WO2021160104A1 - Folium apocyni veneti extract, and preparation method therefor and use thereof - Google Patents
Folium apocyni veneti extract, and preparation method therefor and use thereof Download PDFInfo
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- WO2021160104A1 WO2021160104A1 PCT/CN2021/076120 CN2021076120W WO2021160104A1 WO 2021160104 A1 WO2021160104 A1 WO 2021160104A1 CN 2021076120 W CN2021076120 W CN 2021076120W WO 2021160104 A1 WO2021160104 A1 WO 2021160104A1
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- osteoporosis
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- ethanol solution
- venetum leaf
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Definitions
- 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.
- Osteoporosis 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.
- OP treatment drugs include estrogen, bone resorption inhibitors, bone formation promoters and bone mineral compounds.
- hormone replacement therapy 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.
- 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.
- 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.
- 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.
- One aspect of the present invention relates to a method for preparing apocynum venetum leaf extract, including the following steps:
- step (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) ,
- macroporous resin column for example, macroporous resin D101, D101-I, DA201, DM301, HPD100 or DM130
- 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).
- 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;
- 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 heating temperature is to keep the extraction system or the ethanol solution in a boiling state.
- step (2) 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;
- the first concentrate and/or the second concentrate is an extract.
- 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;
- the dilution factor is 2-10 times or 4-5 times.
- step (4-1) the preparation method, wherein, in step (4-1),
- the concentration of the ethanol solution is 50%-75%, 55%-70%, 55%-65% or 60%.
- step (4-1) the preparation method, wherein, in step (4-1),
- the amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
- step (4-2) 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.
- step (4-3) the preparation method, 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.
- 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.
- the apocynum venetum leaf extract is used to prepare a medicine for treating and/or preventing osteoporosis;
- the osteoporosis is primary osteoporosis or secondary osteoporosis;
- the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
- the Apocynum venetum leaf extract wherein the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.
- the Apocynum venetum leaf extract wherein the drug for treating and/or preventing osteoporosis does not cause estrogen-like effects.
- 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
- a pharmaceutical composition comprising the Apocynum venetum leaf extract of the present invention
- the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;
- the pharmaceutical composition further comprises one or more selected from the following drugs:
- 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,
- 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:
- 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;
- the apocynum venetum leaf ethanol extract is the apocynum venetum leaf extract of the present invention
- the osteoporosis is primary osteoporosis or secondary osteoporosis;
- the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
- the use, wherein the treatment and/or prevention of osteoporosis does not cause estrogen-like effects.
- 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;
- the osteoporosis is primary osteoporosis or secondary osteoporosis;
- the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;
- the method of treating and/or preventing osteoporosis does not cause estrogen-like effects.
- the ethanol solution involved in the present invention refers to an aqueous solution of ethanol.
- the concentration of the ethanol or the concentration of the ethanol solution is the volume percentage concentration (v/v)%.
- 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.
- 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.
- 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.
- dosage form suitable for oral administration for example, 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
- a dosage form suitable for rectal administration for example, as a suppository
- 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.
- 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.
- 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 (25 ⁇ g/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 (25 ⁇ g/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 (25 ⁇ g/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 (25 ⁇ g/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 (25 ⁇ g/mL) treated for 7 days;
- Figure 5H is a statistical analysis diagram of the number of osteoclasts in Figures 5A-5G.
- Apocynum venetum leaf herbal medicine 60% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
- step (2) The product obtained in step (1) is suction filtered and combined to obtain an extract
- step (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.
- 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).
- step (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;
- 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).
- 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%
- the ratio of Apocynum venetum leaf extract 7 is It is 2.02%.
- Apocynum venetum leaf Chinese herbal medicine pieces 20% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
- step (2) The product obtained in step (1) is suction filtered and combined to obtain an extract
- step (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.
- Apocynum venetum leaf herbal medicine 40% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
- step (2) The product obtained in step (1) is suction filtered and combined to obtain an extract
- step (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.
- Apocynum venetum leaf herbal medicine 80% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
- step (2) The product obtained in step (1) is suction filtered and combined to obtain an extract
- step (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.
- Apocynum venetum leaf herbal medicine 95% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
- step (2) The product obtained in step (1) is suction filtered and combined to obtain an extract
- step (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.
- Chloral hydrate, paraformaldehyde, decalcification solution, hematoxylin and eosin were purchased from Shanghai Shenggong;
- ⁇ -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
- 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.
- 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.
- sham operation group SHAM
- model group OX
- model-estradiol group OVX-E 2
- model-Apocynum venetum leaf extract 1 group OVX-Apocynum venetum Leaf extract 1
- 6 rats 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.
- 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.
- 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.
- 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:
- 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.
- 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.
- the bone calcium content of the left femur was measured by atomic spectrophotometry, as shown in Table 2.
- 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.
- 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.
- the uterine index can be obtained by dividing the wet weight of the uterus by the weight.
- 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.
- 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.
- 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 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.
- the model group had lower bone density, lower bone calcium content, and lower uterine index, indicating that the model was successful.
- the estradiol group and the apocynum venetum leaf extract group 1 can significantly increase femoral bone density and increase bone calcium content.
- 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.
- 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.
- 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.
- SHAM sham operation group
- 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.
- SHAM sham operation group
- 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.
- 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).
- Chloral hydrate was purchased from Shanghai Shenggong.
- Three-month-old female rats were given intragastric administration one month after ovariectomy.
- Three rats in each group 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.
- 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.
- 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 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.
- 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.
- 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.
- the experimental sample is Apocynum venetum leaf extract 8-11 prepared in Preparation Example 8-11.
- 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.
- mice 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.
- tibia Prepare the ⁇ -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.
- M-CSF 50ng/mL
- 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.
- M-CSF 50ng/mL
- RANKL 50ng/mL
- the cells While processing M-CSF and RNAKL, the cells were treated with Apocynum venetum leaf extract 8-11 (25 ⁇ g/mL) respectively. After the cells were cultured for 7 days, they were observed under a microscope.
- Osteoclasts have a larger volume and obvious cytoplasmic structure.
- a mature osteoclast usually has 3-100 nuclei and is a multinucleated fusion cell.
- 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.
- the extracted monocytes ( Figure 5A, blank control group) are small in size and cannot be stained red by the TRAP staining kit.
- 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.
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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
本发明属于医药领域,涉及一种罗布麻叶提取物、其制备方法及用途。具体地,本发明涉及罗布麻叶提取物的医药用途。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.
骨质疏松(osteoporosis,OP)是由多因素引起的一种以单位体积内骨量减少和骨组织微结构破坏为特征,从而导致骨脆性增加和易于骨折的全身性骨代谢性疾病。美国国立卫生研究院定义骨质疏松是一种以骨强度受损,骨组织显微结构退化(松质骨骨小梁变细、断裂、数目减少)和骨折危险性增加的骨骼疾病,骨强度是骨密度和骨质量的综合反映。骨质疏松可分为原发性OP和继发性OP两大类,绝经后OP和老年性OP均属于原发性OP,骨质疏松患者一般无特殊临床表现,但严重时常发生髋部、椎体或桡骨远端等处骨折。目前常用的OP治疗药物包括雌激素、骨吸收抑制剂、骨形成促进剂和骨矿化物等。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.
对于绝经后骨质疏松症来说,激素替代治疗(hormone replacement therapy,HRT)是传统的作为绝经后妇女防治骨质疏松性骨折的“金标准”方法。HRT能够防止骨丢失,并且降低骨折的发生率。此外HRT还能改善绝经症状和预防结肠癌。但是,HRT长期使用时会导致雌激素样作用,主要表现为导致乳腺癌的危险性增加,在不添加孕激素的情况下子宫内膜癌的危险性增加。其它副作用还包括液体潴留、乳房胀痛、头痛及阴道不规则流血。在动物水平实验中,雌激素样作用最直接的证据是卵巢切除后,雌激素分泌大幅度减少,大鼠子宫应迅速萎缩,子宫指数降低,补充雌激素后子宫指数会大幅度提高。这些不良反应限制了HRT的应用。一般情况下中药引起的雌激素样作用远低于雌激素替代疗法,而且中药治疗骨质疏松症具有多年的历史依据,所以中药在预防和治疗骨质疏松中扮演者越来越重要的角色。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.
罗布麻叶是夹竹桃科植物罗布麻(拉丁名:Apocynum.Venetum L.)的叶,是我国传统中药之一。现代药理活性显示,罗布麻叶主要有保肝、抗抑郁、降血压、降血糖、调节血脂等作用。中医认为罗布麻叶味苦、性凉,既有平抑肝阳之功,又有清泻肝热之效,临床上主要应用罗布麻叶治疗高血压、水肿、心力衰竭等疾病。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.
目前,尚需要开发新的防治骨质疏松症的药物或手段。At present, there is still a need to develop new drugs or methods for preventing and treating osteoporosis.
发明内容Summary of the invention
本发明人经过深入的研究和创造性的劳动,惊奇地发现,罗布麻叶或者罗布麻叶提取物(例如罗布麻叶的乙醇提取物)具有良好的防治骨质疏松症特别是绝经后骨质疏松症的效果。本发明人还进一步发现,其用于防治骨质疏松症并不引起雌激素样作用。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.
由此提供了下述发明:This provides the following inventions:
本发明的一个方面涉及一种制备罗布麻叶提取物的方法,包括下述步骤:One aspect of the present invention relates to a method for preparing apocynum venetum leaf extract, including the following steps:
(1)使用20%-95%的乙醇溶液对罗布麻叶进行提取,得到提取液;(1) Extracting Apocynum venetum leaves with a 20%-95% ethanol solution to obtain an extract;
(2)将提取液进行浓缩,得到第一浓缩物;(2) Concentrating the extract to obtain the first concentrate;
优选地,还包括步骤(3)、(4-1)和(5),步骤(3)、(4-2)和(5),或者步骤(3)、(4-2)、(4-3)和(5):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)将步骤(1)的提取液或者将步骤(2)的第一浓缩物用水稀释后上样大孔树脂柱(例如大孔树脂D101、D101-Ⅰ、DA201、DM301、HPD100或DM130),(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)使用25%-95%的乙醇溶液进行洗脱,得到洗脱液;(4-1) Perform elution with a 25%-95% ethanol solution to obtain an eluate;
(4-2)依次用水、20%-30%乙醇溶液(优选25%乙醇溶液)洗脱之后,(4-2) After eluting with water and 20%-30% ethanol solution (preferably 25% ethanol solution),
用35%-60%乙醇溶液(优选50%乙醇溶液)进行洗脱,得到洗脱液,Elution is carried out with 35%-60% ethanol solution (preferably 50% ethanol solution) to obtain the eluent,
(4-3)用60%-80%乙醇溶液(优选75%乙醇溶液)进行洗脱,得到洗脱液;(4-3) Perform elution with 60%-80% ethanol solution (preferably 75% ethanol solution) to obtain an eluate;
(5)将洗脱液进行浓缩,得到第二浓缩物。(5) Concentrate the eluate to obtain a second concentrate.
所述第一浓缩物或者第二浓缩物均可作为本发明的罗布麻叶提取物。The first concentrate or the second concentrate can be used as the Apocynum venetum leaf extract of the present invention.
步骤(5)中的洗脱液为步骤(4-1)得到的洗脱液,步骤(4-2)得到的洗脱液,步骤(4-3)得到的洗脱液,或者将步骤(4-2)得到的洗脱液与步骤(4-3)得到的洗脱液合并后的洗脱液。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).
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(1)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (1),
乙醇溶液的浓度为50%-75%、55%-70%、55%-65%或者60%;The concentration of the ethanol solution is 50%-75%, 55%-70%, 55%-65% or 60%;
乙醇溶液的用量为罗布麻叶的5-20倍量、8-15倍量或者10-12倍量;The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times the amount of Apocynum venetum leaves;
罗布麻叶为粉碎的或未经粉碎的罗布麻叶;Apocynum venetum leaf is crushed or not crushed apocynum venetum leaf;
所述提取为回流提取;The extraction is reflux extraction;
提取次数为1次或多次,例如1-4次;优选地,合并每次提取得到的提取液;The number of extractions is one or more times, such as 1-4 times; preferably, the extracts obtained from each extraction are combined;
和/或and / or
每次提取的时间为至少0.2小时、至少0.5小时、至少1小时、1-24小时、1-10小时或者1-5小时。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.
本领域技术人员知悉,回流提取时,加热的温度为保持提取体系或乙醇溶液为沸腾状态。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.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(2)和/或步骤(5)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (2) and/or step (5),
所述浓缩为减压浓缩;The concentration is concentration under reduced pressure;
浓缩的温度为40℃-60℃,优选50℃;The temperature of concentration is 40°C-60°C, preferably 50°C;
优选地,所述第一浓缩物和/或所述第二浓缩物为浸膏。Preferably, the first concentrate and/or the second concentrate is an extract.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(3)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (3),
重复上样至少1次、1-10次、1-6次、1-3次或者1-2次;Repeat loading at least once, 1-10 times, 1-6 times, 1-3 times or 1-2 times;
和/或and / or
稀释的倍数为2-10倍或者4-5倍。The dilution factor is 2-10 times or 4-5 times.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(4-1)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (4-1),
乙醇溶液的浓度为50%-75%、55%-70%、55%-65%或者60%。The concentration of the ethanol solution is 50%-75%, 55%-70%, 55%-65% or 60%.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(4-1)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (4-1),
乙醇溶液的用量为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(4-2)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (4-2),
乙醇溶液的用量独立地为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is independently 1-10 times the column volume, preferably 4-5 times the column volume.
在本发明的一个或多个实施方式中,所述的制备方法,其中,步骤(4-3)中,In one or more embodiments of the present invention, the preparation method, wherein, in step (4-3),
乙醇溶液的用量为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
本发明的另一方面一种罗布麻叶提取物,其由本发明中任一项所述的制备方法制得。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.
在本发明的一个或多个实施方式中,所述的罗布麻叶提取物,其用于制备治疗和 /或预防骨质疏松症的药物;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;
优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;
优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症。Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
在本发明的一个或多个实施方式中,所述的罗布麻叶提取物,其中,所述治疗和/或预防骨质疏松症不引起雌激素样作用。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.
在本发明的一个或多个实施方式中,所述的罗布麻叶提取物,其中,所述治疗和/或预防骨质疏松症的药物不引起雌激素样作用。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.
在本发明的一个或多个实施方式中,所述的罗布麻叶提取物,其用于治疗和/或预防骨质疏松症且不引起雌激素样作用。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.
本发明的再一方面涉及一种药物组合物,其包含本发明的罗布麻叶提取物;Another aspect of the present invention relates to a pharmaceutical composition comprising the Apocynum venetum leaf extract of the present invention;
可选地,所述药物组合物还包含一种或多种药学上可接受的辅料;Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;
优选地,所述药物组合物还包含选自如下药物中的一种或多种:Preferably, the pharmaceutical composition further comprises one or more selected from the following drugs:
钙剂、维生素D、双磷酸盐、雌激素例如雌二醇、雌激素受体调节剂、降钙素、甲状旁腺激素和骨保护素;Calcium, vitamin D, bisphosphonates, estrogen such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone and osteoprotegerin;
优选地,所述药物组合物用于治疗和/或预防骨质疏松症。Preferably, the pharmaceutical composition is used to treat and/or prevent osteoporosis.
本发明的再一方面涉及一种组合药物产品,其包含独立包装的第一产品和第二产品,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,
其中,in,
所述第一产品包含本发明的罗布麻叶提取物;The first product contains the Apocynum venetum leaf extract of the present invention;
所述第二产品包含选自如下药物中的一种或多种:The second product contains one or more selected from the following drugs:
钙剂、维生素D、双磷酸盐、雌激素例如雌二醇、雌激素受体调节剂、降钙素、甲状旁腺激素和骨保护素;Calcium, vitamin D, bisphosphonates, estrogen such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone and osteoprotegerin;
优选地,所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料。Preferably, the first product and the second product also independently include one or more pharmaceutically acceptable excipients.
本发明的再一方面涉及罗布麻叶或者罗布麻叶乙醇提取物在制备治疗和/或预防骨质疏松症的药物中的用途;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;
优选地,所述罗布麻叶乙醇提取物为本发明的罗布麻叶提取物;Preferably, the apocynum venetum leaf ethanol extract is the apocynum venetum leaf extract of the present invention;
优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;
优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症。Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
在本发明的一个或多个实施方式中,所述的用途,其中,所述治疗和/或预防骨质疏松症不引起雌激素样作用。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.
在本发明的一个或多个实施方式中,所述的用途,其中,所述治疗和/或预防骨质疏松症的药物不引起雌激素样作用。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.
本发明的再一方面涉及一种治疗和/或预防骨质疏松症的方法,包括给予有需求的受试者(例如人)以有效量的本发明的罗布麻叶提取物的步骤;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;
优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;
优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症;Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;
优选地,所述治疗和/或预防骨质疏松症的方法不引起雌激素样作用。Preferably, the method of treating and/or preventing osteoporosis does not cause estrogen-like effects.
本发明中涉及的乙醇溶液,如果没有特别说明,均指乙醇的水溶液。The ethanol solution involved in the present invention, unless otherwise specified, refers to an aqueous solution of ethanol.
本发明中,如果没有特别说明,所述乙醇的浓度或乙醇溶液的浓度为体积百分比浓度(v/v)%。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)%.
通常本发明的药物组合物含有作为主药的0.1重量%-90重量%的本发明的罗布麻叶提取物。药物组合物可根据本领域已知的方法制备。用于此目的时,如果需要,可将主药与一种或多种固体或液体药物赋形剂和/或辅剂结合,制成可作为人用的适当的施用形式或剂量形式。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.
术语“辅料”在本申请中是指用以将主药给药的赋形剂或者媒介物,其包括但不限于稀释剂、崩解剂、沉淀抑制剂、表面活性剂、助流剂、粘合剂、润滑剂、包衣材料等。辅料在E.W.Martin的“Remington's Pharmaceutical Sciences”中被一般性描述。辅料的实例包括但不限于单硬脂酸铝、硬脂酸铝、羧甲基纤维素、羧甲基纤维素钠、交聚维酮、异硬脂酸甘油酯、单硬脂酸甘油酯、羟基乙基纤维素、羟基甲基纤维素、羟基硬脂酸羟基二十八酯、羟基丙基纤维素、羟基丙基甲基纤维素、乳糖、乳糖一水合物、硬脂酸镁、甘露醇、微晶纤维素等。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.
本发明的罗布麻叶提取物可配制成药物制剂,包括适用于口服给药的剂型,适用 于胃肠外注射(例如静脉注射、皮下注射)的剂型(例如作为溶液剂),适用于表面给药的剂型(例如作为软膏剂、贴剂或者乳膏剂),以及适用于直肠给药的剂型(例如作为栓剂)等。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.
取决于待治疗的疾病和患者以及给药途径,本发明的药物制剂可以以不同剂量每日一次或者多次给药。给药剂量取决于许多因素,例如所要治疗或辅助治疗的骨质疏松症的严重程度,患者的性别、年龄、体重及个体反应,给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个,例如二、三、四个剂量形式给药。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.
可改变本发明药物组合物中各主药的实际剂量水平,以便能有效针对具体患者、组合物和给药方式得到所需的治疗反应。剂量水平须根据给药途径、所治疗病况的严重程度以及待治疗患者的病况和既往病史来选定。但是,本领域的做法是,给药剂量从低于为得到所需治疗效果而要求的水平开始,逐渐增加剂量,直到得到所需的效果。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.
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。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.
发明的有益效果The beneficial effects of the invention
本发明制得的罗布麻叶提取物具有如下技术效果中的任意一项或者多项:The Apocynum venetum leaf extract prepared by the present invention has any one or more of the following technical effects:
(1)提高骨骼例如股骨和/或胫骨的骨密度;(1) Improve bone density of bones such as femur and/or tibia;
(2)增加骨骼例如股骨和/或胫骨的骨钙含量;(2) Increase bone calcium content of bones such as femur and/or tibia;
(3)改善骨骼例如股骨和/或胫骨的骨组织微结构;(3) Improve the bone tissue microstructure of bones such as femur and/or tibia;
(4)不引起雌激素样作用;(4) Does not cause estrogen-like effects;
(5)具有明显的预防和/或治疗骨质疏松的效果;(5) It has obvious effects of preventing and/or treating osteoporosis;
(6)相对于雌二醇,能够更有效地提高骨小梁厚度;(6) Compared with estradiol, it can increase the thickness of trabecular bone more effectively;
(7)有效地抑制破骨细胞的形成。(7) Effectively inhibit the formation of osteoclasts.
图1:大鼠体重变化曲线。Figure 1: The change curve of rat body weight.
图2A-图2H:股骨micro-CT图,其中图2A-图2D为股骨远心端的纵切面,图2E-图2H为股骨远心端的横切面。其中: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:
图2A为假手术组股骨的横切面图;Figure 2A is a cross-sectional view of the femur in the sham operation group;
图2B为模型组股骨的横切面图;Figure 2B is a cross-sectional view of the femur in the model group;
图2C为雌二醇组股骨的横切面图;Figure 2C is a cross-sectional view of the femur in the estradiol group;
图2D为罗布麻叶提取物1组股骨的横切面图;Figure 2D is a cross-sectional view of a group 1 femur of Apocynum venetum leaf extract;
图2E为假手术组股骨的纵切面图;Figure 2E is a longitudinal section view of the femur in the sham operation group;
图2F为模型组股骨的纵切面图;Figure 2F is a longitudinal section view of the femur in the model group;
图2G为雌二醇组股骨的纵切面图;Figure 2G is a longitudinal section view of the femur in the estradiol group;
图2H为罗布麻叶提取物1组股骨的纵切面图。Fig. 2H is a longitudinal section view of a group 1 femur of Apocynum venetum leaf extract.
图3A-图3F:股骨远心端石蜡切片图。其中:Figure 3A-Figure 3F: Paraffin section of the distal end of the femur. in:
图3A为假手术组股骨的石蜡切片图;Figure 3A is a paraffin section of the femur in the sham operation group;
图3B为OVX组股骨的石蜡切片图;Figure 3B is a paraffin section of the femur in the OVX group;
图3C为罗布麻叶提取物1组股骨的石蜡切片图;Figure 3C is a paraffin section of the femur of the first group of Apocynum venetum leaf extract;
图3D为假手术组股骨的石蜡切片局部放大4倍图;Figure 3D is a 4 times enlarged view of the paraffin section of the femur in the sham operation group;
图3E为OVX组股骨的石蜡切片局部放大4倍图;Figure 3E is a 4 times enlarged view of the paraffin section of the femur in the OVX group;
图3F为罗布麻叶提取物1组股骨的石蜡切片局部放大4倍图。Fig. 3F is a 4 times enlarged view of the paraffin section of the femur of group 1 of Apocynum venetum leaf extract.
图4A-图4F:胫骨近心端石蜡切片图。其中:Figure 4A-4F: Paraffin section of the proximal tibia. in:
图4A为假手术组胫骨的石蜡切片图;Figure 4A is a paraffin section of the tibia in the sham operation group;
图4B为OVX组胫骨的石蜡切片图;Figure 4B is a paraffin section of the tibia in the OVX group;
图4C为罗布麻叶提取物1组胫骨的石蜡切片图;Fig. 4C is a paraffin section of tibia in group 1 of Apocynum venetum leaf extract;
图4D为假手术组胫骨的石蜡切片局部放大4倍图;Figure 4D is a 4 times enlarged view of the paraffin section of the tibia in the sham operation group;
图4E为OVX组胫骨的石蜡切片局部放大4倍图;Figure 4E is a 4 times enlarged view of the paraffin section of the tibia in the OVX group;
图4F为罗布麻叶提取物1组胫骨的石蜡切片局部放大4倍图。Fig. 4F is a 4 times enlarged view of the paraffin section of tibia in group 1 of Apocynum venetum leaf extract.
图5A-图5H:破骨细胞分化图。其中:Figure 5A-Figure 5H: Osteoclast differentiation map. in:
图5A为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理10天后的形态图;Figure 5A is a morphological diagram of the extracted monocyte-macrophage precursor cells treated with M-CSF (50ng/mL) for 10 days;
图5B为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL)和RANKL(50ng/mL)共处理7天后的形态图;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;
图5C为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL),RANKL(50ng/mL)和罗布麻叶20%乙醇提取物(25μg/mL)共处理7天后的形态图;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;
图5D为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL),RANKL(50ng/mL)和罗布麻叶40%乙醇提取物(25μg/mL)共处理7天后的形态图;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;
图5E为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL),RANKL(50ng/mL)和罗布麻叶60%乙醇提取物(25μg/mL)共处理7天后的形态图;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;
图5F为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL),RANKL(50ng/mL)和罗布麻叶80%乙醇提取物(25μg/mL)共处理7天后的形态图;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;
图5G为提取的单核-巨噬细胞前体细胞经M-CSF(50ng/mL)处理3天,然后经M-CSF(50ng/mL),RANKL(50ng/mL)和罗布麻叶95%乙醇提取物(25μg/mL)共处理7天后的形态图;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;
图5H为图5A-5G中破骨细胞数量的统计分析图。Figure 5H is a statistical analysis diagram of the number of osteoclasts in Figures 5A-5G.
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。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.
制备例1:罗布麻叶提取物1的制备Preparation Example 1: Preparation of Apocynum venetum leaf extract 1
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片,60%(v/v)乙醇溶液,粉碎机,布氏漏斗,抽滤瓶。Apocynum venetum leaf herbal medicine, 60% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
2.罗布麻叶提取物1制备步骤:2. Apocynum venetum leaf extract 1 preparation steps:
(1)将500g罗布麻叶中药饮片粉碎,使用10倍量(重量/体积,单位是mL/g,下同)的60%(v/v)的乙醇溶液浸泡30分钟,加热至沸腾(约为81℃),回流提取,提取2次,每次回流1小时;(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)将步骤(1)得到的产物抽滤,合并,得到提取液;(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;
(3)将步骤(2)得到的提取液在50℃的温度下进行减压浓缩,得到罗布麻叶提 取物1。(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.
制备例2-7:罗布麻叶提取物2-7的制备Preparation example 2-7: Preparation of Apocynum venetum leaf extract 2-7
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片,60%(v/v)乙醇溶液,25%(v/v)乙醇溶液,50%(v/v)乙醇溶液,75%(v/v)乙醇溶液,95%(v/v)乙醇溶液,大孔树脂D101(粒径:0.3-1.25mm,平均孔径:
),大孔树脂柱(柱长×内径:150cm×15cm)。
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.罗布麻叶提取物2-7的制备步骤:2. The preparation steps of Apocynum venetum leaf extract 2-7:
(1)将20kg罗布麻叶中药饮片使用10-12倍量的60%(v/v)的乙醇溶液加热沸腾(约为81℃),回流提取,提取4次,每次回流1小时;(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)将步骤(1)得到的产物合并,过滤,得到提取液;(2) Combine the products obtained in step (1) and filter to obtain an extract;
(3)将步骤(2)得到的提取液在40-60℃的温度下进行减压浓缩,得到浸膏,为罗布麻叶提取物2;(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)将步骤(3)得到的罗布麻叶提取物2取出约2.5kg加入10L水,得到罗布麻叶提取物2的水溶液;(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)使用大孔树脂D101进行分段分离,首先将10kg的大孔树脂D101装入大孔树脂柱中,使用95%(v/v)乙醇溶液浸泡大孔树脂D101,浸泡12小时,使其充分溶胀,接着用水冲洗大孔树脂柱,替换其中的乙醇;之后将步骤(4)中得到的罗布麻叶提取物2的水溶液反复上样6次,目的是使大孔树脂D101尽量充分吸附罗布麻叶提取物2的水溶液;(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)使用100L水对大孔树脂D101进行洗脱,洗脱液在40-60℃的温度下进行减压浓缩,得到罗布麻叶提取物3;(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)接着使用100L 25%(v/v)的乙醇溶液对大孔树脂D101进行洗脱,洗脱液在40-60℃的温度下进行减压浓缩,得到罗布麻叶提取物4;(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)接着使用100L 50%(v/v)的乙醇溶液对大孔树脂D101进行洗脱,洗脱液在40-60℃的温度下进行减压浓缩,得到罗布麻叶提取物5;(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)接着使用100L 75%(v/v)的乙醇溶液对大孔树脂D101进行洗脱,洗脱液在40-60℃的温度下进行减压浓缩,得到罗布麻叶提取物6;(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)接着使用100L 95%(v/v)的乙醇溶液对大孔树脂D101进行洗脱,洗脱 液在40-60℃的温度下进行减压浓缩,得到罗布麻叶提取物7。(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.
最终得到的罗布麻叶提取物3的重量占罗布麻叶提取物2上样量(2.5kg)的比例为14.31%(说明:计算这个比例主要是为了确定下一步动物灌胃的剂量,为了让进一步大孔树脂分离得到的样品的灌胃剂量与总提取物相应成分灌胃的剂量相同)。同理计算,得到的罗布麻叶提取物4的比例为32.34%、罗布麻叶提取物5的比例为17.10%、罗布麻叶提取物6的比例为2.45%、罗布麻叶提取物7的比例为2.02%。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%.
制备例8:罗布麻叶提取物8的制备Preparation Example 8: Preparation of Apocynum venetum Leaf Extract 8
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片、20%(v/v)乙醇溶液、粉碎机、布氏漏斗、抽滤瓶。Apocynum venetum leaf Chinese herbal medicine pieces, 20% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
2.罗布麻叶提取物8制备步骤:2. 8 preparation steps of Apocynum venetum leaf extract:
(1)将500g罗布麻叶中药饮片粉碎,使用10倍量的20%(v/v)的乙醇溶液浸泡30分钟,加热沸腾(约为87℃),回流提取,提取2次,每次回流1小时;(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;
(2)将步骤(1)得到的产物抽滤,合并,得到提取液;(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;
(3)将步骤(2)得到的提取液在50℃的温度下进行减压浓缩,得到罗布麻叶提取物8。(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.
制备例9:罗布麻叶提取物9的制备Preparation Example 9: Preparation of Apocynum venetum Leaf Extract 9
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片、40%(v/v)乙醇溶液、粉碎机、布氏漏斗、抽滤瓶。Apocynum venetum leaf herbal medicine, 40% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
2.罗布麻叶提取物9制备步骤:2. 9 preparation steps of Apocynum venetum leaf extract:
(1)将500g罗布麻叶中药饮片粉碎,使用10倍量的40%(v/v)的乙醇溶液浸泡30分钟,加热沸腾(约为83℃),回流提取,提取2次,每次回流1小时;(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;
(2)将步骤(1)得到的产物抽滤,合并,得到提取液;(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;
(3)将步骤(2)得到的提取液在50℃的温度下进行减压浓缩,得到罗布麻叶提取物9。(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.
制备例10:罗布麻叶提取物10的制备Preparation Example 10: Preparation of Apocynum venetum Leaf Extract 10
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片、80%(v/v)乙醇溶液、粉碎机、布氏漏斗、抽滤瓶。Apocynum venetum leaf herbal medicine, 80% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
2.罗布麻叶提取物10制备步骤:2. 10 preparation steps of Apocynum venetum leaf extract:
(1)将500g罗布麻叶中药饮片粉碎,使用10倍量的80%(v/v)的乙醇溶液浸泡30分钟,加热沸腾(约为80℃),回流提取,提取2次,每次回流1小时;(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;
(2)将步骤(1)得到的产物抽滤,合并,得到提取液;(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;
(3)将步骤(2)得到的提取液在50℃的温度下进行减压浓缩,得到罗布麻叶提取物10。(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.
制备例11:罗布麻叶提取物11的制备Preparation Example 11: Preparation of Apocynum venetum Leaf Extract 11
1.原料、试剂和仪器1. Raw materials, reagents and instruments
罗布麻叶中药饮片、95%(v/v)乙醇溶液、粉碎机、布氏漏斗、抽滤瓶。Apocynum venetum leaf herbal medicine, 95% (v/v) ethanol solution, pulverizer, Buchner funnel, suction filter bottle.
2.罗布麻叶提取物11制备步骤:2. 11 preparation steps of Apocynum venetum leaf extract:
(1)将500g罗布麻叶中药饮片粉碎,使用10倍量的95%(v/v)的乙醇溶液浸泡30分钟,加热沸腾(约为78℃),回流提取,提取2次,每次回流1小时;(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;
(2)将步骤(1)得到的产物抽滤,合并,得到提取液;(2) The product obtained in step (1) is suction filtered and combined to obtain an extract;
(3)将步骤(2)得到的提取液在50℃的温度下进行减压浓缩,得到罗布麻叶提取物11。(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.
实验例1:罗布麻叶提取物1抗骨质疏松的活性的检测Experimental example 1: Detection of anti-osteoporosis activity of Apocynum venetum leaf extract 1
1.实验动物、试剂和仪器1. Laboratory animals, reagents and instruments
制备例1制得的罗布麻叶提取物1。Apocynum venetum leaf extract 1 prepared in Preparation Example 1.
SD清洁级大鼠,雌性,3月龄,购于厦门大学实验动物中心;SD clean rat, female, 3 months old, purchased from the Laboratory Animal Center of Xiamen University;
水合氯醛、多聚甲醛、脱钙液、苏木素、伊红购自上海生工;Chloral hydrate, paraformaldehyde, decalcification solution, hematoxylin and eosin were purchased from Shanghai Shenggong;
β-雌二醇(E
2)购自sigma,micro-CT为Siemens Inveon PET/CT;
β-estradiol (E 2 ) was purchased from sigma, and the micro-CT was Siemens Inveon PET/CT;
石墨炉火焰原子分光光度计为日本岛津;The graphite furnace flame atomic spectrophotometer is Shimadzu of Japan;
固体密度仪为Germany;The solid density meter is Germany;
(上面已经描述)分析天平。(Described above) Analytical balance.
2.实验方法2. Experimental method
2.1大鼠造模2.1 Rat model
动物模型采用去卵巢雌性大鼠骨质疏松模型,原理是雌性大鼠卵巢被切除后,骨代谢增强,骨吸收和骨形成均增强,但骨吸收增强的速度远大骨形成的速度,经过一定时间的积累,导致骨量丢失增多,骨密度减少,其中以松质骨减少最为明显。文献报道建立模型1月后,松质骨密度可减少至60%-70%,补充受试样品,抑制破骨细胞或促进成骨细胞活性等骨代谢的调节作用,观察受试品在增加骨密度和骨钙含量的效果,分析骨组织微结构的变化,评价受试品对骨质疏松的调节作用。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分组和给药2.2 Grouping and administration
大鼠经腹腔注射30mg/kg水合氯醛溶液,固定后延背中线距离尾骨3-4cm剃毛,消毒后,切开皮肤2cm。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.
模型组:行背腹部卵巢切除术,切除双侧卵巢,结扎输卵管;假手术组:仅切除部分脂肪。缝合消毒,保证无菌操作。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.
术后1个月进行分组,分为假手术组(SHAM)、模型组(OVX)、模型-雌二醇组(OVX-E
2),模型-罗布麻叶提取物1组(OVX-罗布麻叶提取物1),每组6只大鼠。OVX-E
2组给药剂量为1mg/kg/d,模型-罗布麻叶提取物1组给药剂量为500mg/kg/d,溶剂为0.5%羧甲基纤维素钠溶液,假手术组及模型组分别给予同等体积的0.5%羧甲基纤维素钠溶液,至第90天处死大鼠,检测骨质疏松相关指标。
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检测指标2.3 Testing indicators
2.3.1阿基米德浮力法检测股骨的骨密度:105℃将骨头烘干至恒重,进行检测和计算。骨骼在空气和水的重量之差为骨骼在水中的浮力,即骨骼排水的重量,根据水和空气的密度可计算出骨骼密度,单位为g/mm
3。
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 .
2.3.2X射线(micro-CT)检测骨密度:取左侧股骨中段至下1/3处(远心端),采用X射线计算机断层扫描成像,计算骨密度,单位为HU。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骨恒重:105℃烘箱烘干12h后,每隔1h精确称量股骨,直至每间隔1h,称量股骨的重量变化小于0.0003g,即为骨恒重。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骨钙含量测定,使用原子吸收法对灰化的左侧股骨进行测定。马弗炉550℃彻底灰化股骨,准确称取0.3000g的灰化骨粉,置于150mL的消解管中,加入混合酸(硝酸:高氯酸=4:1),电热器加热消化至透明无色,去离子水稀释,容量瓶定容,使用炉火焰原子分光光度计仪的钙离子检测灯测量溶液中的钙离子浓度。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子宫指数:大鼠称重,颈椎脱臼处死后,取出子宫,剥离子宫附近脂肪组织, 称量。子宫指数=子宫湿重/体重。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股骨组织形态计量学参数:取右侧股骨,按照常规固定,脱钙,切片,HE染色,采用正置显微镜进行拍照记录,多功能真彩色病理图像分析软件统计和分析股骨的病理学参数。各指标的涵义和计算公式如表1所示: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:
表1:骨组织微结构的参数和计算公式Table 1: Parameters and calculation formulas of bone tissue microstructure
骨组织形态计量学参数和计算公式Bone tissue morphometric parameters and calculation formulas
2.3.7胫骨骨组织形态计量学参数:取右侧胫骨,按照常规固定,脱钙,切片,HE染色,采用正置显微镜进行拍照记录,多功能真彩色病理图像分析软件统计和分析股骨的病理学参数。各指标的涵义和计算公式如表1所示。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.实验结果3. Experimental results
3.1固体密度仪测定骨密度:结果如表2的骨密度1所示。3.1 Determination of Bone Density by Solid Density Meter: The results are shown in Bone Density 1 in Table 2.
结果:浮力法测定的骨密度的值为骨密度1,由表2可知,假手术组和模型组比较,模型组骨密度显著降低(P<0.05),表明去卵巢引起的大鼠骨质疏松造模成功。给药组与模型组比较,雌二醇组和罗布麻叶提取物1组均能提高骨密度,表明罗布麻叶提取物能有效防止骨质疏松大鼠的骨密度降低。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.
3.2micro-CT测定松质骨密度:结果如表2的骨密度2所示。3.2 Micro-CT measurement of cancellous bone density: The results are shown in Table 2 Bone Density 2.
结果:X射线断层扫描的松质骨密度的值为骨密度2,由表2可知,假手术组与模型组比较,模型组松质骨密度显著降低(P<0.001),表明去卵巢引起的大鼠骨质疏松造模成功。与模型组比较,雌二醇组和罗布麻叶提取物1组均能提高松质骨密度(P<0.01),表明罗布麻叶提取物能有效防止骨质疏松大鼠松质骨密度的降低。具体CT图详见图2A-图2H。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.
另外,观察骨髓腔内骨小梁(micro-CT图中呈白色网线状分布,图2A-图2H)还显示,与假手术组(SHAM)相比,模型组(OVX)大鼠的股骨骨小梁的结构不完 整,数量减少;与模型组相比,雌二醇组(OVX-E
2)和罗布麻叶提取物1组(OVX-罗布麻叶提取物1)大鼠股骨的骨小梁结构完整性提高,骨小梁数量增加。说明罗布麻叶提取物1可以有效改善骨质疏松大鼠的骨小梁结构。
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股骨恒重:结果如表2的骨恒重所示。3.3 Constant weight of femur: The results are shown in Table 2 for the constant weight of the femur.
结果:雌二醇组骨恒重与模型组有统计学差异(P<0.05),但罗布麻叶提取物1组无统计学差异,暗示罗布麻叶提取物1和雌二醇的作用机制不同。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原子分光光度法测定左侧股骨的骨钙含量,如表2的骨钙含量所示。3.4 The bone calcium content of the left femur was measured by atomic spectrophotometry, as shown in Table 2.
结果:假手术组与模型组比较,模型组左侧股骨骨钙含量显著降低(P<0.05),表明去卵巢引起的大鼠骨质疏松造模成功。与模型组比较,雌二醇组能提高骨钙含量(P<0.05),罗布麻叶提取物1组能提高骨钙含量(P<0.01),但罗布麻叶提取物1提高骨钙含量的作用更强,具有一定的优势。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子宫指数:结果如表2的子宫指数所示。3.5 Uterine Index: The results are shown in Table 2 for Uterine Index.
通过子宫湿重的重量除以体重可得子宫指数。与假手术组比较,模型组左侧股骨骨钙含量显著降低(P<0.001),子宫内皮增生明显减少,表明去卵巢引起的大鼠骨质疏松造模成功。与模型组比较,雌二醇组能提高子宫指数(P<0.001),雌二醇发挥雌激素样作用,刺激子宫内皮增生。罗布麻叶提取物1组的子宫指数与模型组比较则无统计学差异,暗示罗布麻叶提取物1并不会发挥雌激素样作用,不会刺激子宫内膜增生,减少子宫内膜癌、乳腺癌发生的风险。此外,本发明人还发现,本发明制备的罗布麻叶提取物特别是罗布麻叶提取物2-7均不会发挥雌激素样作用。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.
表2.:罗布麻叶提取物1对大鼠Table 2. Apocynum venetum leaf extract 1 to rats
骨密度、骨恒重、骨钙含量、子宫指数的影响The influence of bone density, bone constant weight, bone calcium content, uterine index
其中:模型组(OVX)相较于假手术组(SHAM),
#为P<0.05,
##为P<0.01,
###为P<0.001;给药组(OVX-E
2和OVX-罗布麻叶提取物1)相较于模型组(OVX),
*为P<0.05,
**为P<0.01,
***为P<0.001,N=6。
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.
结果显示:与假手术组比较,模型组骨密度降低,骨钙含量减少,子宫指数降低,表明造模成功。给药组与模型组比较,雌二醇组和罗布麻叶提取物1组均能显著性增 加股骨密度,提高骨钙含量。但罗布麻叶提取物1组对骨钙密度作用更强,且对子宫刺激作用无统计学差异,所以在预防和治疗骨质疏松方面,罗布麻叶提取物1相较于雌二醇组具有一定的优势。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大鼠体重:结果如图1的体重所示。3.6 Rat body weight: The results are shown in the body weight of Figure 1.
图1显示,假手术组(SHAM)体重相对稳定,模型组(OVX)体重逐步上升,说明卵巢切除后大鼠体重增加;与模型组相比,雌二醇组(OVX-E
2)大鼠体重下降,说明雌二醇在OVX模型中具有下调体重的作用;与模型组相比,罗布麻叶提取物1(OVX-罗布麻叶提取物1)体重无显著性变化,说明罗布麻叶提取物1在OVX模型中不具有明显调节体重的作用。
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罗布麻叶提取物1对大鼠股骨组织微结构和胫骨组织微结构的影响3.7 Effect of Apocynum venetum leaf extract 1 on the microstructure of femur tissue and tibia tissue in rats
结果如表3、图3A-图3F以及图4A-图4F所示。The results are shown in Table 3, Figure 3A-Figure 3F and Figure 4A-Figure 4F.
表3:罗布麻叶提取物对大鼠股骨组织微结构的影响Table 3: Effect of Apocynum venetum leaf extract on the microstructure of rat femur tissue
其中:模型组(OVX)相较于假手术组(SHAM),
#为P<0.05,
##为P<0.01,
###为P<0.001;给药组(OVX-E
2和OVX-罗布麻叶提取物1)相较于模型组(OVX),
*为P<0.05,
**为P<0.01,
***为P<0.001,N=6。
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.
图3A-图3F显示,与假手术组(SHAM)相比,模型组(OVX)大鼠的股骨骨小梁的结构不完整,数量减少;与模型组相比,罗布麻叶提取物1组(OVX-罗布麻叶提取物1)大鼠股骨的骨小梁结构完整性提高,骨小梁数量增加。说明罗布麻叶提取物1可以有效改善骨质疏松大鼠的骨小梁结构。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.
图4A-图4F显示,与假手术组(SHAM)相比,模型组(OVX)大鼠的胫骨骨小梁的结构不完整,数量减少;与模型组相比,罗布麻叶提取物1组(OVX-罗布麻叶提取物1)大鼠胫骨的骨小梁结构完整性提高,骨小梁数量增加。说明罗布麻叶提取物1可以有效改善骨质疏松大鼠的骨小梁结构。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.
由以上结果可见,模型组与假手术组比较,模型组的骨小梁面积、骨小梁厚度和数目均显著减少且骨小梁分离度显著增加,显示造模成功。给药组与模型组比较,雌二醇和罗布麻叶提取物1均能有效提高骨小梁面积、骨小梁厚度、骨小梁数目,降低 骨小梁分离度,具有防治骨质疏松症的效果。但罗布麻叶提取物1相较于雌二醇在提高骨小梁厚度上具有优势(P<0.01)。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).
实验例2:罗布麻叶提取物2-7活性检测Experimental example 2: Apocynum venetum leaf extract 2-7 activity detection
1.实验动物、试剂和仪器1. Laboratory animals, reagents and instruments
制备例2-7制得的罗布麻叶提取物2-7。Apocynum venetum leaf extract 2-7 prepared in Preparation Example 2-7.
SD清洁级大鼠,雌性,3月龄,购于厦门大学实验动物中心;SD clean rat, female, 3 months old, purchased from the Laboratory Animal Center of Xiamen University;
水合氯醛购自上海生工。Chloral hydrate was purchased from Shanghai Shenggong.
2.实验方法2. Experimental method
假手术组仅切除卵巢附近脂肪,模型组进行卵巢切除术,术后1个月,进行分组和给药。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.
3月龄雌性大鼠行卵巢切除术后1个月开始灌胃,每组3只,假手术组(SHAM)、手术模型组(OVX),以及模型给药组(罗布麻叶提取物2-7)。罗布麻叶提取物2给药剂量为500mg/kg/d,溶剂为0.5%羧甲基纤维素钠溶液,假手术组及模型组分别给予同等体积的0.5%羧甲基纤维素钠溶液。罗布麻叶提取物3-7,按照表4所占总提取物的比例,按比例给药。给药至第90天处死大鼠,检测骨质疏松相关指标,micro-CT检测松质骨密度值。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.实验结果3. Experimental results
X射线断层扫描的松质骨密度的值为骨密度,由表4可知,假手术组(SHAM)与模型组(OVX)比较,模型组骨密度显著降低(P<0.001),表明去卵巢引起的大鼠骨质疏松造模成功。相较于模型组,OVX-罗布麻叶提取物2组骨密度值显著升高(P<0.01),OVX-罗布麻叶提取物3组骨密度无显著性差异,OVX-罗布麻叶提取物4组和OVX-罗布麻叶提取物7组骨密度显著升高(P<0.05),OVX-罗布麻叶提取物5组和OVX-罗布麻叶提取物6组骨密度显著升高(P<0.001)。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).
表4:罗布麻叶粗提取物的分段分离部分占比及活性检测Table 4: Fractional separation and activity detection of crude extract of Apocynum venetum leaf
罗布麻叶粗提取物的水溶液进行分段分离及活性检测Aqueous solution of crude extract of Apocynum venetum leaves for segmented separation and activity detection
表4:模型组相较于假手术组,
###为P<0.001;给药组相较于模型组,
*为P<0.05,
**为P<0.01,
***为P<0.001,其中SHAM和OVX组为6只大鼠,其余每组为3只大鼠。
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.
结果显示,罗布麻叶提取物2具有增强骨密度的作用(P<0.01),且其进一步分离产物显示罗布麻叶提取物5和6效果更好(P<0.001),能够显著增加骨密度,表明罗布麻叶提取物中的有效成分可能为25%-95%乙醇洗脱部分,其中50%乙醇洗脱部分(提取物5)和75%乙醇洗脱部分(提取物6)的效果更好。提取物7是95%乙醇洗脱部分,应该也含有一定量的有效成分。但是有效成分应该主要集中在50%和75%洗脱的部分。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.
另外,结果还显示,水洗脱的部分(罗布麻叶提取物3)并没有很好的效果,因此,本发明人推测,在制备例2-7中的步骤(5)之后,如果直接用50%-75%乙醇洗脱,可能会包含更多非有效成分。用制备例5-6为代表的本发明的方法,可以更有效地富集有效成分。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.
实验例3:罗布麻叶提取物8-11抗骨质疏松的活性的检测Experimental Example 3: Detection of Anti-osteoporosis Activity of Apocynum venetum Leaf Extract 8-11
1.实验动物、细胞、试剂和仪器1. Laboratory animals, cells, reagents and instruments
如下面的表5所示。As shown in Table 5 below.
表5:本实验涉及的部分实验动物、细胞、试剂和仪器Table 5: Some experimental animals, cells, reagents and instruments involved in this experiment
实验样品为制备例8-11制得的罗布麻叶提取物8-11。The experimental sample is Apocynum venetum leaf extract 8-11 prepared in Preparation Example 8-11.
2.实验方法2. Experimental method
2.1破骨细胞分化模型构建2.1 Construction of osteoclast differentiation model
破骨细胞的过度激活是骨质疏松症发病的主要机制之一,目前临床上使用的药物绝大多数为破骨细胞抑制剂,所以破骨细胞的抑制剂具有潜在的预防或治疗骨质疏松症的效果。在机体中,破骨细胞来源于血系单核-巨噬细胞,骨髓中含有大量单核-巨噬细胞前体细胞。体外分离的单核-巨噬细胞前体细胞在M-CSF的诱导下可形成单核-巨噬细胞,再在M-CSF和RANKL的共同诱导下可以分化成破骨细胞。在体外建立此破骨细胞分化模型可用于骨质疏松症体外药物的筛选和作用评价。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小鼠单核-巨噬细胞前体细胞的提取2.2 Extraction of mouse monocyte-macrophage precursor cells
选取6-8周龄的C57BL/6J小鼠,通过断颈法处死小鼠,并将其浸入70%乙醇中5分钟进行消毒灭菌,在生物安全柜中,无菌操作分离小鼠的股骨和胫骨。配置含10%FBS和1%青霉素混合液的α-MEM培养基,无菌注射器中吸满培养基,使用注射器将整个骨髓细胞从股骨和胫骨上冲出,并以5×10
4/cm
2的密度接种在10cm的培养皿中,加入M-CSF(50ng/mL)培养过夜,然后收集上层的悬浮细胞。
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破骨细胞的分化2.3 Differentiation of osteoclasts
收集的细胞继续在M-CSF(50ng/mL)的处理下培养3天,可成为小鼠单核-巨噬细胞,小鼠单核巨噬细胞为具有多分化潜能的破骨前体细胞。将上述单核-巨噬细胞以 5×10
4/cm
2的密度种植在96孔细胞培养板中,同时处理M-CSF(50ng/mL)和RANKL(50ng/mL)7天,诱导分化成破骨细胞。
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提取物处理2.4 Extract processing
细胞在处理M-CSF和RNAKL的同时,分别用罗布麻叶提取物8-11(25μg/mL)处理,细胞培养7天后,镜下观察。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指标检测2.5 Index detection
破骨细胞具有更大体积和明显的细胞质结构,一个成熟的破骨细胞通常会具有3-100个细胞核,属于多核融合细胞。同时破骨细胞会表达大量抗酒石酸酸性磷酸酶,会被TRAP染色试剂盒染成红色。将细胞培养板在室温下用4%多聚甲醛固定10分钟,然后在37摄氏度下用抗酒石酸酸性磷酸酶染色试剂盒染色1小时,使用倒置显微镜拍照,观察并计数多核融合破骨细胞。采用Graph Prism 8.0软件分析数据,使用t检验分析差异性,***为P<0.001,代表模型组和提取物处理组间的显著性差异。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.实验结果3. Experimental results
如图5A-5H所示,提取的单核细胞(图5A,空白对照组)体积较小且不能被TRAP染色试剂盒染成红色。经M-CSF和RANKL处理后的细胞中(图5B,模型组),出现大量多核融合的破骨细胞,细胞体积显著增大,具有明显的细胞质结构(箭头所示),且可以被TRAP染色试剂盒染成红色。这表明破骨细胞分化模型构建成功。细胞在M-CSF和RANKL诱导分化的同时,用不同浓度乙醇提取的罗布麻叶提取物(25μg/mL)处理,破骨细胞的数量显著减少(图5C-5H)。结果表明,罗布麻叶20%乙醇提取物、40%乙醇提取物、60%乙醇提取物、80%乙醇提取物和95%乙醇提取物均具有抑制破骨细胞形成的作用,具有潜在的抗骨质疏松作用。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.
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。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)
- 一种制备罗布麻叶提取物的方法,包括下述步骤:A method for preparing apocynum venetum leaf extract includes the following steps:(1)使用20%-95%的乙醇溶液对罗布麻叶进行提取,得到提取液;(1) Extracting Apocynum venetum leaves with a 20%-95% ethanol solution to obtain an extract;(2)将提取液进行浓缩,得到第一浓缩物;(2) Concentrating the extract to obtain the first concentrate;优选地,还包括步骤(3)、(4-1)和(5),步骤(3)、(4-2)和(5),或者步骤(3)、(4-2)、(4-3)和(5):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)将步骤(1)的提取液或者将步骤(2)的第一浓缩物用水稀释后上样大孔树脂柱(例如大孔树脂D101、D101-Ⅰ、DA201、DM301、HPD100或DM130),(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)使用25%-95%的乙醇溶液进行洗脱,得到洗脱液;(4-1) Perform elution with a 25%-95% ethanol solution to obtain an eluate;(4-2)依次用水、20%-30%乙醇溶液(优选25%乙醇溶液)洗脱之后,(4-2) After eluting with water and 20%-30% ethanol solution (preferably 25% ethanol solution),用35%-60%乙醇溶液(优选50%乙醇溶液)进行洗脱,得到洗脱液,Elution is carried out with 35%-60% ethanol solution (preferably 50% ethanol solution) to obtain the eluent,(4-3)用60%-80%乙醇溶液(优选75%乙醇溶液)进行洗脱,得到洗脱液;(4-3) Perform elution with 60%-80% ethanol solution (preferably 75% ethanol solution) to obtain an eluate;(5)将洗脱液进行浓缩,得到第二浓缩物。(5) Concentrate the eluate to obtain a second concentrate.
- 根据权利要求1所述的制备方法,其中,步骤(1)中,The preparation method according to claim 1, wherein, in step (1),乙醇溶液的浓度为50%-75%、55%-70%、55%-65%或者60%;The concentration of the ethanol solution is 50%-75%, 55%-70%, 55%-65% or 60%;乙醇溶液的用量为罗布麻叶的5-20倍量、8-15倍量或者10-12倍量;The amount of ethanol solution is 5-20 times, 8-15 times or 10-12 times the amount of Apocynum venetum leaves;罗布麻叶为粉碎的或未经粉碎的罗布麻叶;Apocynum venetum leaf is crushed or not crushed apocynum venetum leaf;所述提取为回流提取;The extraction is reflux extraction;提取次数为1次或多次,例如1-4次;优选地,合并每次提取得到的提取液;The number of extractions is one or more times, such as 1-4 times; preferably, the extracts obtained from each extraction are combined;和/或and / or每次提取的时间为至少0.2小时、至少0.5小时、至少1小时、1-24小时、1-10小时或者1-5小时。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.
- 根据权利要求1至2中任一权利要求所述的制备方法,其中,步骤(2)和/或步骤(5)中,The preparation method according to any one of claims 1 to 2, wherein in step (2) and/or step (5),所述浓缩为减压浓缩;The concentration is concentration under reduced pressure;浓缩的温度为40℃-60℃,优选50℃;The temperature of concentration is 40°C-60°C, preferably 50°C;优选地,所述第一浓缩物和/或所述第二浓缩物为浸膏。Preferably, the first concentrate and/or the second concentrate is an extract.
- 根据权利要求1至3中任一权利要求所述的制备方法,其中,步骤(3)中,重复上样至少1次、1-10次、1-6次、1-3次或者1-2次;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;和/或and / or稀释的倍数为2-10倍或者4-5倍。The dilution factor is 2-10 times or 4-5 times.
- 根据权利要求1至4中任一权利要求所述的制备方法,其中,步骤(4-1)中,The preparation method according to any one of claims 1 to 4, wherein, in step (4-1),乙醇溶液的浓度为50%-75%、55%-70%、55%-65%或者60%;The concentration of the ethanol solution is 50%-75%, 55%-70%, 55%-65% or 60%;乙醇溶液的用量为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
- 根据权利要求1至4中任一权利要求所述的制备方法,其中,步骤(4-2)中,The preparation method according to any one of claims 1 to 4, wherein, in step (4-2),乙醇溶液的用量独立地为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is independently 1-10 times the column volume, preferably 4-5 times the column volume.
- 根据权利要求1至4中任一权利要求所述的制备方法,其中,步骤(4-3)中,The preparation method according to any one of claims 1 to 4, wherein, in step (4-3),乙醇溶液的用量为1-10倍柱体积,优选为4-5倍柱体积。The amount of the ethanol solution is 1-10 times the column volume, preferably 4-5 times the column volume.
- 一种罗布麻叶提取物,其由权利要求1至7中任一权利要求所述的制备方法制得。An apocynum venetum leaf extract, which is prepared by the preparation method according to any one of claims 1 to 7.
- 一种药物组合物,其包含权利要求8所述的罗布麻叶提取物;A pharmaceutical composition comprising the Apocynum venetum leaf extract according to claim 8;可选地,所述药物组合物还包含一种或多种药学上可接受的辅料;Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;优选地,所述药物组合物还包含选自如下药物中的一种或多种:Preferably, the pharmaceutical composition further comprises one or more selected from the following drugs:钙剂、维生素D、双磷酸盐、雌激素例如雌二醇、雌激素受体调节剂、降钙素、甲状旁腺激素和骨保护素;Calcium, vitamin D, bisphosphonates, estrogen such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone and osteoprotegerin;优选地,所述药物组合物用于治疗和/或预防骨质疏松症。Preferably, the pharmaceutical composition is used to treat and/or prevent osteoporosis.
- 一种组合药物产品,其包含独立包装的第一产品和第二产品,A combination drug product, which comprises a first product and a second product that are individually packaged,其中,in,所述第一产品包含权利要求8所述的罗布麻叶提取物;The first product comprises the Apocynum venetum leaf extract of claim 8;所述第二产品包含选自如下药物中的一种或多种:The second product contains one or more selected from the following drugs:钙剂、维生素D、双磷酸盐、雌激素例如雌二醇、雌激素受体调节剂、降钙素、甲状旁腺激素和骨保护素;Calcium, vitamin D, bisphosphonates, estrogen such as estradiol, estrogen receptor modulators, calcitonin, parathyroid hormone and osteoprotegerin;优选地,所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料。Preferably, the first product and the second product also independently include one or more pharmaceutically acceptable excipients.
- 罗布麻叶或者罗布麻叶乙醇提取物在制备治疗和/或预防骨质疏松症的药物中的用途;Use of apocynum venetum leaf or apocynum venetum leaf ethanol extract in the preparation of a medicine for treating and/or preventing osteoporosis;优选地,所述罗布麻叶乙醇提取物为权利要求8所述的罗布麻叶提取物;Preferably, the ethanol extract of apocynum venetum leaf is the extract of apocynum venetum leaf according to claim 8;优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症。Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis.
- 根据权利要求11所述的用途,其中,所述治疗和/或预防骨质疏松症或者所述治疗和/或预防骨质疏松症的药物不引起雌激素样作用。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.
- 根据权利要求8所述的罗布麻叶提取物,其用于制备治疗和/或预防骨质疏松症的药物;The Apocynum venetum leaf extract according to claim 8, which is used to prepare a medicine for treating and/or preventing osteoporosis;优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症;Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;优选地,,所述治疗和/或预防骨质疏松症的药物不引起雌激素样作用。Preferably, the drug for treating and/or preventing osteoporosis does not cause estrogen-like effects.
- 一种治疗和/或预防骨质疏松症的方法,包括给予有需求的受试者以有效量的权利要求8所述的罗布麻叶提取物的步骤;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;优选地,所述骨质疏松症为原发性骨质疏松症或继发性骨质疏松症;Preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis;优选地,所述骨质疏松症为绝经后骨质疏松症或老年性骨质疏松症;Preferably, the osteoporosis is postmenopausal osteoporosis or senile osteoporosis;优选地,所述治疗和/或预防骨质疏松症的方法不引起雌激素样作用。Preferably, the method of treating and/or preventing osteoporosis does not cause estrogen-like effects.
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