An extract of carob pods, a preparation method thereof, and applications thereof

Abstract

The present invention relates to the technical field of plant extracts, and particularly relates to a carob pod extract, a preparation method thereof and an application. The preparation method of the carob pod extract provided by the present invention uses the seedless pods of carob tree as raw materials, and includes: refluxing and extracting the raw materials with an aqueous ethanol solution, adsorbing with resin, recovering non-saccharide small molecules, extracting the non-saccharide small molecules with petroleum ether, extracting the aqueous layer obtained by petroleum ether extraction with ethyl acetate, and recovering the ethyl acetate layer; subjecting the ethyl acetate layer to silica gel column chromatography separation, Sephadex LH-20 separation and semi-preparative liquid phase separation in sequence twice. This preparation method can obtain an extract with high purity and excellent anti-osteoporosis efficacy, provides a new way for the development and utilization of carob tree resources, and provides a valuable product for the prevention and treatment of osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and particularly relates to a carob pod extract, a preparation method thereof, and an application thereof. Background Art

[0002] The carob tree (Ceratonia siliqua Linn.) belongs to the leguminous long bean genus and is native to the eastern Mediterranean. It has now been introduced and cultivated artificially in China. Carob has very high nutritional value, is rich in minerals and trace elements, and the tannins and polyphenolic substances contained therein have medicinal value and can improve the respiratory, metabolic, and immune systems. Carob extracts can be used as food flavors, cocoa substitute powders, and are also used in products for improving women's health.

[0003] As people age, bone density inevitably decreases in the human body. The causes of bone mass loss are complex and diverse. Currently, calcium supplementation remains the basis for preventing and treating reduced bone density. The calcium intake can be improved by adjusting the diet structure, but more people choose to quickly supplement calcium through calcium-containing dietary supplements. Traditional Chinese medicine for calcium supplementation often requires a long time of conditioning, while hormone-based calcium supplementation has the problem of relatively large side effects. Therefore, it is of great significance to provide a non-calcium and non-hormonal supplement that can more effectively improve bone density as a natural dietary supplement for preventing and treating bone density decline. Summary of the Invention

[0004] The first object of the present invention is to provide a preparation method of a carob pod extract.

[0005] The second object of the present invention is to provide a carob pod extract prepared by the above method.

[0006] The third object of the present invention is to provide the uses of the above preparation method and carob pod extract.

[0007] At present, in the prior art, the seeds, leaves, gums and other tissues of the carob tree are mostly used for the development of food or functional substances, and the utilization of the seedless pods of the carob tree is less. After the seeds are taken out and utilized, the seedless pods can only be used as raw materials for low-value products such as feed or directly discarded as waste, and are rarely used for the development of active ingredients. During the research and development process of the present invention, it was unexpectedly found that the extract of the seedless pods contains anti-osteoporosis active ingredients and can be used for the development of anti-osteoporosis products. However, the seedless pods contain more impurities such as non-target active ingredients, which need to be removed to the greatest extent during the extraction process, and the physicochemical properties and stabilities of the anti-osteoporosis active ingredients and impurity components in the pods are also unknown, which all increase the difficulty of developing the functional extract derived from the carob tree pods. In order to make the extract have a high content of anti-osteoporosis components, the present invention has developed a method that can obtain a high content of anti-osteoporosis active ingredients for the raw material of the carob tree seedless pods.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing an extract of carob tree pods, which uses the seedless pods of the carob tree as a raw material and includes the following steps: after reflux extraction of the raw material with an aqueous ethanol solution, resin adsorption is carried out to recover non-saccharide small molecules, the non-saccharide small molecules are extracted with petroleum ether, and the aqueous layer obtained by petroleum ether extraction is extracted with ethyl acetate, and the ethyl acetate layer is recovered;

[0010] The ethyl acetate layer is successively subjected to two silica gel column chromatographic separations, Sephadex LH-20 separation, and semi-preparative liquid phase separation.

[0011] The present invention has found that after the seedless pods of the carob tree are subjected to the above extraction, the content of anti-osteoporosis active ingredients in the extract can be significantly increased, and the anti-osteoporosis effect of the extract can be improved. The present invention has continuously screened and optimized the extraction method, and has found that during the extraction process, the method of two-step extraction with petroleum ether and ethyl acetate has a significantly better recovery effect on anti-osteoporosis active ingredients and a better removal effect on non-target active ingredients than other extractants and extraction methods.

[0012] In the above method, the seedless pods of the carob tree are dry pods.

[0013] Before the reflux extraction, it further includes the step of crushing the dry seedless pods. Preferably, the crushed pods are passed through a 60-100 mesh medicinal sieve to obtain carob tree pod coarse powder, and then the coarse powder is subjected to reflux extraction with an aqueous ethanol solution.

[0014] In the above-described method, in the first silica gel column chromatography separation, the silica gel column is gradient eluted first with petroleum ether - ethyl acetate with a volume ratio of 1:1, 1:3, 0:1, and then with dichloromethane - methanol with a volume ratio of 7:1, 1:1, 0:1. After detection by thin-layer chromatography (TLC), similar fractions are combined to obtain fractions concentrated with syringaresinol.

[0015] In the above gradient elution, equal aliquots are collected for each elution gradient. The collected fractions are detected by thin-layer chromatography (TLC), similar fractions are combined, and the fractions concentrated with syringaresinol are recovered for the second silica gel column chromatography separation.

[0016] After the first silica gel column chromatography separation, the fractions concentrated with syringaresinol collected in the first silica gel column chromatography separation are subjected to the second silica gel column chromatography separation; in the second silica gel column chromatography separation, the silica gel column is gradient eluted with petroleum ether - ethyl acetate with a volume ratio of 3:1, 2:1, 1:1, 0:1. After detection by thin-layer chromatography (TLC), similar fractions are combined to obtain fractions concentrated with syringaresinol.

[0017] In the above gradient elution, equal aliquots are collected for each elution gradient. The collected fractions are detected by thin-layer chromatography (TLC), similar fractions are combined, and the fractions concentrated with syringaresinol are recovered for Sephadex LH-20 separation.

[0018] The present invention discovers that by using the above silica gel column chromatography elution mobile phase and gradient elution program, the content of anti-osteoporosis active ingredients in the extract can be significantly increased, while the content of impurity components can be reduced.

[0019] The above dichloromethane - methanol with a volume ratio of 0:1 means that the proportion of dichloromethane is 0 and the proportion of methanol is 100%.

[0020] The above petroleum ether - ethyl acetate with a volume ratio of 0:1 means that the proportion of petroleum ether is 0 and the proportion of ethyl acetate is 100%.

[0021] Preferably, the chromatographic column used in the silica gel column chromatography separation is a silica gel column with 200 - 300 mesh.

[0022] Preferably, the Sephadex LH-20 separation is eluted with a mobile phase of dichloromethane - methanol with a volume ratio of 1:(0.8 - 1.2).

[0023] In the above preparation method, the mobile phase used in the semi-preparative liquid phase separation is methanol and water with a volume ratio of (45 - 55):(45 - 55).

[0024] Preferably, in the semi-preparative liquid separation, first separate using a YMC-Pack ODS-A chromatographic column with an inner diameter of 18-22 mm, and then separate using a YMC-Pack ODS-A chromatographic column with an inner diameter of 8-12 mm.

[0025] The present invention discovers that using the above semi-preparative liquid separation and purification method can effectively remove impurities such as inactive components, improve the content and purity of the anti-osteoporosis active components in the extract, and further improve the anti-osteoporosis activity of the extract.

[0026] Preferably, first separate using a YMC-Pack ODS-A chromatographic column with an inner diameter of 18-22 mm with a methanol aqueous solution having a volume ratio of methanol to water of (50-52):48, and then separate using a YMC-Pack ODS-A chromatographic column with an inner diameter of 8-12 mm with a methanol aqueous solution having a volume ratio of methanol to water of 48:(50-52).

[0027] Preferably, the YMC-Pack ODS-A chromatographic column has a column length of 250 mm and a particle size of 5 μm.

[0028] In the above preparation method, the reflux extraction is as follows: first reflux extract 2-3 times with an ethanol aqueous solution of 90-95%, and then reflux extract 1-2 times with an ethanol aqueous solution of 70-75%, and combine the extracts of each time and concentrate to obtain an extract.

[0029] Preferably, the time for each reflux extraction is 1-2 h.

[0030] Preferably, during the reflux extraction process, the mass ratio of the crude powder of the carob pod to the ethanol aqueous solution is 2:(1-2).

[0031] In the above preparation method, the resin adsorption is as follows: disperse the concentrated extract with water and then use macroporous resin, first elute with water to remove impurities, and then elute with an ethanol aqueous solution of 90-98% to recover the non-carbohydrate small molecule part.

[0032] For the type of macroporous resin, it is preferably to use macroporous adsorption resin D101.

[0033] For the macroporous resin elution, the preferred elution volume is 2-4 column volumes.

[0034] In the above preparation method, after dispersing the recovered non-carbohydrate small molecule part with a 50-60% methanol aqueous solution, first extract with petroleum ether 4-6 times, and then extract the water layer obtained by petroleum ether extraction with ethyl acetate 4-6 times, and recover the ethyl acetate layer.

[0035] In the second aspect, the present invention provides a carob pod extract prepared by the above preparation method.

[0036] Animal experiments have proven that the carob pod extract obtained by the above preparation method has good anti-osteoporosis activity, can significantly increase bone density and bone calcium content, and can be used for the treatment and prevention of osteoporosis.

[0037] Preferably, the carob pod extract contains syringaresinol, and its structural formula is shown in formula (I):

[0038]

[0039] Preferably, in the carob pod extract, the mass percentage content of syringaresinol is ≥80%; more preferably ≥90%; more preferably ≥95%.

[0040] In a third aspect, the present invention provides any one of the following applications of the preparation method of the carob pod extract or the carob pod extract or syringaresinol prepared by this preparation method:

[0041] (1) Application in the preparation of foods, drugs, health products or feeds for increasing bone density and / or bone calcium content.

[0042] (2) Application in the preparation of foods, drugs, health products or feeds for preventing and / or treating osteoporosis.

[0043] In a fourth aspect, the present invention provides a method for preparing syringaresinol from carob pods as raw materials, and the method is the same as the above-mentioned preparation method of carob pod extract.

[0044] The main reported effects of syringaresinol are anti-tumor, antioxidant, anti-inflammatory and immunomodulatory. The present invention extracts syringaresinol from the carob pods without seeds, and the extraction rate of this extraction method is relatively high, and the purity of syringaresinol in the prepared extract can reach more than 99.50%. Verified by animal experiments, the carob pod extract has a good effect on improving osteoporosis.

[0045] In a fifth aspect, the present invention provides a product, and this product contains the carob pod extract.

[0046] Preferably, the product is a food, drug, health product or feed.

[0047] The carob pod extract provided by the present invention can be further prepared into products such as foods, drugs, health products or feeds, and excipients allowed in the above product fields can be added during the preparation process.

[0048] The beneficial effects of the present invention are as follows: The preparation method of the carob pod extract provided by the present invention uses carob pods as raw materials, and can obtain an extract with excellent anti-osteoporosis effects. The extract prepared by this method can significantly increase bone density and bone calcium content, reduce the bone loss rate, and play an effective function in preventing and treating osteoporosis. This method and the extract provide a new way for the development and utilization of carob resources, and provide valuable products for the prevention and treatment of osteoporosis. Description of the Drawings

[0049] Figure 1 1H NMR spectrum of the extract in Example 1 of the present invention 1 1H NMR

[0050] Figure 2 13C NMR spectrum of the extract in Example 1 of the present invention 13 13C NMR Detailed Embodiments

[0051] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0052] The detection conditions of HPLC in the following examples are as follows: The chromatographic column is an ACE 5C18 column (4.6×250mm, 5μm); the mobile phase system is water (A) - methanol (B); the flow rate is 1.0 mL / min; the column temperature is 30°C, the injection volume is 10 μL, the detection wavelength is 254 nm; gradient elution (A:B is 9:1 at 0 - 10 min, 7:3 at 10 - 40 min, and 0.5:9.5 at 40 - 50 min).

[0053] Example 1

[0054] This example provides a preparation method of a carob pod extract, and the specific steps are as follows:

[0055] (1) Take 20 parts by weight of dried carob pods without seeds, crush them and pass through a medicinal sieve to obtain a coarse powder with a mesh size of ≥60. Add 10 times the weight of the coarse powder of 95% ethanol to the coarse powder and reflux extract twice, each time for 1.5 hours. Then, reflux extract with 70% ethanol once, and the reflux extraction time is 1.5 hours. Combine the extraction solutions of each time and concentrate to obtain 12 parts by weight of an extract.

[0056] (2) Disperse the extract obtained in step (1) with 12 parts by weight of water, adsorb it using macroporous adsorption resin D101, wash it with distilled water for 3 column volumes to remove impurities such as sugars, and then elute it with 95% ethanol for 3 column volumes. After recovering the solvent, 0.5 part by weight of a non-sugar small molecule fraction is obtained.

[0057] (3) Disperse the non - carbohydrate small - molecule fraction obtained in step (2) with 0.8 parts by weight of 50% methanol. First, extract it with petroleum ether 5 times, then extract the aqueous layer obtained from the petroleum - ether extraction with ethyl acetate 5 times. Recover the ethyl - acetate layer to obtain 0.05 parts by weight of the petroleum - ether fraction, 0.15 parts by weight of the ethyl - acetate fraction, and 0.30 parts by weight of the water fraction respectively.

[0058] (4) Separate the ethyl - acetate fraction collected in step (3) by silica - gel column chromatography with 200 meshes. First, perform gradient elution with petroleum ether (P) - ethyl acetate (E). The gradient - elution program is P:E = 1:1, P:E = 1:3, P:E = 0:1. Then perform gradient elution with dichloromethane (C) - methanol (M). The gradient - elution program is C:M = 7:1, C:M = 1:1, C:M = 0:1. Collect samples in equal portions, and a total of 108 fractions are collected. After detection by thin - layer chromatography (TLC), combine similar fractions to obtain 10 fractions (A - J). Detect the content of the target component syringaresinol in each fraction by HPLC, and recover the fraction D with the highest concentration of syringaresinol.

[0059] Separate the recovered fraction D again by silica - gel column chromatography with 200 meshes, and perform gradient elution with petroleum ether - ethyl acetate with a volume ratio of 3:1, 2:1, 1:1, 0:1. Collect samples in equal portions. After detection and identification by TLC, combine similar fractions to obtain 4 fractions (D1 - D4). Detect the content of the target component syringaresinol in each fraction by HPLC, and recover the fraction D3 with the highest concentration of syringaresinol.

[0060] Elute the fraction D3 collected from the second silica - gel column chromatography with Sephadex LH - 20 using dichloromethane - methanol with a volume ratio of 1:1 as the mobile phase. First, separate and purify the collected eluate with semi - preparative liquid chromatography (column I, YMC - Pack ODS - A (250×20mm, 5μm), mobile phase: methanol: pure water = 52:48, absorption wavelength: 210nm); then purify it with semi - preparative liquid chromatography (column II, YMC - Pack ODS - A (250×10mm, 5μm), mobile phase: methanol: pure water = 48:52, absorption wavelength: 210nm) to obtain the extract of Ceratonia siliqua fruit pods.

[0061] After detection, the purity of the compound in the above - prepared extract of Ceratonia siliqua fruit pods reaches 99.8%. The nuclear magnetic resonance spectrum of this compound is as Figure 1 and Figure 2 shown. According to the nuclear magnetic resonance spectrum, this compound is determined to be syringaresinol.

[0062] This example also provides an extract of Ceratonia siliqua fruit pods prepared by the above - mentioned preparation method.

[0063] Example 2

[0064] This embodiment provides a method for preparing an extract of Ceratonia siliqua fruit pods, and the specific steps are as follows:

[0065] (1) Take 20 parts by weight of dried Ceratonia siliqua fruit pods without seeds, crush them and pass through a medicinal sieve to obtain a coarse powder of ≥60 mesh. Add 10 times the weight of the coarse powder of 90% ethanol to the coarse powder and reflux extract twice, each time for 1 hour, then use 75% ethanol to reflux extract once, with the reflux extraction time being 1 hour. Combine the extraction solutions of each time and concentrate to obtain 10 parts by weight of an extract.

[0066] (2) Disperse the extract obtained in step (1) with 12 parts by weight of water, adsorb it using macroporous adsorption resin D101, wash it with distilled water for 3 column volumes to remove impurities such as sugars, and then elute it with 95% ethanol for 3 column volumes. After recovering the solvent, obtain 0.42 parts by weight of a non-sugar small molecule fraction.

[0067] (3) Disperse the non-sugar small molecule fraction obtained in step (2) with 1.0 part by weight of 60% methanol. First, extract it with petroleum ether 5 times, and then extract the aqueous layer obtained by petroleum ether extraction with ethyl acetate 5 times. Recover the ethyl acetate layer to obtain 0.04 parts by weight of a petroleum ether fraction, 0.12 parts by weight of an ethyl acetate fraction, and 0.24 parts by weight of an aqueous fraction respectively.

[0068] (4) Separate the ethyl acetate fraction collected in step (3) by silica gel column chromatography with 300 mesh. First, perform gradient elution with petroleum ether (P) - ethyl acetate (E), and the gradient elution program is P:E = 1:1, P:E = 1:3, P:E = 0:1. Then perform gradient elution with dichloromethane (C) - methanol (M), and the gradient elution program is C:M = 7:1, C:M = 1:1, C:M = 0:1. Collect samples in equal portions, and a total of 108 fractions are collected; after TLC detection, combine similar fractions to obtain 10 fractions (A - J). Use HPLC to detect the content of the target component syringaresinol in each fraction, and recover the fraction D with the highest concentration (highest content) of syringaresinol.

[0069] Separate the recovered fraction D again by silica gel column chromatography with 300 mesh, and perform gradient elution with petroleum ether - ethyl acetate at a volume ratio of 3:1, 2:1, 1:1, 0:1. Collect samples in equal portions, identify the obtained fractions by TLC detection, combine similar fractions to obtain 4 fractions (D1 - D4). Use HPLC to detect the content of the target component syringaresinol in each fraction, and recover the fraction D3 with the highest concentration (highest content) of syringaresinol.

[0070] The fractions D3 collected from the second silica gel column chromatography were further eluted with Sephadex LH-20 using dichloromethane-methanol with a volume ratio of 1:1 as the mobile phase. The collected eluate was first separated and purified by semi-preparative liquid chromatography (column I, YMC-Pack ODS-A (250×20 mm, 5 μm), mobile phase: methanol: pure water = 52:48, absorption wavelength: 210 nm); then purified by semi-preparative liquid chromatography (column II, YMC-Pack ODS-A (250×10 mm, 5 μm), mobile phase: methanol: pure water = 48:52, absorption wavelength: 210 nm) to obtain the Bauhinia purpurea L. fruit pod extract.

[0071] After detection, the purity of the compound in the above-prepared Bauhinia purpurea L. fruit pod extract reached 99.5%, and the compound was identified as syringaresinol by nuclear magnetic resonance spectroscopy.

[0072] This example also provides the Bauhinia purpurea L. fruit pod extract prepared by the above preparation method.

[0073] Example 3

[0074] This example provides a preparation method of Bauhinia purpurea L. fruit pod extract, and the specific steps are as follows:

[0075] (1) Take 20 parts by weight of dried Bauhinia purpurea L. fruit pods without seeds, crush them and pass through a medicinal sieve to obtain a coarse powder of ≥60 mesh. Add 10 times the weight of the coarse powder of 95% ethanol to the coarse powder and reflux extract for 2 times, 2 hours each time, then reflux extract with 70% ethanol for 1 time, and the reflux extraction time is 2 hours. Combine the extraction solutions each time and concentrate to obtain 13 parts by weight of an extract.

[0076] (2) Disperse the extract obtained in step (1) with 15 parts by weight of water, adsorb it with macroporous adsorption resin D101, wash it with distilled water for 4 column volumes to remove impurities such as sugars, and then elute it with 95% ethanol for 4 column volumes. After recovering the solvent, 0.6 parts by weight of the non-sugar small molecule part is obtained.

[0077] (3) Disperse the non-sugar small molecule part obtained in step (2) with 1.0 part by weight of 50% methanol. First, extract it with petroleum ether 6 times, and then extract the water layer obtained by petroleum ether extraction with ethyl acetate 6 times. Recover the ethyl acetate layer to obtain 0.06 parts by weight of the petroleum ether part, 0.22 parts by weight of the ethyl acetate part, and 0.32 parts by weight of the water part respectively.

[0078] (4) The ethyl acetate fraction collected in step (3) was separated by silica gel column chromatography with 200 mesh. First, gradient elution was carried out using petroleum ether (P) - ethyl acetate (E), and the gradient elution program was P:E = 1:1, P:E = 1:3, P:E = 0:1. Then, gradient elution was carried out using dichloromethane (C) - methanol (M), and the gradient elution program was C:M = 7:1, C:M = 1:1, C:M = 0:1. Equal portions were sampled, and a total of 106 fractions were collected. After TLC detection, similar fractions were combined to obtain 10 fractions (A - J). The content of the target component, syringaresinol, in each fraction was detected by HPLC, and the fraction D with the highest concentration (highest content) of syringaresinol was recovered.

[0079] The recovered fraction D was separated again by silica gel column chromatography with 200 mesh, and gradient elution was carried out using petroleum ether - ethyl acetate with a volume ratio of 3:1, 2:1, 1:1, 0:1. Equal portions were sampled. After the obtained fractions were detected and identified by TLC, similar fractions were combined to obtain 4 fractions (D1 - D4). The content of the target component, syringaresinol, in each fraction was detected by HPLC, and the fraction D3 with the highest concentration (highest content) of syringaresinol was recovered.

[0080] The fraction D3 collected from the second silica gel column chromatography was further eluted with a mobile phase of dichloromethane - methanol with a volume ratio of 1:1 by Sephadex LH - 20. The collected eluate was first separated by semi - preparative liquid chromatography (column I, YMC - Pack ODS - A (250×20mm, 5μm), mobile phase: methanol: pure water = 52:48, absorption wavelength: 210nm); then, it was separated and purified by semi - preparative liquid chromatography (column II, YMC - Pack ODS - A (250×10mm, 5μm), mobile phase: methanol: pure water = 48:52, absorption wavelength: 210nm) to obtain the extract of Ceratonia siliqua fruit pods.

[0081] After detection, the purity of the compound in the extract of Ceratonia siliqua fruit pods prepared above reached 99.5%. This compound was identified as syringaresinol by nuclear magnetic resonance spectrum.

[0082] This example also provides an extract of Ceratonia siliqua fruit pods prepared by the above preparation method.

[0083] Comparative Example 1

[0084] This comparative example provides a preparation method of an extract of Ceratonia siliqua fruit pods. The difference from the preparation method of Example 1 is only that: in step (4), methanol in the mobile phase of the two - step semi - preparative liquid chromatography was replaced by ethanol.

[0085] After detection, the purity of syringaresinol in the extract of Ceratonia siliqua fruit pods prepared by the above method was only 36%.

[0086] Comparative Example 2

[0087] This comparative example provides a method for preparing an extract of Caesalpinia coriaria pods, and the difference from the preparation method of Example 1 is only that: in step (4), semi-preparative liquid separation is only separated and recovered by column I and does not go through the separation and purification of column II.

[0088] After testing, the purity of syringaresinol in the extract of Caesalpinia coriaria pods prepared by the above method is only 52%.

[0089] Comparative Example 3

[0090] This comparative example provides a method for preparing an extract of Caesalpinia coriaria pods, and the difference from the preparation method of Example 1 is only that: in step (3), the extractant is replaced by a mixed solution of benzene and chloroform with a volume ratio of 1:1 instead of petroleum ether.

[0091] After testing, the purity of syringaresinol in the extract of Caesalpinia coriaria pods prepared by the above method is only 68%.

[0092] Experimental Example Functional Detection of Extract of Caesalpinia coriaria Pods

[0093] The function of the extracts of Caesalpinia coriaria pods prepared in Examples 1-3 and Comparative Examples 1-3 in improving osteoporosis was detected, and the specific method is as follows:

[0094] 1. Experimental animals: 90 SPF-grade Wistar rats at 9 weeks of age were randomly divided into a sham operation group (10 rats) and an ovariectomized model group (80 rats), with a body weight of 250-300 g. The rats were raised in an environment with a relative humidity of 50% - 60% and a temperature of 18 - 22 °C, with a 12-hour day-night cycle, and free access to food and water.

[0095] 2. The method for establishing the osteoporosis model refers to the "Technical Specifications for the Inspection and Evaluation of Health Foods" (2003 edition).

[0096] 3. The rats were randomly grouped according to body weight: 10 rats in the ovariectomy model group, 10 rats in the estradiol valerate group, 10 rats in each of the groups of Examples 1-3 and Comparative Examples 1-3, and 10 rats in the sham operation group.

[0097] 4. The rats in each administration group were given drugs by gavage: the rats in the example groups and the comparative example groups were respectively given the extract of Caesalpinia coriaria pods prepared in Examples 1-3 or Comparative Examples 1-3 at a concentration of 1 g / 100 g body weight; the rats in the estradiol valerate group were given an estradiol valerate drug solution at a concentration of 0.01 mg / mL, and the administration volume was 1 mL / 100 g body weight; the sham operation group and the model group were both given an equal volume of normal saline. The above administration and water supply were carried out once a day for 90 consecutive days.

[0098] The bone mineral density of the femurs and lumbar vertebrae of rats in each group was measured using a dual-energy X-ray absorptiometer, and the calcium content was determined by atomic absorption spectrophotometry. The experimental results are shown in Table 1.

[0099] Table 1 Detection results of BMD (bone mineral density) and calcium content of rats in each group

[0100] Group <![CDATA[Femoral BMD (g / cm 2 )]]> Femoral calcium content (g / g) <![CDATA[Lumbar BMD (g / cm 2 )]]> Lumbar vertebra calcium content (g / g) Sham operation group 0.257±0.011 0.327±0.016 0.234±0.015 0.311±0.014 Ovariectomized model <![CDATA[0.215±0.017 △ > <![CDATA[0.228±0.018 △ > <![CDATA[0.194±0.012 △ > 0.188±0.016△ Estradiol valerate group 0.244±0.031* 0.269±0.033** 0.230±0.02*** 0.245±0.021*** Example 1 0.253±0.014*** 0.288±0.019*** 0.228±0.021*** 0.257±0.022*** Example 2 0.251±0.010*** 0.275±0.021*** 0.225±0.012*** 0.248±0.016*** Example 3 0.249±0.020** 0.267±0.022*** 0.226±0.015*** 0.233±0.03*** Comparative example 1 0.218±0.019 0.231±0.031 0.198±0.025 0.192±0.006 Comparative example 2 0.223±0.008* 0.235±0.013 0.204±0.022 0.207±0.018 Comparative example 3 0.228±0.020 0.242±0.014 0.212±0.013 0.211±0.045

[0101] Note: Compared with the sham operation group △ p < 0.001, compared with the ovariectomized model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0102] The results showed that the BMD value of the femurs of rats in the ovariectomized model group decreased by 16.3%, and the BMD value of the lumbar bones decreased by 17.09%, indicating that the osteoporosis model was successfully established. In the rats of the Example 1 group, the BMD of the femurs increased by 17.7% compared with the ovariectomized model, the calcium content increased by 26.3%, the BMD of the lumbar vertebrae increased by 17.5%, and the calcium content increased by 36.7%. The BMD value and calcium content of the rats in the Example group given the syringaresinol extracted from the carob pod prepared in the Example were better than those in the hormone intervention group and were similar to those in the sham operation group. The BMD and calcium content of each comparative example group were slightly higher than or equivalent to those in the ovariectomized model group and were significantly lower than those in each example group. The above results prove that the carob pod extract provided by the present invention has a high efficacy of increasing bone mineral density and improving bone calcium content and can be used for the prevention and treatment of osteoporosis.

[0103] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A preparation method of a carob pod extract with anti-osteoporosis activity, characterized in that, Using the de-seeded pods of the carob tree as raw materials, including: after reflux extraction of the raw materials with an aqueous ethanol solution, resin adsorption is carried out to recover non-carbohydrate small molecules. After dispersing the recovered non-carbohydrate small molecule fraction with a 50-60% aqueous methanol solution, it is first extracted with petroleum ether, and then the aqueous layer obtained from the petroleum ether extraction is extracted with ethyl acetate, and the ethyl acetate layer is recovered; The ethyl acetate layer is successively subjected to two silica gel column chromatographic separations, Sephadex LH-20 separation, and semi-preparative liquid separation; Among them, the reflux extraction is as follows: first, reflux extraction is carried out 2-3 times with a 90-95% aqueous ethanol solution, and then reflux extraction is carried out 1-2 times with a 70-75% aqueous ethanol solution. The extracts from each time are combined and concentrated to obtain an extract; The resin adsorption is as follows: the extract obtained by concentration is dispersed with water and then macroporous resin is used. First, it is eluted with water to remove impurities, and then eluted with a 90-98% aqueous ethanol solution to recover the non-carbohydrate small molecule fraction; In the first silica gel column chromatographic separation, the silica gel column is first gradient eluted with petroleum ether-ethyl acetate with a volume ratio of 1:1, 1:3, 0:1, and then gradient eluted with dichloromethane-methanol with a volume ratio of 7:1, 1:1, 0:

1. After detection by thin layer chromatography (TLC), similar fractions are combined to obtain fractions concentrated in syringaresinol; The fractions concentrated in syringaresinol collected from the first silica gel column chromatographic separation are subjected to a second silica gel column chromatographic separation; In the second silica gel column chromatographic separation, the silica gel column is gradient eluted with petroleum ether-ethyl acetate with a volume ratio of 3:1, 2:1, 1:1, 0:

1. After detection by thin layer chromatography (TLC), similar fractions are combined to obtain fractions concentrated in syringaresinol; The chromatographic column used for the silica gel column chromatographic separation is a 200-300 mesh silica gel column; The Sephadex LH-20 separation is eluted with a mobile phase of dichloromethane-methanol with a volume ratio of 1:(0.8-1.2); The mobile phase used for the semi-preparative liquid separation is methanol and water with a volume ratio of (45-55):(45-55); In the semi-preparative liquid separation, separation is first carried out using a YMC-Pack ODS-A chromatographic column with an inner diameter of 18-22 mm, and then separation is carried out using a YMC-Pack ODS-A chromatographic column with an inner diameter of 8-12 mm; In the carob tree pod extract, the mass percentage content of syringaresinol is ≥95%; 2. The preparation method according to claim 1, characterized in that, The time for each reflux extraction is 1-2 h.

3. The preparation method according to claim 1 or 2, characterized in that, After dispersing the recovered non-carbohydrate small molecule fraction with a 50-60% aqueous methanol solution, it is first extracted with petroleum ether 4-6 times, and then the aqueous layer obtained from the petroleum ether extraction is extracted with ethyl acetate 4-6 times, and the ethyl acetate layer is recovered.

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