CN101773490B - Active part of Sambucus williamsii Hance for reducing risk of bone-related diseases of menopausal women and application thereof - Google Patents

Active part of Sambucus williamsii Hance for reducing risk of bone-related diseases of menopausal women and application thereof Download PDF

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CN101773490B
CN101773490B CN2010101037236A CN201010103723A CN101773490B CN 101773490 B CN101773490 B CN 101773490B CN 2010101037236 A CN2010101037236 A CN 2010101037236A CN 201010103723 A CN201010103723 A CN 201010103723A CN 101773490 B CN101773490 B CN 101773490B
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sambuci williamsii
ramulus sambuci
active site
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dextrorotation
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CN101773490A (en
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黄文秀
姚新生
肖辉辉
戴毅
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Jinan University
HKUST Shenzhen Research Institute
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HKUST Shenzhen Research Institute
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Abstract

The invention discloses an active part of Sambucus williamsii Hance with the effect of reducing bone mass loss after menopause. The active part has definite chemical components and mainly comprises seven compounds: vanillic acid, coniferol, levo erythro form guaiacyl glycerol-beta-O-4'-coniferyl alcohol ether, dextro threo form-1-(4-hydroxy-3-methoxyphenyl)-2-[-4-(3-hydroxypropyl)-2-hydroxyphenoxy]-1,3-propanediol, dextro threo form guaiacyl glycerol-beta-O-4'-coniferyl alcohol ether, coniferyl alcohol dehydrogenase and dihydro-coniferyl alcohol dehydrogenase. The active part can remarkably reduce the bone loss of ovariectomized mice, increase bone density of thighbone and shinbone, increase bone volume, does not increase the weight of uterus, and is a component for safely and effectively relieving and improving various symptoms of the bone-related diseases of the menopausal women. The invention has the advantages of providing the active part of the Sambucus williamsii Hance which can be used for preparing a medicament, food or a food additive for preventing and treating the bone-related diseases after menopause.

Description

Have the Ramulus Sambuci Williamsii active site and the application thereof that reduce menopausal women bone photo related disorders onset risk
Technical field
The present invention relates to the Ramulus Sambuci Williamsii active site to menopause after the protective effect of bone, the specifically medicine of Ramulus Sambuci Williamsii active site and contained active component bone photo related disorders after preparation prevention or treatment menopause or the application in the health product.
Background technology
20th century, the postclimacteric life span of women had significant prolongation along with the progress of medical level, the prolongation of human longevity.The sickness rate of the diseases such as the climacteric syndrome that therefore, is caused by menopause, obesity, osteoporosis, Urogenital atrophy, diabetes, cardiovascular disease and senile dementia has also had significant increase.These diseases have not only affected patient individual's quality of life, have brought white elephant also for society and family.
In 1～10 year, losing fast of calcium can occur in the body after women's menopause, be easy to occur osteoporosis.Osteoporosis is that a kind of bone amount reduces, and the osseous tissue microstructure destroys, and skeleton fragility increases, thus the general skeletal diseases [1] that is easy to fracture.The demonstration of WHO data, osteoporosis occupy the 6th [2] of old people's commonly encountered diseases, frequently-occurring disease.In Europe, Japan and the U.S., there are every year 7500 ten thousand people of surpassing to suffer from osteoporosis.50% women can fracture because of osteoporosis; According to statistics, the mortality rate due to the osteoporotic fracture surpasses the summation [3-6] of breast carcinoma, cervical cancer and carcinoma of endometrium.China is the maximum country of the whole world aged, and the elderly population that is subject at present the osteoporosis threat has 8,400 ten thousand, and along with the aged tendency of population aggravation, this numeral is also progressively increasing [7].
In recent decades, the drug development for the treatment of postmenopausal osteoporosis is rapid.Used medicine is mainly synthetic drug at present, comprises the anti-bone absorption medicines such as estrogen, calcitonin, diphosphate, the promoting bone growing medicines such as fluoride, the bone mineralising medicines such as calcium preparation, vitamin D.Many-sided shortcomings such as these medicines are the generation of prevention of osteoporosis disease to some extent, alleviates ailing symptom, reduces the fracture incidence rate, and toxic and side effects is large, late result is not affirmed but also exist, and is expensive.So far there is no and a kind ofly integrate that efficient, safety, toxic and side effects are little, the ideal medicament of economic dispatch advantage.
In the traditional medicine works " Plain Questions " of China ancients before more than 2000 years, just the men and women is described in detail in the variation of growth promoter different phase bone.Traditional medicine is thought: the kidney being the origin of congenital constitution, and the kidney generating marrow and dominating bone, powerful, weakly all has substantial connection with the Sheng sorrow of kidney essense at the growth of bone, growth.Deficiency of the kidney, the bone marrow weary source of growing then, skeleton loses supports, and bone mineral content descends, and bone density descends, and then can produce " bone is withered ", " bone exhaustion ", " atrophic debility of bones ", i.e. the osteoporosis of modern medicine definition.Chinese medicine dialectical executed to control also bone closely linked to each other with kidney, proposes the Therapeutic Method such as the kidney invigorating and essence nourishing, kidney-supplementing liver-boosting, kidney and spleen invigorating, invigorating kidney, promoting blood circulation QI invigorating.Chinese herbal medicine resource is abundant, toxic and side effects is little, relative low price and abundant theory of Chinese medical science is arranged and the clinical practice support, has become the important source of seeking the treatment medicine for treating osteoporosis present age.
Ramulus Sambuci Williamsii (the Sambucus williamsii Hance) beginning is stated from Tang Materia Medica, another name symplectic bone wood (" detailed outline "), Schisandra propinqua (Wall.) Baill. Var. intermedia A. Smith (" Zhiwu Mingshi Tukao "), Cortex torricelliae tiliifoliae (" vegetation is the side just ") etc. are the dry stem branch of Caprifoliaceae Ramulus Sambuci Williamsii.Its sweet in the mouth, hardship, property is flat, and is nontoxic, returns Liver Channel.Ramulus Sambuci Williamsii has the effect of expelling wind and removing dampness, promoting blood circulation and stopping pain, reunion of bone, is mainly used in treating the diseases [8] such as rheumatism bones and muscles pain, lumbago, treating swelling and pain by traumatic injury, fracture, wound hemorrhage.Modern pharmacology studies show that the contained part lignans of Ramulus Sambuci Williamsii can promote Osteoblast like Cells and ALP activity external.Yet the research for mentioned component lacks animal pharmacodynamics experimental evidence in the body, and Ramulus Sambuci Williamsii anti-osteoporosis activity position and the mechanism of action thereof are also not yet distinct simultaneously., be necessary its extract and fraction thereof for this reason, adopt internationally recognized pharmacodynamics animal model, carry out deep research.
Summary of the invention
The technical problem to be solved in the present invention is to determine Ramulus Sambuci Williamsii active site and contained main active.
Another technical problem that the present invention will solve provides a kind of Ramulus Sambuci Williamsii active site, medicine, food or food additive for the preparation of bone photo related disorders after prevention or the treatment menopause, and the Ramulus Sambuci Williamsii active site with have medicine, Chinese medicine, the natural product of alleviating and improve the bone photo related disorders and mix, preparation has medicine, food or the food additive of preventing and treating bone photo related disorders effect after the menopause.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
The invention discloses the Ramulus Sambuci Williamsii active site, and contained main active, the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure GSA00000008941700031
Described Ramulus Sambuci Williamsii active site by weight percentage this active site is comprised of following chemical composition:
Vanillic acid (1) 0.5%-5%
4-hydroxy-3-methoxycinnamic alcohol (2) 0.5%-10%
Left-handed erythro form guaiacyl glycerol-β-O-4 '-pine and cypress alcohol radical ether (3) 1.0%-15%
Dextrorotation Su Shi-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)
-2-hydroxyphenoxy]-1,3-PD (4) 1.0%-15%
The formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether (5) 1.0%-15%
Dehydrodiconiferylalcohol (6) 2.0%-20%
Dihydro-dehydrodiconiferylalcohol (7) 2.0%-20%
Other component surpluses
Described other components mainly are other Phenylpropanoid Glycosides, lignanoid, phenolic acid and the glucosides class materials that obtains in the leaching process.
The HPLC finger printing of described Ramulus Sambuci Williamsii active site comprises 10 main chromatographic peaks, wherein the chemical constitution of 7 chromatographic peaks is accurately pointed out, relative retention time take the retention time of dehydrodiconiferylalcohol (peak 9) as 1 each chromatographic peak that calculates, be respectively 0.40 ± 0.02 (vanillic acid), 0.55 ± 0.02 (4-hydroxy-3-methoxycinnamic alcohol), 0.67 ± 0.02 (left-handed erythro form guaiacyl glycerol-β-0-4 '-pine and cypress alcohol radical ether), 0.69 ± 0.02 (dextrorotation Su Shi-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-hydroxyphenoxy]-1,3-PD), 0.71 ± 0.02 (the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether), (1.00 dehydrodiconiferylalcohol), 1.02 ± 0.02 (dihydro-dehydrodiconiferylalcohol).
The HPLC finger printing of described Ramulus Sambuci Williamsii active site is to adopt reversed phase high-performance liquid chromatography to set up, and the condition of described reversed phase high-performance liquid chromatography is: take octadecyl siliconoxygen bond and silica gel as immobile phase; As mobile phase, gradient elution: flow velocity is 0.8mL/min with the methanol-water solution that contains 0.1% acetic acid; The detection wavelength is 280nm; Chromatogram column temperature is 35 ℃, with the dehydrodiconiferylalcohol theory of computation number of plates, is not less than 2000.
The invention discloses a kind of preparation method of Ramulus Sambuci Williamsii active site, it is characterized in that realizing by following steps:
(1) after suitable pulverizing of the dry stem branch of Ramulus Sambuci Williamsii, with methanol, ethanol or water, adopts different extraction times and time, extract or the supersound extraction extraction by cold extraction, heat, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, water or 30%-95% ethanol elution are collected eluent, and decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
In the said extracted step, preferred 60% ethanol with 10 times of amounts in step (1), heating and refluxing extraction 3 times, each 2 hours; In step (2), preferably use 50% ethanol elution.
The invention discloses the application of Ramulus Sambuci Williamsii active site in preparing the medicine of alleviating and improve menopausal women bone photo related disorders.Described medicine is pharmaceutically acceptable dosage form, and described dosage form includes but not limited to tablet, capsule, oral liquid.
The invention discloses the Ramulus Sambuci Williamsii active site and alleviate and improve the food of menopausal women bone photo related disorders or the application in the food additive in preparation.
With aforementioned Ramulus Sambuci Williamsii active site, directly mix with appropriate amount of drug pharmaceutical adjunct or food, make the medicine, food or the food additive that are suitable for taking.Or aforementioned Ramulus Sambuci Williamsii active site and one or more are had medicine, Chinese medicine, natural product (Herba Epimedii, Rhizoma Curculiginis, Fructus Ligustri Lucidi, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Alismatis etc.) and the appropriate amount of auxiliary materials of alleviating or improve the bone photo related disorders mix, make medicine, food or food additive.The medicine that contains the Ramulus Sambuci Williamsii active site, food or the food additive that makes carried out the activity rating of ovariectomized mouse bone photo related disorders, the result shows with the pathological model group and compares, the femur that above-mentioned medicine, food or the food additive that contains the Ramulus Sambuci Williamsii active site can inhibition removal ovary in various degree causes and the bone loss of tibia, and on uterus index without impact.Show above-mentioned medicine, food or the food additive that contains the Ramulus Sambuci Williamsii active site have alleviation in various degree and improve menopause after bone loss, improve femur and tibial bone density, increase the bone volume, and do not increase uterus weight, point out above-mentioned medicine, food or food additive can alleviate safely and effectively and improve the various symptoms of postmenopausal women's bone photo related disorders.
To sum up, Ramulus Sambuci Williamsii active site disclosed by the invention can significantly improve the bone-loss of ovariectomized mouse, improves femur and tibial bone density, increases the bone volume, and not increasing uterus weight, is a kind of composition of alleviating safely and effectively and improving the various symptoms of menopausal women bone photo related disorders.The invention has the beneficial effects as follows provides a kind of Ramulus Sambuci Williamsii active site, can be for the preparation of having medicine, food or the food additive of bone photo related disorders effect after the control menopause.
Description of drawings
Fig. 1 analyzes the Ramulus Sambuci Williamsii 50% ethanol elution finger printing partly that liquid phase is determined by HPLC;
Fig. 2 is the chemical structural drawing that separates 7 chemical compounds that obtain from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 3 separates the chromatographic peak of chemical compound 1 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 4 separates the chromatographic peak of chemical compound 2 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 5 separates the chromatographic peak of chemical compound 3 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 6 separates the chromatographic peak of chemical compound 4 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 7 separates the chromatographic peak of chemical compound 5 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 8 separates the chromatographic peak of chemical compound 6 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Fig. 9 separates the chromatographic peak of chemical compound 7 under identical HPLC condition that obtains to point out collection of illustrative plates from Ramulus Sambuci Williamsii 50% ethanol elution part;
Figure 10 is that each fraction of Ramulus Sambuci Williamsii of the present invention is to the impact analysis figure of mouse uterine weight;
Figure 11 is that each fraction of Ramulus Sambuci Williamsii of the present invention is to the impact analysis figure of mouse femur and tibial bone density;
Figure 12 is that each fraction of Ramulus Sambuci Williamsii of the present invention is to the impact analysis figure of mouse femur and tibial bone volume.
The specific embodiment
Data below in conjunction with embodiment and applied research, checking are further set forth technical scheme of the present invention, but summary of the invention is not limited to listed embodiment.
Embodiment 1: the preparation method of Ramulus Sambuci Williamsii active site
Get 50 kilograms in the dry stem branch of Ramulus Sambuci Williamsii, after suitably pulverizing, with the 60% alcohol heating reflux extraction of 10 times of amounts 3 times, each 2 hours.Merge extractive liquid,, the pressure reducing and steaming solvent obtains Ramulus Sambuci Williamsii total extract 2160 grams.Get subsequently Ramulus Sambuci Williamsii total extract 1250 grams, dissolve with suitable quantity of water, carry out the open column chromatography of macroporous resin, use successively the water, 30%, 50% of 5 times of bed volumes, 95% ethanol-water solution gradient elution, collect the each several part eluent, the difference decompression and solvent recovery, obtain water elution part 712.5 grams, 30% ethanol elution part, 160 grams, 50% ethanol elution part, 125 grams and 95% ethanol elution part, 111 grams, wherein 50% ethanol elution partly is Ramulus Sambuci Williamsii active site (the screening active ingredients data are referring to embodiment 4,5).
Embodiment 2: the Isolation and Identification of main component in the Ramulus Sambuci Williamsii active site
Ramulus Sambuci Williamsii 50% ethanol elution of preparation part has been determined its finger printing by HPLC analysis liquid phase among the embodiment 1, sees Fig. 1.Process ODS column chromatography under finger printing instructs, HW 40 column chromatographies, RP-HPLC prepares the separation means such as liquid phase, pass through HPLC, MS, NMR etc. analyze authentication method, 7 chemical compounds have been determined, vanillic acid (1), 4-hydroxy-3-methoxycinnamic alcohol (2), the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether (3), dextrorotation erythro form-1-(4-hydroxyl-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-hydroxyphenoxy]-1, ammediol (4), dextrorotation erythro form guaiacyl glycerol-β-O-4 '-pine and cypress alcohol radical ether (5), dehydrodiconiferylalcohol (6), Dihydrodehydodiconiferyl alcohol (7).
Separation obtains structural formula of compound and sees Fig. 2.Under the chromatographic condition identical with Ramulus Sambuci Williamsii 50% ethanol elution part HPLC finger printing, the chemical compound that separation obtains to be pointed out, the process of specifically pointing out is seen Fig. 3.
2.1 concrete separation process is as follows
Ramulus Sambuci Williamsii 50% ethanol elution part (SWC) 20.33g, the dissolving of 60% alcohol-water, filtration, filtrate is carried out the separation of ODS mesolow column chromatography, and (φ 2.2 * 30cm), 30%, 50%, 100% methanol-water gradient elution, obtain three fraction: SWC1 (5.20g), SWC2 (7.68g), SWC3 (4.19g).(φ 3 * 60cm) for the separation of SWC1 process HW 40 column chromatographies, 20%, 40%, 100% methanol-water gradient elution obtains 6 fractions (fraction A-F), wherein fraction C (40% methanol-water eluate) separates through the ODS column chromatography again, 30%, 50%, 100% methanol-water gradient elution obtains 7 fractions (subfractionC1-7), the fraction SubfractionC2 (30% methanol-water eluate) that obtains is prepared the type high performance liquid chromatogram to be separated, with 28% methanol-water eluting, obtain chemical compound 2 (7.2mg); (30% methanol-water eluate) is prepared the separation of type high performance liquid chromatogram to SubfractionC3, and 30% methanol-water eluting further by behind the Sephadex LH 20 column chromatography purification, obtains chemical compound 3 (15.3mg) subsequently.Same fraction D (40% methanol-water eluate) is through the ODS column chromatography, 30%, 50%, 100% methanol-water gradient elution and preparation type high performance liquid chromatogram separate, 30% methanol-water eluting obtains chemical compound 1 (31.0mg), 4 (15.2mg), 5 (15.4mg).(φ 3 * 60cm) for the separation of SWC2 process HW 40 column chromatographies, 20%, 40%, 100% methanol-water gradient elution obtains 7 fractions (fraction A-G), wherein fraction C (20% methanol-water eluate) is prepared the separation of type high performance liquid chromatogram, 40% methanol-water eluting obtains chemical compound 7 (180.0mg); (φ 3 * 60cm) for the separation of fractionD (40% methanol-water eluate) process ODS column chromatography, 30%, obtains 5 fractions (subfraction D 1-5) behind 50%, the 100% methanol-water gradient elution, wherein subfraction D3 further carries out the ODS column chromatography and separates that (φ 1 * 30cm), obtain 4 fractions (subfraction D3a-d) behind the 30% methanol-water isocratic elution, wherein subfraction D3c is prepared the separation of type high performance liquid chromatogram, 38% methanol-water eluting obtains chemical compound 6 (22.7mg).
2.2 compound structure resolving is as follows
2.21 chemical compound 1
White powder (methanol).The ESI-MS experiment provides m/z 167[M-H] -, pointing out this is that a molecular weight is 168 micromolecular compound.
Chemical compound 1 1H NMR collection of illustrative plates is simpler, and low place only has three groups of signals, δ 7.55 (1H, d, J=8.7,1.9Hz, H-6); δ 7.54 (1H, d, J=1.9Hz H-2); δ 6.83 (1H, d, J=8.7Hz, H-5) and, be typical phenyl ring ABX coupled system hydrogen signal.High field region has a methoxyl group signal δ 3.88 (3H, s, 3-OMe).
Chemical compound 13C NMR shows a carbonyl signal δ 170.1, because chemical shift illustrates that with respect to aldehyde carbonyl deflection High-Field this is a sour carbonyl or ester carbonyl group.6 carbon signal on the phenyl ring: δ 152.7 (C-4) also appear in this external low place, δ 148.7 (C-3), and δ 125.3 (C-6), δ 123.2 (C-1), δ 113.9 (C-2) reach at high field region and 1 methoxyl group carbon signal occurs.
Through and document [9] relatively, the structure of chemical compound 1 is defined as vanillic acid, i.e. vanillic acid, vanillic acid, chemical compound 13C NMR is referring to table 1.
2.22 chemical compound 2
Faint yellow gluey solid (methanol).The ESI-MS experiment provides m/z 179[M-H] -, pointing out this is that a molecular weight is 180 micromolecular compound.
Be presented at low place one group of typical phenyl ring ABX coupling hydrogen signal δ 6.98 (1H, d, J=1.9Hz, H-2) is arranged; δ 6.83 (1H, dd, J=8.1,1.9Hz, H-6); δ 6.72 (1H, d, J=8.1Hz, H-5) and a pair of trans alkene hydrogen signal δ 6.52 (1H, d, J=15.8Hz, H-7); δ 6.49 (1H, dt, J=15.8,5.9Hz, H-8).High field region shows a methylene hydrogen signal δ 4.18 (2H, dd, J=5.9,1.4Hz, H-9), and it is connected on two keys as can be known to split minute situation by it, also has in addition a methoxyl group hydrogen signal δ 3.85 (3H, s, H-10).
At chemical compound 13On C NMR and the DEPT-135 collection of illustrative plates, can be clearly seen that the carbon signal at the unsaturated double-bond at δ 132.1,121.0 places, the methoxyl group carbon signal at the mesomethylene carbon signal at δ 63.9 places and δ 56.4 places, all the other are carbon signal on the phenyl ring.Therefore determine that chemical compound 2 structures are 4-hydroxy-3-methoxycinnamic alcohol (Coniferylalcohol).The nuclear magnetic data of chemical compound is referring to table 1.
Table 1 chemical compound 1 and 2 1H NMR (400MHz, CD 3OD) and 13C NMR (100MHz, CD 3OD) data
Figure GSA00000008941700091
2.23 chemical compound 3
Yellow oily (methanol), [α] 27 D-1.5 ° (c=0.67, MeOH).In conjunction with 1H NMR, 13C NMR and DEPT-135 determine that the molecular formula of chemical compound is C 20H 24O 7, calculating its degree of unsaturation is 9.
At chemical compound 3 1H NMR (400MHz, in CD 3OD) on the collection of illustrative plates, low place provides 8 groups of hydrogen signals, and one of them unimodal integrated value is 2.Although signal overlap is arranged, still can judge 2 ABX coupled systems of existence and 1 trans double bond coupling according to chemical displacement value and coupling constant, the ABX coupled system is respectively δ 7.01 (1H, d, J=1.8Hz, H-2), δ 6.72 (1H, d, J=8.1Hz, H-5) and δ 6.83 (1H, dd, J=8.1,1.8Hz, H-6); δ 6.98 (1H, d, J=1.9Hz, H-2 '), δ 6.86 (1H, H-5 ') and δ 6.86 (1H, H-6 '), trans double bond is δ 6.50 (1H, d, J=16.0Hz, H-7 ') and δ 6.22 (1H, dt, J=15.8,5.8Hz, H-8 ').
1H NMR collection of illustrative plates has 2 obvious methoxyl group signal: δ 3.86 (3H, s, 3-OMe) at high field region, δ 3.82 (3H, s, 3 '-OMe); 2 company's oxygen methine hydrogen signals: δ 4.82 (1H, d, J=5.8Hz, H-7), δ 4.35 (1H, m, H-8); 1 company's Oxymethylene hydrogen signal: δ 4.18 (2H, dd, J=5.8,1.4Hz, H-9 ') and 1 company's Oxymethylene hydrogen signal of being covered by methoxyl group hydrogen: δ 3.81 (2H, o, H-9).
Chemical compound 3 13C NMR (100MHz, in CD3OD) collection of illustrative plates provides 20 carbon signals, in conjunction with the DEPE-135 collection of illustrative plates, show that there is the quaternary carbon signal of 6 SP2 hydridization: δ 151.9 (C-3 ') low place, (148.9 C-4 '), 148.7 (C-3), 147.0 (C-4), 134.1 (C-1), (133.1 C-1 '), 8 SP2 hydridization tertiary carbon signal: δ 131.4 (C-7 '), (128.5 C-8 '), 121.0 (C-6), (120.7 C-6 '), (118.9 C-5 '), 115.7 (C-5), 111.9 (C-2), (111.4 C-2 '); The methine carbon signal of 2 company's oxygen: δ 86.2 (C-8), 74.1 (C-7) also have 2 even mesomethylene carbon signals of oxygen: δ 63.7 (C-9 '), 62.2 (C-9) and 2 methoxyl group carbon signal δ 56.5 (3-OMe), 56.3 (3 '-OMe).Nuclear magnetic data and document [10,11] nuclear magnetic data of report is basically identical, chemical compound 3 is defined as left-handed erythro form guaiacyl glycerol-β-O-4 '-pine and cypress alcohol radical ether, i.e. (-)-erythro-guaiacylglycerol-β-O-4 '-conifery ether.Nuclear magnetic data sees Table 2.
2.24 chemical compound 4
Yellow oily (methanol), [α] 26 D° 11.3 (c=0.67, MeOH).ESI-MS provides m/z387[M+Na] +, 363[M-H] -The molecular weight of prompting chemical compound is 364.In conjunction with 1H NMR, 13C NMR and DEPT-135 determine that the molecular formula of chemical compound is C 19H 24O 7, calculating its degree of unsaturation is 8.
Chemical compound 4 exists with chemical compound 3 1HNMR (400MHz, in CD 3OD) trace analysis of low place is similar, except the hydrogen that has lacked 1 trans ethylene linkage coupling, 2 ABX coupled system: δ 7.00 (1H is arranged similarly also, d, J=1.8Hz, H-2), δ 6.75 (1H, d, J=8.1Hz, H-5) and δ 6.84 (1H, dd, J=8.2,1.8Hz, H-6); δ 6.68 (1H, d, J=2.0Hz, H-2 '), δ 6.90 (1H, d, J=8.2Hz H-5 ') and δ 6.55 (1H, dd, J=8.3,2.1Hz H-6 ').
At High-Field, except only having a methoxyl group hydrogen signal and occurring 2 methylene hydrogen signals at minimum, all the other hydrogen signals are also similar with chemical compound 3, methoxyl group hydrogen signal: δ 3.80 (3H, s, 3-OMe); 2 methylene hydrogen signal: δ 2.54 (2H, t, J=7.4Hz, H-7 '), δ 1.77 (2H, m, H-8 '); 2 company's oxygen methine hydrogen signals: δ 4.89 (1H, d, J=6.2Hz, H-7), δ 4.07 (1H, dd, J=10.5,4.8Hz, H-8); 2 Oxymethylene hydrogen signal: δ 3.72 (1H, dd, J=11.8,4.1Hz, H-9 ' b), δ 3.47 (1H, dd, J=11.8,5.0Hz, H-9 ' a), δ 3.53 (2H, t, J=6.5Hz H-9) even.
Chemical compound 4 13C NMR (100MHz, in CD 3OD) collection of illustrative plates provides 19 carbon signals, in conjunction with the DEPE-135 collection of illustrative plates, can find out with chemical compound 3 fewer a methoxyl group carbon signal and two olefinic carbon signals, all the other carbon signals are similar.The quaternary carbon signal of 6 the SP2 hydridization in low place: δ 149.3 (C-3 '), 146.0 (C-4 '), 148.9 (C-3), 147.3 (C-4), 134.0 (C-1), 138.6 (C-1 '), 6 SP2 hydridization tertiary carbon signal: 120.8 (C-6), 120.6 (C-6 '), 119.5 (C-5 '), 117.2 (C-2 '), 116.0 (C-5), 111.5 (C-2); High field region has 2 even methine carbon signals of oxygen: δ 87.8 (C-8), 74.2 (C-7) also have 2 even mesomethylene carbon signals of oxygen: δ 62.3 (C-9 '), 61.8 (C-9) and 1 methoxyl group carbon signal δ 56.3 (3-OMe).
Because the chemical displacement value δ 87.8 (C-8) of C-8 compares to low field displacement with δ 86.2 (C-8) in the chemical compound 3 in the chemical compound 4, thus deduction, this Compound Phase is trans to being configured as.Reference literature [12], determine that chemical compound 4 is dextrorotation Su Shi-1-(4-hydroxyl-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-hydroxyphenoxy]-1, ammediol, i.e. (+)-thero-1-(4-hydroxy-3-methoxyphenyl)-2-[-4-(3-hydroxypropanyl)-2-hydroxy-phenoxy]-1,3-propanediol.
Nuclear magnetic data sees Table 2.
2.25 chemical compound 5
Yellow oily (methanol), [α] 27 D° 11.9 (c=0.67, MeOH).ESI-MS provides m/z399[M+Na] +, 775[2M+Na] +The molecular weight of prompting chemical compound is 376.In conjunction with 1H NMR, 13C NMR and DEPT-135 determine that the molecular formula of chemical compound is C 20H 24O 7, calculating its degree of unsaturation is 9.
Chemical compound 5 with chemical compound 3 1H NMR (400MHz, in CD 3OD) trace analysis is similar, provides 8 groups of hydrogen signals in low place, can determine also to exist two ABX coupled systems and a trans double bond coupling according to the chemical shiftsum coupling constant, the ABX coupled system is: δ 7.02 (1H, d, J=1.8Hz, H-2), δ 6.75 (1H, d, J=8.1Hz, H-5) and δ 6.85 (1H, dd, J=8.1,1.8Hz, H-6); δ 7.04 (1H, d, J=1.9Hz, H-2 '), δ 6.99 (1H, d, J=8.3Hz H-5 ') and δ 6.90 (1H, dd, J=8.3,1.9HzH-6 '), trans double bond is δ 6.52 (1H, d, J=16.0Hz, H-7 ') and δ 6.24 (1H, dt, J=16.0,5.7Hz, H-8 '), but the H-5 here and H-6 are underlapped, have had well to split minute.
At high field region 2 obvious methoxyl group signal: δ 3.86 (3H, s, 3-OMe) are arranged, δ 3.82 (3H, s, 3 '-OMe); 2 company's oxygen methine hydrogen signal: δ 4.89 (1H, d, J=5.8Hz, H-7), δ 4.28 (1H, m, H-8) and 1 company's Oxymethylene hydrogen signal: some variations have occured in δ 4.19 (2H, dd, J=5.8,1.4Hz, H-9 '); And 1 the methylene hydrogen signal that is buried in the methoxyl group is moved to δ 3.72 (1H, dd, J=11.9,4.1Hz, H-9b) to High-Field in this chemical compound, δ 3.47 (1H, dd, J=11.9,5.4Hz H-9a).
Chemical compound 5 and chemical compound 3 13C NMR data are more approaching, only be the chemical displacement value δ 86.2 (C-8) of C-8 in the chemical compound 3 in chemical compound 5 to low field displacement to δ 87.2 (C-8).Infer that according to nuclear magnetic data chemical compound 5 is identical with 3 structures, only spatial configuration is different, contrast with document [11], affirmed this supposition, determine that chemical compound 5 is the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, i.e. (+)-thero-guaiacylglycerol-β-O-4 '-conifery ether.Nuclear magnetic data sees Table 2.
Table 2 chemical compound 3,4 and 5 1H NMR (400MHz, CD 3OD) and 13C NMR (100MHz, CD 3OD) data
2.26 chemical compound 6
Yellow oily, [α] 26 D° 10.4 (c=0.67, MeOH).In conjunction with 1H NMR, 13C NMR and DEPT-135 determine that the molecular formula of chemical compound is C 20H 22O 6, calculating its degree of unsaturation is 10.
At chemical compound 6 1H NMR (400MHz, in CD 3OD) on the collection of illustrative plates, low shows 8 groups of hydrogen signals.Infer that according to chemical displacement value and coupling constant containing 1 ABX coupled system, 1 quaternary phenyl ring and 1 trans double bond is coupled.ABX coupled system: δ 6.94 (1H, d, J=1.8Hz, H-2), δ 6.81 (1H, d, J=8.1Hz, H-5) and δ 6.82 (1H, dd, J=8.1,1.8Hz, H-6); Four substituted benzene rings: δ 6.95 (1H, s, H-2 '), δ 6.93 (1H, s, H-6 '); Trans double bond coupling: δ 6.53 (1H, d, J=15.9Hz, H-7 '), δ 6.21 (1H, dt, J=15.8,5.9Hz, H-8 ').
1The high field region of H NMR collection of illustrative plates can be seen two obvious methoxyl group signal: δ 3.86 (3H, s, 3-OMe), δ 3.80 (3H, s, 3 '-OMe); 2 methine hydrogen signal: δ 5.51 (1H, d, J=6.3Hz, H-7), δ 3.48 (1H, dd, J=12.3,6.2Hz, H-8); 1 company's Oxymethylene hydrogen signal: δ 4.19 (2H, dd, J=5.9,1.3Hz, H-9 ') and 1 company's Oxymethylene hydrogen signal of being covered by methoxyl group hydrogen: δ 3.73-3.85 (2H, o, H-9).
Chemical compound 6 13C NMR (100MHz, in CD 3OD) collection of illustrative plates provides 20 carbon signals, in conjunction with the DEPE-135 collection of illustrative plates, show that there are the quaternary carbon signal of 7 SP2 hydridization: 149.2 (C-4 ') low place, δ 145.5 (C-3 '), 149.1 (C-3), 147.6 (C-4), 134.5 (C-1), (132.5 C-5 '), (130.3 C-1 '), 7 SP2 hydridization tertiary carbon signal: δ 132.0 (C-7 '), (127.5 C-8 '), 119.8 (C-6), (116.5 C-6 '), 116.2 (C-5), (112.1 C-2 '), 110.5 (C-2); The methine carbon signal of 1 company's oxygen: 1 methine carbon signal δ 55.1 (C-8); The mesomethylene carbon signal of 2 company's oxygen: δ 64.8 (C-9), 63.9 (C-9 ') and 2 methoxyl group carbon signal δ 56.7 (3-OMe), 56.4 (3 '-OMe).The nuclear magnetic data of nuclear magnetic data and document [13] report is basically identical, and chemical compound 6 is defined as dehydrodiconiferylalcohol, i.e. dehydrodiconiferylalcohol.Nuclear magnetic data sees Table 3.
2.27 chemical compound 7
Yellow oily, [α] 26 D° 17.3 (c=0.67, MeOH).ESI-MS provides m/z 383[M+Na] +, 359[M-H] -The molecular weight of prompting chemical compound is 360.In conjunction with 1H NMR, 13C NMR and DEPT-135 determine that the molecular formula of chemical compound is C 20H 24O 6, calculating its degree of unsaturation is 9.
Chemical compound 7 1H NMR (400MHz, in CD 3OD) collection of illustrative plates, show in low place a typical ABX coupled system and integrated value be 2 unimodal, ABX coupled system: δ 6.94 (1H, d, J=1.8Hz, H-2), δ 6.83 (1H, dd, J=8.2,1.7Hz, H-6) and δ 6.75 (1H, d, J=8.1Hz, H-5); Unimodal: δ 6.71 (1H, s, H-2 '), δ 6.71 (1H, s, H-6 ').
1The high field region of H NMR collection of illustrative plates can be seen two obvious methoxyl group hydrogen signal: δ 3.80 (3H, s, 3-OMe), δ 3.84 (3H, s, 3 '-OMe); Also has in addition 1 even Oxymethylene hydrogen signal: δ 3.56 (2H, t, J=6.5Hz, H-9 '); 2 methylene signals: δ 2.60 (2H, t, J=8.0Hz, H-7 '), δ 1.80 (2H, m, H-8 '); 2 methine hydrogen signal: δ 5.48 (1H, d, J=6.2Hz, H-7), δ 3.46 (1H, dd, J=12.4,6.2Hz, H-8) and 2 single hydrogen signal: δ 3.82 (1H, m), δ 3.76 (1H, m).
Chemical compound 7 13C NMR (100MHz, in CD 3OD) collection of illustrative plates is more similar to chemical compound 6, and high field region had increased by two secondary carbon, all the other were more approaching except having lacked two olefinic carbons in low place, infers that thus chemical compound 7 is the derivants after 6 pairs of key hydrogenations of chemical compound.Nuclear magnetic data and document [10,14] are basically identical, and this chemical compound is defined as Dihydrodehydodiconiferyl alcohol, i.e. dihydrodehydodiconiferyl alcohol.Nuclear magnetic data sees Table 3.
Table 3 chemical compound 6 and 7 1H NMR (400MHz, CD 3OD) and 13C NMR (100MHz, CD 3OD) data
Figure GSA00000008941700161
Embodiment 3: the pointing out of the foundation of finger printing and each chemical compound
3.1 the foundation of finger printing
Get above-mentioned Ramulus Sambuci Williamsii 50% ethanol elution part 40mg, be dissolved in and cross the ODS solid-phase extraction column behind 2mL 60% methanol-water, the 10mL methanol-eluted fractions behind the eluent evaporate to dryness, is dissolved in 2mL methanol, after 0.45 μ m filter membrane, namely gets the need testing solution of Ramulus Sambuci Williamsii active site.
Precision is got above-mentioned need testing solution 10 μ L, with reference to the high efficient liquid phase analysis method of stipulating in the state-promulgated pharmacopoeia appendix, injects high performance liquid chromatograph and carries out chromatography, record chromatogram (seeing Fig. 1).Liquid phase chromatogram condition is: adopting octadecyl silica bonded silica gel is immobile phase; With the methanol-water solution that contains 0.1% acetic acid as mobile phase, gradient elution; Flow velocity is 0.8mL/min; The detection wavelength is 280nm; Chromatogram column temperature is 35 ℃.The Ramulus Sambuci Williamsii active site high performance liquid chromatogram standard finger-print that obtains has 10 chromatographic peaks, relative retention time take the chromatographic retention at No. 9 peaks as 1.00 each chromatographic peak that calculates is respectively 0.40 ± 0.02 (peak 1), 0.55 ± 0.02 (peak 2), 0.67 ± 0.02 (peak 3), 0.69 ± 0.02 (peak 4), 0.71 ± 0.02 (peak 5), 0.73 ± 0.02 (peak 6), 0.86 ± 0.02 (peak 7), 0.93 ± 0.02 (peak 8), 1.00 (peaks 9), 1.02 ± 0.02 (peaks 10).Calculate with dehydrodiconiferylalcohol (No. 9 peaks), theoretical cam curve is not less than 2000.
3.2 the chromatographic peak of each chemical compound is pointed out
Each chemical compound is dissolved in respectively in the methanol in right amount, excessively behind the filter membrane of 0.45 μ m, makes need testing solution.With reference to stipulating to get colleges and universities' liquid phase analysis method in the state-promulgated pharmacopoeia appendix, inject successively high performance liquid chromatograph and carry out chromatography, record respectively chromatogram (seeing Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9), chromatographic condition is identical with embodiment 3.1.Thereby the chemical compound 1-7 that obtains by separation is product in contrast, finished the chemistry of main chromatographic peak in the HPLC characteristic spectrum is pointed out.
Embodiment 4: the Ramulus Sambuci Williamsii total extract that embodiment 1 makes and each eluting fraction of total extract are on the impact of class skeletonization UMR 106 cells of In vitro culture
Propagation and ALP with rat skeletonization sample UMR 106 cells are active, obtain the In Vitro Anti osteoporosis activity of chemical compound in the Evaluation operation example 1.
4.1 laboratory sample
Get the fraction that embodiment 1 makes, namely refer to Ramulus Sambuci Williamsii total extract, Ramulus Sambuci Williamsii total extract water elution part, 30% ethanol elution part, 50% ethanol elution part and 95% ethanol elution part that embodiment 1 makes.
4.2 experimental technique
UMR 106 cells are inoculated in 96 orifice plates with the density of 5000 cells/well after trypsinization disperses, place 37 ℃, humidity 95%, contain 5%CO 2Incubator in cultivate.In 10%FBS (hyclone)-DMEM culture fluid, cultivate after 2 days, cultivated again 1 day with 1%Charcoal Stripped FBS (charcoal treatment is removed the hyclone of hormone)-Phenol red-free DMEM (removing phenol red DMEM).Fraction described in the embodiment 1 is diluted to variable concentrations (0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, 100 μ g/mL) with Phenol red-free DMEM joins in 96 orifice plates, cultivated 24 or 48 hours in 4 multiple holes of each concentration again.The positive matched group of 17 beta estradiols (E2,10nM), the cell that n.s adds is normal group.
4.3 cell-proliferation activity detects
Cell detection: cell is removed culture fluid after cultivating 24 or 48 hours under the effect of medicine, after PBS cleans, adds MTS/PMS reagent and cultivates 3 hours, measures absorbance in the 490nm place, estimates the propagation situation of cell with absorbance.Experimental result is expressed as through one factor analysis of variance: mean ± S.E.M..
Experimental result: see Table 4.
Each fraction of table 4 is on the impact of UMR 106 cell proliferation
Figure GSA00000008941700181
Figure GSA00000008941700191
Annotate: compare with normal group, *P＜0.01, * *P＜0.001
Conclusion: compare with normal group, the Ramulus Sambuci Williamsii total extract has the difference of significance to the propagation of cell when 1 μ g/mL, and water elution part and 50% ethanol elution part are that the compared with normal group has very significant difference at 0.1 μ g/mL, and 50% ethanol elution partly shows the activity of stronger promotion cell proliferation, and the active component in the prompting total extract is enriched to 50% ethanol elution part mostly.
Detect 4.4 cell ALP is active
ALP is active to be detected: ALP (alkaline phosphatase), alkali phosphatase, a kind of enzyme of osteoblast differentiation, it also is the key protein that participates in bone metabolism, by decomposing phosphate ester, increase the concentration of local Phos, thereby promote the calcification [15] of bone matrix.Cell is cultivated after 24 hours under drug effect, remove culture fluid, PBS cleans twice, 3 multiple holes respectively add 20 μ L lysates, 5 multiple holes add 100 μ LALP reagent (4-NPP), 96 orifice plates place incubator to cultivate 15,30,45 minutes, and 405nm measures the ALP absorption value, and 595nm measures the absorption value of albumen.The ALP activity represents with the rate of change of unit albumen alkaline phosphatase activities.Experimental result is expressed as through one factor analysis of variance: mean ± S.E.M..
Experimental result: see Table 5.
Each fraction of table 5 is on the impact of UMR 106 cell ALP activity
Figure GSA00000008941700201
Figure GSA00000008941700211
Annotate: compare with normal group, *P＜0.05, *P＜0.01, * *P＜0.001
Conclusion: can find out from experimental result, compare with normal group, the Ramulus Sambuci Williamsii total extract shows significant difference at 0.1 μ g/mL, and 30% ethanol elution part and 50% ethanol elution part can promote the differentiation of cell extremely significantly in this concentration, and 50% is stronger than the activity of 30% ethanol elution part.
Comprehensively the experiment in vitro result can find out, no matter active to propagation or the ALP of cell, 50% ethanol elution part all shows the strongest activity.
Embodiment 5: each eluting fraction of Ramulus Sambuci Williamsii total extract and total extract is on the impact of removal ovary osteoporosis model mice
5.1 laboratory sample and reagent
Each fraction that embodiment 1 makes namely refers to Ramulus Sambuci Williamsii total extract, Ramulus Sambuci Williamsii total extract water elution part, 30% ethanol elution part and 50% part that embodiment 1 makes (partly merged form by 50% and 95% ethanol elution).17 beta estradiols (Sigma, USA)
5.2 laboratory animal
A cleaning level C57BL/6J mice, female, 3 monthly ages are available from Hong Kong Chinese University's Experimental Animal Center.
5.3 experimental technique
Grouping and administration: the position of bowing behind the mice etherization is fixing, at back spinal column place otch, extracts bilateral ovaries as the Osteoporotic Model mice under aseptic condition, sews up in contrast behind the otch of spinal column both sides, sham operated rats back again.In two weeks of postoperative recovery, 55 of the mices of the health of surviving are divided into 7 groups at random, and gavage gives following medicine respectively.Mice gives distilled water and removes estrogenic Mus grain (AIN93M) every day.
Sham operated rats (Sham, n=8) 0.1% ethanol water 10mL/Kg body weight/d
Pathological model group (OVX, n=8) 0.1% ethanol water 10mL/Kg body weight/d
Positive controls (OVX+E2, n=8) 17 beta estradiol 3.2mg/Kg body weight/d
Be subjected to the reagent group
Ramulus Sambuci Williamsii total extract (OVX+SWT, n=7): 1g/Kg body weight/d
Water elution part (OVX+SWA, n=8): 570mg/Kg body weight/d
30% ethanol elution part (OVX+SWB, n=8): 128mg/Kg body weight/d
50%+95% ethanol elution part (OVX+SWC, n=8): 189mg/Kg body weight/d
5.4 the impact on mouse uterine weight
Gastric infusion is after three months continuously, and the mice etherization behind the abdominal aortic blood, extracts the uterus, weighs rapidly, calculates the uterus organ coefficient.Experimental result is expressed as through one factor analysis of variance: mean ± S.E.M..
Experimental result: see Table 6 and accompanying drawing 10.
Each fraction of table 6 is on the impact of Mouse Uterus coefficient
Figure GSA00000008941700221
Annotate: compare with model group, * *P＜0.001
Conclusion: behind the mice removal ovary, pathological model OVX group compares with sham-operation Sham group, and the uterus coefficient significantly descends, and shows the modeling success.Compare with the OVX group, the estrogen group makes the rapid increase of uterus index, can find out estrogenic side effect highly significant.Compare each fraction uterus coefficient of Ramulus Sambuci Williamsii and model group zero difference with the OVX group, each fraction no estrogen sample side effect of Ramulus Sambuci Williamsii is described.
5.5 the mensuration of bone density and bone volume
The mice etherization, behind the abdominal aortic blood, take out femur and the tibia in mice left side, reject muscle and cartilage, with having infiltrated binding up with gauze of PBS, adopt bone density and the histomorphometry parameters bone volume (BV/TV) of Micro-CT technical measurement distal femur and proximal tibia.Experimental result is expressed as through the T check analysis: mean ± S.E.M..
Experimental result sees Table 7, Figure 11 and Figure 12:
Each fraction of table 7 is on the impact of mouse femur and tibial bone density and bone volume
Figure GSA00000008941700231
Annotate: compare with model group, *P＜0.05, *P＜0.01, * *P＜0.001
Conclusion: the Micro-CT scanning result shows, model group rat femur and tibial bone density and sham-operation Sham organize apparent in view reduction.Compare with the OVX group, SWT (Ramulus Sambuci Williamsii ethanol total extract), SWA (water elution part) and SWC (50%+95% ethanol elution part) can promote the bone density of femur and tibia significantly, can increase significantly the bone volume simultaneously.The wherein effect of SWC approaches or is better than SWT.
Embodiment 6: the preparation of Ramulus Sambuci Williamsii active site related preparations
6.1 the preparation of Ramulus Sambuci Williamsii active site tablet
Prescription: Ramulus Sambuci Williamsii active site (50% ethanol elution part), 20 grams; Amylum pregelatinisatum, 20 grams; 50% syrup, an amount of; Magnesium stearate, 1%.
Preparation method: Ramulus Sambuci Williamsii 50% ethanol elution part is dried in 60 ℃ of-80 ℃ of baking ovens, wear into fine powder and cross 80 mesh sieves, add 50% syrup and make suitable soft material, granulate 60 ℃ of dryings, granulate (16 order) with 16 eye mesh screens, add the magnesium stearate mix homogeneously, tabletting, and get final product.
6.2 the preparation of Ramulus Sambuci Williamsii active site capsule
Prescription: Ramulus Sambuci Williamsii active site (50% ethanol elution part), 45 grams; Starch, 2 grams; Microcrystalline Cellulose, 3 grams; 75% ethanol, an amount of.
Preparation method: Ramulus Sambuci Williamsii 50% ethanol elution part is dried in 60 ℃ of-80 ℃ of baking ovens, be ground into fine powder and cross 80 mesh sieves, 60 ℃ of dryings.Add in proportion medicated powder, microcrystalline Cellulose, beat starch and 75% appropriate amount of ethanol after sticking with paste, make suitable soft material, 40 eye mesh screens are granulated, 70 ℃ of oven dry, and granulate, 60 mesh sieves remove fine powder, the fill capsule, and get final product.
6.3 the preparation of Ramulus Sambuci Williamsii active site oral liquid
Prescription: Ramulus Sambuci Williamsii active site (50% ethanol elution part), 100 grams; Flos Chrysanthemi is brilliant, 10 grams; Benzoic acid, 1 gram; Ethyl hydroxybenzoate, 0.3 gram; 1% chitin dilute acetic acid solution, an amount of.
Preparation method: get Ramulus Sambuci Williamsii 50% ethanol elution part, be dissolved in an amount of 60% ethanol, stir, be statically placed in 2 ℃ of-8 ℃ of freezers precipitations 48 hours, filter filtrate recycling ethanol, to without the alcohol flavor, add Flos Chrysanthemi crystalline substance, benzoic acid, the ethyl hydroxybenzoate of suitable quantity of water and prescribed dose, stir, add again 1% chitin dilute acetic acid solution an amount of, filter packing, gland, 100 ℃ of flowing steam sterilizations 30 minutes, and get final product.
6.4 the preparation of Ramulus Sambuci Williamsii compound recipe medicated wine
Ingredients: Ramulus Sambuci Williamsii active site 10 grams, Herba Epimedii 10 grams, Rhizoma Curculiginis 10 grams, Fructus Ligustri Lucidi 8 grams, Radix Rehmanniae Preparata 8 grams, Radix Angelicae Sinensis 8 grams, Rhizoma Alismatis 8 grams, Poria 8 grams, Chinese liquor an amount of (more than 50 degree).
Preparation method: Ramulus Sambuci Williamsii 50% ethanol elution part is dried in 60 ℃ of-80 ℃ of baking ovens, be ground into fine powder; Medical material Rhizoma Curculiginis, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Alismatis, Poria are cut into the tablet of 3 millimeter thins; Herba Epimedii is cut into 2～3 centimetres section; Fructus Ligustri Lucidi is pulverized.Medical material after the processing places in the wine jar by the prescription proportioning cloth bag of packing into, adds 8～12 times of Chinese liquor, seals, and soaks under room temperature 15～25 days, and get final product.
6.5 the preparation of Ramulus Sambuci Williamsii Medulla Bovis grunniens bone meal
Ingredients: Ramulus Sambuci Williamsii active site (50% ethanol elution part), yak bone meal, Lentinus Edodes, Semen Armeniacae Amarum, Semen Coicis, Semen Glycines.
Preparation method: Ramulus Sambuci Williamsii 50% ethanol elution part is dried in 60 ℃ of-80 ℃ of baking ovens, be ground into fine powder and cross 80 mesh sieves.Lentinus Edodes, Semen Armeniacae Amarum, Semen Coicis, the equal crushed after being dried of Semen Glycines are crossed 80 mesh sieves.Ramulus Sambuci Williamsii 50% ethanol elution part, yak bone meal, Lentinus Edodes, Semen Armeniacae Amarum, Semen Coicis, Semen Glycines were by 1: 2: 1: 2: 3: 3 mix homogeneously, and get final product.
Embodiment 7: Ramulus Sambuci Williamsii active site preparation is on the impact of osteoporosis model Mouse Bone
7.1 animal experiment method
Animal experiment method is with embodiment 5.
Group is as follows:
Sham operated rats distilled water 10mL/Kg body weight/d
The blank tablet 10mL/Kg of pathological model group body weight/d
Tablet formulation winding bone woodwork position tablet 300mg/Kg body weight/d
Capsule prescription winding bone woodwork position capsule 300mg/Kg body weight/d
Oral liquid prescription winding bone woodwork position oral liquid 15ml/Kg body weight/d
Medicated wine prescription winding bone woodwork position medicated wine 15ml/Kg body weight/d
Medulla Bovis grunniens bone meal prescription group Ramulus Sambuci Williamsii Medulla Bovis grunniens bone meal 300mg/Kg body weight/d
7.2 laboratory sample
Tablet: get respectively blank tablet and Ramulus Sambuci Williamsii active site tablet, grind, be suspended in the distilled water, gastric infusion.
Capsule: get respectively blank capsules agent and Ramulus Sambuci Williamsii active site capsule, take out content, be suspended in the distilled water gastric infusion.
Oral liquid: take out oral liquid and be made into suitable concentration, gastric infusion with distilled water.
Medicated wine: according to the dosage gastric infusion.
Medulla Bovis grunniens bone meal: take out an amount of Ramulus Sambuci Williamsii Medulla Bovis grunniens bone meal, boiling water furnishing pasty state, gastric infusion.
7.3 experimental result
Experimental result sees Table 8.
Table 8 gavage gives the impact of the various preparations of Ramulus Sambuci Williamsii active site on Mouse Bone density
Figure GSA00000008941700261
Annotate: compare with model group, *P＜0.01, * *P＜0.001
7.4 conclusion
Experimental result shows, compare with model group, Ramulus Sambuci Williamsii active site tablet, capsule and oral liquid that embodiment 6 makes all can significantly improve because the loss of the caused mouse femur of removal ovary and tibial bone amount can be used as the drug use for the treatment of bone amount loss relevant disease.The result shows simultaneously, and capsule is best to the protective effect of bone, secondly is tablet, is oral liquid again.Although Ramulus Sambuci Williamsii active site medicated wine group and bone marrow bone meal group do not have statistical significance to the effect of bone, show and suppress the trend that the bone amount runs off, the health product that can be used as prevention of osteoporosis are used.
Embodiment 8: the assay of Ramulus Sambuci Williamsii active site and preparation main active thereof
Get 20 in Ramulus Sambuci Williamsii active site tablet, take by weighing 40mg after the grinding, be dissolved in and cross the ODS solid-phase extraction column behind 2mL 60% methanol-water, the 10mL methanol-eluted fractions behind the eluent evaporate to dryness, is dissolved in 2mL methanol, after 0.45 μ m filter membrane, namely get the need testing solution of Ramulus Sambuci Williamsii active site tablet.
Get respectively Ramulus Sambuci Williamsii capsule 's content and each 40mg of Medulla Bovis grunniens bone meal, make the need testing solution of Ramulus Sambuci Williamsii capsule and Medulla Bovis grunniens bone meal with method same as described above.
Get respectively each 4mL of Ramulus Sambuci Williamsii oral liquid and medicated wine, behind the evaporate to dryness, be dissolved in 2mL 60% methanol-water, make the need testing solution of Ramulus Sambuci Williamsii oral liquid and medicated wine with method same as described above.
Liquid-phase condition carries out liquid phase analysis described in the employing embodiment 3.1.Calculate the content of each chemical compound with reference to turning towards of the area method in the assay under the high-efficient liquid phase technique item in 2005 editions pharmacopeia appendix, the assay result is as follows:
The content of each active component in table 9 Ramulus Sambuci Williamsii active site and the preparation thereof
Chemical compound Active site (%) Tablet (%) Capsule (%) Oral liquid (%) Medicated wine (%) Medulla Bovis grunniens bone meal (%) Content range (%)
1 2.90 3.10 3.74 2.64 1.04 0.54 0.5-5.0
2 3.23 5.43 7.82 3.02 1.38 0.73 0.5-10.0
3 7.15 8.36 10.21 7.03 2.96 1.42 1.0-15.0
4 7.36 8.71 10.44 7.27 3.05 1.55 1.0-15.0
5 7.56 10.98 11.37 7.45 3.11 1.62 1.0-15.0
6 15.80 17.49 19.05 15.58 7.37 2.37 2.0-20.0
7 13.63 16.28 17.46 13.43 6.53 2.16 2.0-20.0
Almost do not have at present commercially available good effect, side effect is little and price is relatively low is used for the treatment of the diseases related medicine of bone after the menopause; the invention provides a kind of Ramulus Sambuci Williamsii active site extract; in the coalition and experiment in vitro proved that this active site has no side effect to the uterus and bone is had good protective effect; use simultaneously this active site and prepared multiple dosage form, for medicine and the health product of bone protective effect after fully exploitation, the development menopause provide foundation.
[list of references]
[1]Consensus Development Conference.Prophilxis and treatment of osteoporosis[J]Am J Med，1991，90：107-110
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Claims (11)

1. a Ramulus Sambuci Williamsii active site is preparing the medicine of alleviating and improving menopausal women osteoporosis disease, purposes in food and the food additive, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000011
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
2. purposes in medicine, food and the food additive of menopausal women osteoporosis disease is alleviated and improved to Ramulus Sambuci Williamsii active site according to claim 1 in preparation, and it is characterized in that: this active site is comprised of following chemical composition by weight percentage:
Figure FSB00000957951000021
Described other component mainly is other Phenylpropanoid Glycosides, lignanoid, phenolic acid and the glucosides class material that obtains in the leaching process.
3. Ramulus Sambuci Williamsii active site according to claim 1 alleviates and improves the medicine of menopausal women osteoporosis disease in preparation, purposes in food and the food additive, it is characterized in that: the HPLC finger printing of described Ramulus Sambuci Williamsii active site comprises 10 main chromatographic peaks, wherein the chemical constitution of 7 chromatographic peaks is accurately pointed out, the retention time of 9 dehydrodiconiferylalcohols is the relative retention time of 1 each chromatographic peak that calculates take the peak, the retention time that is respectively vanillic acid is 0.40 ± 0.02, the retention time of 4-hydroxy-3-methoxycinnamic alcohol is 0.55 ± 0.02, the retention time of left-handed erythro form guaiacyl glycerol-β-O-4 '-pine and cypress alcohol radical ether is 0.67 ± 0.02, dextrorotation Su Shi-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-hydroxyphenoxy]-retention time of 1,3-PD is 0.69 ± 0.02, the retention time of the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether is 0.71 ± 0.02, the retention time of dehydrodiconiferylalcohol is 1.00, the retention time of dihydro-dehydrodiconiferylalcohol is 1.02 ± 0.02.
4. purposes in medicine, food and the food additive of menopausal women osteoporosis disease is alleviated and improved to Ramulus Sambuci Williamsii active site according to claim 1 in preparation, it is characterized in that: the HPLC finger printing of described Ramulus Sambuci Williamsii active site is to adopt reversed phase high-performance liquid chromatography to set up, and the condition of described reversed phase high-performance liquid chromatography is: take octadecyl siliconoxygen bond and silica gel as immobile phase; As mobile phase, gradient elution: flow velocity is 0.8mL/min with the methanol-water solution that contains 0.1% acetic acid; The detection wavelength is 280nm; Chromatogram column temperature is 35 ℃, with the dehydrodiconiferylalcohol theory of computation number of plates, is not less than 2000.
5. purposes in medicine, food and the food additive of menopausal women osteoporosis disease is alleviated and improved to Ramulus Sambuci Williamsii active site according to claim 1 in preparation, it is characterized in that: described medicine is pharmaceutically acceptable dosage form, and described dosage form includes but not limited to tablet, capsule, oral liquid.
6. application in the medicine of menopausal women osteoporosis disease is alleviated and improved to mixture with the medicine alleviating or improve the osteoporosis disease and suitable excipient substance of Ramulus Sambuci Williamsii active site and one or more in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000031
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
7. application in the medicine of menopausal women osteoporosis disease is alleviated and improved to mixture with the Chinese medicine alleviating or improve the osteoporosis disease and suitable excipient substance of Ramulus Sambuci Williamsii active site and one or more in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000041
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
8. application in the medicine of menopausal women osteoporosis disease is alleviated and improved to mixture with the natural product alleviating or improve the osteoporosis disease and suitable excipient substance of Ramulus Sambuci Williamsii active site and one or more in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000051
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
9. the food of menopausal women osteoporosis disease and the application in the food additive are alleviated and improved to Ramulus Sambuci Williamsii active site and one or more mixture with medicine of alleviating or improving the osteoporosis disease in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000061
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
10. the food of menopausal women osteoporosis disease and the application in the food additive are alleviated and improved to Ramulus Sambuci Williamsii active site and one or more mixture with Chinese medicine of alleviating or improving the osteoporosis disease in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
11. the food of menopausal women osteoporosis disease and the application in the food additive are alleviated and improved to Ramulus Sambuci Williamsii active site and one or more mixture with natural product of alleviating or improving the osteoporosis disease in preparation, it is characterized in that: the composition of this active site mainly contains 7 chemical compounds: vanillic acid, 4-hydroxy-3-methoxycinnamic alcohol, the left-handed formula guaiacyl glycerol-β of Soviet Union-O-4 '-pine and cypress alcohol radical ether, dextrorotation erythro form-1-(4 hydroxyls-3-anisyl)-2-[-4-(3-hydroxypropyl)-2-methoxy phenoxy]-1, ammediol, the formula guaiacyl glycerol-β of dextrorotation Soviet Union-O-4 '-pine and cypress alcohol radical ether, dehydrodiconiferylalcohol, Dihydrodehydodiconiferyl alcohol, the chemical structural formula of described 7 chemical compounds is respectively:
Figure FSB00000957951000081
The preparation method of described Ramulus Sambuci Williamsii active site realizes by following steps:
(1) after the dry stem branch of Ramulus Sambuci Williamsii is suitably pulverized, with 60% ethanol of 10 times of amounts, heating and refluxing extraction 3 times, each 2 hours, collect extracting solution, will obtain the Ramulus Sambuci Williamsii total extract behind the extracting solution concentrating under reduced pressure;
(2) with suitable quantity of water dissolving Ramulus Sambuci Williamsii total extract, carry out the open column chromatography of macroporous resin, use 50% ethanol elution, collect eluent, decompression and solvent recovery obtains eluting and partly is the Ramulus Sambuci Williamsii active site.
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