;

(3) The extract obtained in step (2) was concentrated under reduced pressure at a temperature of 50°C to obtain Apocynum venetum leaf extract 1.

Preparation example 2-7: Preparation of Apocynum venetum leaf extract 2-7

1. Raw materials, reagents and instruments

Apocynum venetum leaf herbal medicine, 60% (v/v) ethanol solution, 25% (v/v) ethanol solution, 50% (v/v) ethanol solution, 75% (v/v) ethanol solution, 95% (v) /v) Ethanol solution, macroporous resin D101 (particle size: 0.3-1.25mm, average pore size:

), a macroporous resin column (column length×inner diameter: 150cm×15cm).

2. The preparation steps of Apocynum venetum leaf extract 2-7:

(1) Use 10-12 times the amount of 60% (v/v) ethanol solution to boil 20 kg of Apocynum venetum leaf Chinese herbal medicine pieces (approximately 81°C), reflux extraction, extract 4 times, 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 40-60° C. to obtain an extract, which is Apocynum venetum leaf extract 2;

(4) Take out about 2.5 kg of Apocynum venetum leaf extract 2 obtained in step (3) and add 10 L of water to obtain an aqueous solution of Apocynum venetum leaf extract 2;

(5) Use macroporous resin D101 for segmented separation. First, put 10kg of macroporous resin D101 into the macroporous resin column, soak the macroporous resin D101 with a 95% (v/v) ethanol solution, and soak for 12 hours. It is fully swelled, and then the macroporous resin column is washed with water to replace the ethanol in it; then the aqueous solution of Apocynum venetum leaf extract 2 obtained in step (4) is repeatedly loaded 6 times, the purpose is to make the macroporous resin D101 fully adsorb as much as possible Apocynum venetum leaf extract 2 in aqueous solution;

(6) Use 100L of water to elute the macroporous resin D101, and the eluate is concentrated under reduced pressure at a temperature of 40-60°C to obtain Apocynum venetum leaf extract 3;

(7) Then use 100L 25% (v/v) ethanol solution to elute the macroporous resin D101, and the eluate is concentrated under reduced pressure at a temperature of 40-60°C to obtain Apocynum venetum leaf extract 4;

(8) Then use 100L 50% (v/v) ethanol solution to elute the macroporous resin D101, and the eluate is concentrated under reduced pressure at a temperature of 40-60°C to obtain Apocynum venetum leaf extract 5;

(9) Then use 100L 75% (v/v) ethanol solution to elute the macroporous resin D101, and the eluate is concentrated under reduced pressure at a temperature of 40-60°C to obtain Apocynum venetum leaf extract 6;

(10) Next, the macroporous resin D101 was eluted with 100 L 95% (v/v) ethanol solution, and the eluate was concentrated under reduced pressure at a temperature of 40-60°C to obtain Apocynum venetum leaf extract 7.

The final weight of Apocynum venetum leaf extract 3 accounts for 14.31% of the sample amount (2.5kg) of Apocynum venetum leaf extract 2. The gavage dose of the sample obtained by further macroporous resin separation is the same as the gavage dose of the corresponding components of the total extract). By the same calculation, the ratio of Apocynum venetum leaf extract 4 is 32.34%, the ratio of Apocynum venetum leaf extract 5 is 17.10%, the ratio of Apocynum venetum leaf extract 6 is 2.45%, and the ratio of Apocynum venetum leaf extract 7 is It is 2.02%.

Preparation Example 8: Preparation of Apocynum venetum Leaf Extract 8

1. Raw materials, reagents and instruments

Apocynum venetum leaf Chinese herbal medicine pieces, 20% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.

2. 8 preparation steps of Apocynum venetum leaf extract:

(1) Crush 500g of Apocynum venetum leaf herbal medicine pieces, soak in 10 times of 20% (v/v) ethanol solution for 30 minutes, heat to boil (approximately 87°C), reflux extraction, extract twice, reflux each time 1 hour;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 50° C. to obtain Apocynum venetum leaf extract 8.

Preparation Example 9: Preparation of Apocynum venetum Leaf Extract 9

1. Raw materials, reagents and instruments

Apocynum venetum leaf herbal medicine, 40% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.

2. 9 preparation steps of Apocynum venetum leaf extract:

(1) Crush 500g of Apocynum venetum leaf Chinese herbal medicine pieces, soak in 10 times the amount of 40% (v/v) ethanol solution for 30 minutes, heat to boil (about 83°C), reflux extraction, extract twice, reflux each time 1 hour;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 50° C. to obtain Apocynum venetum leaf extract 9.

Preparation Example 10: Preparation of Apocynum venetum Leaf Extract 10

1. Raw materials, reagents and instruments

Apocynum venetum leaf herbal medicine, 80% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.

2. 10 preparation steps of Apocynum venetum leaf extract:

(1) Crush 500g of Apocynum venetum leaf Chinese herbal medicine pieces, soak in 10 times the amount of 80% (v/v) ethanol solution for 30 minutes, heat to boil (approximately 80°C), reflux extraction, extract twice, reflux each time 1 hour;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 50° C. to obtain apocynum venetum leaf extract 10.

Preparation Example 11: Preparation of Apocynum venetum Leaf Extract 11

1. Raw materials, reagents and instruments

Apocynum venetum leaf herbal medicine, 95% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.

2. 11 preparation steps of Apocynum venetum leaf extract:

(1) Crush 500g of Apocynum venetum leaf Chinese herbal medicine pieces, soak in 10 times of 95% (v/v) ethanol solution for 30 minutes, heat to boil (about 78°C), reflux extraction, extract twice, reflux each time 1 hour;

(3) The extract obtained in step (2) is concentrated under reduced pressure at a temperature of 50° C. to obtain Apocynum venetum leaf extract 11.

Experimental example 1: Detection of anti-osteoporosis activity of Apocynum venetum leaf extract 1

1. Laboratory animals, reagents and instruments

Apocynum venetum leaf extract 1 prepared in Preparation Example 1.

SD clean rat, female, 3 months old, purchased from the Laboratory Animal Center of Xiamen University;

Chloral hydrate, paraformaldehyde, decalcification solution, hematoxylin and eosin were purchased from Shanghai Shenggong;

β-estradiol (E ₂ ) was purchased from sigma, and the micro-CT was Siemens Inveon PET/CT;

The graphite furnace flame atomic spectrophotometer is Shimadzu of Japan;

The solid density meter is Germany;

(Described above) Analytical balance.

2. Experimental method

2.1 Rat model

The animal model adopts the osteoporosis model of ovariectomized female rats. The principle is that after the female rat’s ovaries are removed, bone metabolism is enhanced, bone resorption and bone formation are enhanced, but the speed of bone resorption is much greater than the speed of bone formation, after a certain period of time The accumulation of bone mass leads to increased bone loss and decreased bone density, among which cancellous bone is the most obvious. It is reported in the literature that the bone density of cancellous bone can be reduced to 60%-70% after establishing the model for one month. Supplement the test sample to inhibit osteoclasts or promote osteoblast activity and other regulation effects of bone metabolism. Observe that the test product is increasing The effect of bone density and bone calcium content, analysis of changes in bone tissue microstructure, and evaluation of the regulatory effect of the test product on osteoporosis.

2.2 Grouping and administration

Rats were injected with 30 mg/kg chloral hydrate solution into the abdominal cavity, and after fixation, the hair was shaved 3-4 cm away from the tailbone from the midline of the back. After disinfection, the skin was incised 2 cm.

Model group: dorsal-abdominal ovariectomy, removal of both ovaries, and ligation of fallopian tubes; sham operation group: only partial fat removal. Sterilization of sutures to ensure aseptic operation.

One month after the operation, they were divided into sham operation group (SHAM), model group (OVX), model-estradiol group (OVX-E ₂ ), model-Apocynum venetum leaf extract 1 group (OVX-Apocynum venetum) Leaf extract 1), 6 rats in each group. The dosage of OVX-E ₂ group was 1mg/kg/d, the dosage of model-Apocynum venetum leaf extract 1 group was 500mg/kg/d, the solvent was 0.5% sodium carboxymethyl cellulose solution, the sham operation group and The model group was given the same volume of 0.5% sodium carboxymethyl cellulose solution, and the rats were sacrificed on the 90th day to detect osteoporosis-related indicators.

2.3 Testing indicators

2.3.1 Archimedes buoyancy method to detect the bone density of the femur: the bones are dried at 105°C to a constant weight for detection and calculation. The difference between the weight of bones in air and water is the buoyancy of bones in water, that is, the weight of bones that drain water. The density of bones can be calculated based on the density of water and air, and the unit is g/mm ³ .

2.3.2 X-ray (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.

2.3.3 Constant bone weight: After drying in an oven at 105°C for 12 hours, accurately weigh the femur every 1 hour until the weight change of the femur is less than 0.0003g every 1 hour, which is the constant bone weight.

2.3.4 Determination of bone calcium content, using atomic absorption method to determine the left side of the ashing femur. Thoroughly ash the femur in a muffle furnace at 550°C, accurately weigh 0.3000g of ashing bone meal, place it in a 150mL digestion tube, add mixed acid (nitric acid: perchloric acid = 4:1), and heat it with an electric heater to digest until it is transparent. Color, dilute with deionized water, make the volume constant, use the calcium ion detection lamp of the furnace flame atomic spectrophotometer to measure the calcium ion concentration in the solution.

2.3.5 Uterine index: Weigh the rat, and after the cervical vertebrae is sacrificed, remove the uterus, peel off the adipose tissue near the uterus, and weigh. Uterine index=wet weight of uterus/body weight.

2.3.6 Femoral histomorphometric parameters: take the right femur, fix it according to routine, decalcify, slice, HE stain, take pictures and record with an upright microscope, multi-functional true color pathological image analysis software to count and analyze the pathology of the femur parameter. The meaning and calculation formula of each index are shown in Table 1:

Table 1: Parameters and calculation formulas of bone tissue microstructure

Bone tissue morphometric parameters and calculation formulas

2.3.7 Histomorphometric parameters of tibia bone: Take the right tibia, 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 for statistics and analysis of femoral diseases Physical parameters. The meaning and calculation formula of each index are shown in Table 1.

3. Experimental results

3.1 Determination of Bone Density by Solid Density Meter: The results are shown in Bone Density 1 in Table 2.

Results: The value of bone density measured by buoyancy method is bone density 1. It can be seen from Table 2 that compared with the model group, the bone density of the model group was significantly lower in the sham operation group (P<0.05), indicating osteoporosis in rats caused by ovariectomy Modeling was successful. Compared with the model group, the estradiol group and the Apocynum venetum leaf extract group 1 can both increase the bone density, indicating that the apocynum venetum leaf extract can effectively prevent the bone density reduction in osteoporotic rats.

3.2 Micro-CT measurement of cancellous bone density: The results are shown in Table 2 Bone Density 2.

Results: The cancellous bone density value of X-ray tomography is bone density 2. It can be seen from Table 2 that compared with the model group, the cancellous bone density of the model group was significantly lower in the sham operation group (P<0.001), indicating that ovariectomy caused The model of osteoporosis in rats was successfully established. Compared with the model group, both the estradiol group and the apocynum venetum leaf extract 1 group can increase the cancellous bone density (P<0.01), indicating that the apocynum venetum leaf extract can effectively prevent the reduction of the cancellous bone density in osteoporotic rats . The specific CT images are shown in Figure 2A-Figure 2H.

In addition, observation of the bone trabeculae in the bone marrow cavity (white mesh in the micro-CT image, Figure 2A-Figure 2H) also showed that compared with the sham operation group (SHAM), the femoral bones of the model group (OVX) rats The structure of the trabecula is incomplete and the number is reduced; compared with the model group, _{the bones of the femur of rats in the estradiol group (OVX-E 2} ) and apocynum venetum leaf extract 1 group (OVX-Apocynum venetum leaf extract 1) are small The structural integrity of the beam is improved, and the number of trabeculae is increased. It shows that Apocynum venetum leaf extract 1 can effectively improve the trabecular bone structure of osteoporotic rats.

3.3 Constant weight of femur: The results are shown in Table 2 for the constant weight of the femur.

Results: The bone constant weight of the estradiol group was statistically different from that of the model group (P<0.05), but the Apocynum venetum leaf extract 1 group was not statistically different, suggesting that the mechanism of action of Apocynum venetum leaf extract 1 and estradiol is different .

3.4 The bone calcium content of the left femur was measured by atomic spectrophotometry, as shown in Table 2.

Results: Compared with the model group, the sham operation group showed a significant decrease in the bone calcium content of the left femur in the model group (P<0.05), indicating that the model of osteoporosis caused by ovariectomy was successful. Compared with the model group, the estradiol group can increase the bone calcium content (P<0.05), the apocynum venetum leaf extract group 1 can increase the bone calcium content (P<0.01), but the apocynum leaf extract 1 can increase the bone calcium content The effect is stronger and has certain advantages.

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

The uterine index can be obtained by dividing the wet weight of the uterus by the weight. Compared with the sham operation group, the bone calcium content of the left femur in the model group was significantly reduced (P<0.001), and the endothelial proliferation of the uterus was significantly reduced, indicating that the model of osteoporosis caused by ovariectomy was successful. Compared with the model group, the estradiol group can increase the uterine index (P<0.001), and estradiol exerts an estrogen-like effect and stimulates the proliferation of the uterine endothelium. There was no statistical difference between the uterine index of Apocynum venetum leaf extract 1 group and the model group, suggesting that Apocynum venetum leaf extract 1 does not exert an estrogen-like effect, does not stimulate endometrial hyperplasia, and reduces endometrial cancer. The risk of breast cancer. In addition, the inventors also found that the Apocynum venetum leaf extract prepared by the present invention, especially Apocynum venetum leaf extract 2-7, will not exert an estrogen-like effect.

Table 2. Apocynum venetum leaf extract 1 to rats

The influence of bone density, bone constant weight, bone calcium content, uterine index

Among them: the model group (OVX) is compared with the sham operation group (SHAM), ^# is P＜0.05, ^## is P＜0.01, ^### is P＜0.001; administration group (OVX-E ₂ and OVX-Robu Compared with the model group (OVX), the hemp leaf extract 1) ^* is P<0.05, ^** is P<0.01, ^*** is P<0.001, N=6.

The results showed that compared with the sham operation group, the model group had lower bone density, lower bone calcium content, and lower uterine index, indicating that the model was successful. Compared with the model group, the estradiol group and the apocynum venetum leaf extract group 1 can significantly increase femoral bone density and increase bone calcium content. However, Apocynum venetum leaf extract 1 has a stronger effect on bone calcium density and has no statistically significant difference in uterine stimulation. Therefore, Apocynum venetum leaf extract 1 has better effects than estradiol in the prevention and treatment of osteoporosis. Certain advantages.

3.6 Rat body weight: The results are shown in the body weight of Figure 1.

Figure 1 shows that the weight of the sham operation group (SHAM) is relatively stable, and the weight of the model group (OVX) gradually increases, indicating that the weight of the rats after ovariectomy; compared with the model group, the estradiol group (OVX-E ₂ ) rats Weight loss indicates that estradiol has the effect of lowering body weight in the OVX model; compared with the model group, Apocynum venetum leaf extract 1 (OVX-Apocynum venetum leaf extract 1) has no significant changes in body weight, indicating that Apocynum venetum leaf extract In the OVX model, substance 1 does not have a significant effect on regulating body weight.

3.7 Effect of Apocynum venetum leaf extract 1 on the microstructure of femur tissue and tibia tissue in rats

The results are shown in Table 3, Figure 3A-Figure 3F and Figure 4A-Figure 4F.

Table 3: Effect of Apocynum venetum leaf extract on the microstructure of rat femur tissue

Figures 3A-3F show that, compared with the sham operation group (SHAM), the femoral trabecular structure of the model group (OVX) rats is incomplete and the number is reduced; compared with the model group, Apocynum venetum leaf extract group 1 (OVX-Apocynum venetum leaf extract 1) The structural integrity of trabecular bone of rat femur is improved, and the number of trabecular bone is increased. It shows that Apocynum venetum leaf extract 1 can effectively improve the trabecular bone structure of osteoporotic rats.

Figures 4A-4F show that compared with the sham operation group (SHAM), the model group (OVX) rat tibia trabecular structure is incomplete and the number is reduced; compared with the model group, Apocynum venetum leaf extract group 1 (OVX-Apocynum venetum leaf extract 1) The structural integrity of the trabecular bone of rat tibia is improved, and the number of trabecular bone is increased. It shows that Apocynum venetum leaf extract 1 can effectively improve the trabecular bone structure of osteoporotic rats.

It can be seen from the above results that compared with the sham operation group, the trabecular bone area, thickness and number of trabecular bone in the model group are significantly reduced, and the separation of trabecular bone is significantly increased, indicating that the model is successful. Compared with the model group, both estradiol and Apocynum venetum leaf extract 1 can effectively increase the area of trabecular bone, the thickness of trabecular bone, the number of trabecular bone, reduce the separation of trabecular bone, and have the effect of preventing and treating osteoporosis. Effect. But Apocynum venetum leaf extract 1 has an advantage compared with estradiol in increasing the thickness of trabecular bone (P<0.01).

Experimental example 2: Apocynum venetum leaf extract 2-7 activity detection

1. Laboratory animals, reagents and instruments

Apocynum venetum leaf extract 2-7 prepared in Preparation Example 2-7.

Chloral hydrate was purchased from Shanghai Shenggong.

2. Experimental method

In the sham operation group, only the fat near the ovary was removed, and the model group was subjected to ovariectomy. One month after the operation, grouping and administration were performed.

Three-month-old female rats were given intragastric administration one month after ovariectomy. Three rats in each group, sham operation group (SHAM), operation model group (OVX), and model administration group (Apocynum venetum leaf extract 2- 7). The dosage of Apocynum venetum leaf extract 2 was 500 mg/kg/d, and the solvent was 0.5% sodium carboxymethyl cellulose solution. The sham operation group and the model group were given the same volume of 0.5% sodium carboxymethyl cellulose solution. Apocynum venetum leaf extract 3-7, according to the proportion of the total extract in Table 4, administered in proportion. The rats were sacrificed to the 90th day after the administration, and the related indexes of osteoporosis were detected, and the cancellous bone density value was detected by micro-CT.

3. Experimental results

The value of cancellous bone density in X-ray tomography is bone density. Table 4 shows that compared with the model group (OVX) in the sham operation group (SHAM), the bone density in the model group was significantly lower (P<0.001), indicating that ovariectomy caused The model of osteoporosis in rats was successfully established. Compared with the model group, the bone mineral density of the 2 groups of OVX-Apocynum venetum leaf extract was significantly increased (P<0.01). There was no significant difference in bone mineral density between the 3 groups of OVX-Apocynum venetum leaf extract. OVX-Apocynum venetum leaf extract The bone mineral density of the 4 groups and the 7 groups of OVX-Apocynum venetum leaf extract were significantly increased (P<0.05), and the bone mineral density of the OVX-Apocynum venetum leaf extract group 5 and OVX-Apocynum venetum leaf extract group 6 were significantly increased (P<0.05). 0.001).

Table 4: Fractional separation and activity detection of crude extract of Apocynum venetum leaf

Aqueous solution of crude extract of Apocynum venetum leaves for segmented separation and activity detection

Table 4: Compared with the sham operation group in the model group, ^### is P＜0.001; compared with the model group in the administration group, ^* is P＜0.05, ^** is P＜0.01, ^*** is P＜0.001, There were 6 rats in the SHAM and OVX groups, and 3 rats in each group.

The results show that Apocynum venetum leaf extract 2 has the effect of enhancing bone density (P<0.01), and its further separation products show that Apocynum venetum leaf extracts 5 and 6 are more effective (P<0.001), and can significantly increase bone density. It shows that the active ingredient in the extract of Apocynum venetum may be 25%-95% ethanol elution part, and the 50% ethanol elution part (extract 5) and 75% ethanol elution part (extract 6) are more effective . Extract 7 is the 95% ethanol elution fraction and should also contain a certain amount of active ingredients. But the active ingredients should be mainly concentrated in the 50% and 75% eluted parts.

In addition, the results also showed that the water-eluted part (Apocynum venetum leaf extract 3) did not have a very good effect. Therefore, the inventors speculated that after the step (5) in Preparation Examples 2-7, if it is directly used Elution with 50%-75% ethanol may contain more non-active ingredients. The method of the present invention represented by Preparation Examples 5-6 can more effectively enrich the active ingredients.

Experimental Example 3: Detection of Anti-osteoporosis Activity of Apocynum venetum Leaf Extract 8-11

1. Laboratory animals, cells, reagents and instruments

As shown in Table 5 below.

Table 5: Some experimental animals, cells, reagents and instruments involved in this experiment

The experimental sample is Apocynum venetum leaf extract 8-11 prepared in Preparation Example 8-11.

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 neck-breaking method, and they were immersed in 70% 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 cells were cultured for 3 days under the treatment of M-CSF (50ng/mL), and they could become mouse monocytes-macrophages, which 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 processing M-CSF and RNAKL, the cells were treated with Apocynum venetum leaf extract 8-11 (25μg/mL) respectively. After the cells were cultured for 7 days, they were observed under a microscope.

2.5 Index detection

Osteoclasts have a larger volume and obvious cytoplasmic structure. A mature osteoclast usually has 3-100 nuclei and is a multinucleated 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 5A-5H, the extracted monocytes (Figure 5A, 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 5B, model group), a large number of multinucleated fusion osteoclasts appeared, and the cell volume was significantly increased, with obvious cytoplasmic structure (shown by the arrow), and could be stained by TRAP The 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, they were treated with Apocynum venetum leaf extract (25μg/mL) extracted with different concentrations of ethanol, and the number of osteoclasts was significantly reduced (Figure 5C-5H). The results show that the 20% ethanol extract, 40% ethanol extract, 60% ethanol extract, 80% ethanol extract and 95% ethanol extract of Apocynum venetum leaf have the effect of inhibiting the formation of osteoclasts and have potential anti-osteogenesis effects. Porous 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

A method for preparing apocynum venetum leaf extract includes the following steps:

(1) Extracting Apocynum venetum leaves with a 20%-95% ethanol solution to obtain an extract;

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

Preferably, it further includes steps (3), (4-1) and (5), steps (3), (4-2) and (5), or steps (3), (4-2), (4- 3) and (5):

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

(4-1) Perform elution with a 25%-95% ethanol solution to obtain an eluate;

(4-2) After eluting with water and 20%-30% ethanol solution (preferably 25% ethanol solution),

Elution is carried out with 35%-60% ethanol solution (preferably 50% ethanol solution) to obtain the eluent,

(4-3) Perform elution with 60%-80% ethanol solution (preferably 75% ethanol solution) to obtain an eluate;

(5) Concentrate the eluate to obtain a second concentrate.
The preparation method according to claim 1, wherein, in step (1),

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

The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times the amount of Apocynum venetum leaves;

Apocynum venetum leaf is crushed or not crushed apocynum venetum leaf;

The extraction is reflux extraction;

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

and / or

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.
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.
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, 1-6 times, 1-3 times or 1-2 Second-rate;

and / or

The dilution factor is 2-10 times or 4-5 times.
The preparation method according to any one of claims 1 to 4, wherein, in step (4-1),

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

The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
The preparation method according to any one of claims 1 to 4, wherein, in step (4-2),

The amount of the ethanol solution is independently 1-10 times the column volume, preferably 4-5 times the column volume.
The preparation method according to any one of claims 1 to 4, wherein, in step (4-3),

The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
An apocynum venetum leaf extract, which is prepared by the preparation method according to any one of claims 1 to 7.
A pharmaceutical composition comprising the Apocynum venetum leaf extract according to claim 8;

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.
A combination drug product, which comprises a first product and a second product that are individually packaged,

in,

The first product comprises the Apocynum venetum leaf extract of claim 8;

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.
Use of apocynum venetum leaf or apocynum venetum leaf ethanol extract in the preparation of a medicine for treating and/or preventing osteoporosis;

Preferably, the ethanol extract of apocynum venetum leaf is the extract of apocynum venetum leaf according to claim 8;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
The use according to claim 11, wherein the treatment and/or prevention of osteoporosis or the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.
The Apocynum venetum leaf extract according to claim 8, which is used to prepare a medicine for treating and/or preventing osteoporosis;

Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;

Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;

Preferably, the drug for treating and/or preventing osteoporosis does not cause estrogen-like effects.
A method for treating and/or preventing osteoporosis, comprising the step of administering an effective amount of the Apocynum venetum leaf extract according to claim 8 to a subject in need;

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.