Cistanche deserticola extract for improving bone mineral density as well as preparation method and application thereof

Abstract

The invention discloses a cistanche deserticola extract for improving bone mineral density, the total content of echinacoside and verbascoside in the cistanche deserticola extract is greater than 25wt%, and the cistanche deserticola extract is obtained by adding distilled water into cistanche tubulosa coarse powder for extraction, filtration, filtrate centrifugation, concentration, extraction, concentration and impurity removal, dilution, after-treatment, concentration and drying. The total content of echinacoside and verbascoside in the cistanche deserticola extract for improving the bone mineral density is 25-30 wt%, and the cistanche deserticola extract is rich in shikimic acid, phenylpropionic acid and alkaloid components, so that the cistanche deserticola extract can stably play a role in improving the bone mineral density.

Description

Technical Field

[0001] This invention relates to the field of medical preparations, specifically to a Cistanche deserticola extract for improving bone density, its preparation method, and its application. Background Technology

[0002] Cistanche deserticola is a plant belonging to the Orobanchaceae family. Cistanche deserticola YC Ma) or Cistanche tubulosa ( Cistanche tuhulosa The dried, scaly, fleshy stem of *Cistanche deserticola* (Schenk. Wight) possesses various functions, including immunomodulatory, anti-inflammatory, anti-fatigue, antioxidant, neuroprotective, and reproductive system-improving effects. Phenylene glycol glycosides (echinacoside and verbascoside are the main phenylethyl glycol glycosides in *Cistanche deserticola*) are important components of *Cistanche deserticola*. Echinacoside has been shown to increase bone density; however, we have found that the bone density-increasing effect of *Cistanche deserticola* extract is not solely related to the echinacoside content. Simply controlling the echinacoside content does not guarantee a stable bone density-increasing effect. Therefore, developing a stable bone density-increasing extract of *Cistanche deserticola* and its preparation method is crucial.

[0003] Chinese invention patent application CN102441040A discloses a method for preparing total phenylethanoid glycosides from Cistanche deserticola, involving steps such as Cistanche deserticola pulverization, extraction, adsorption with adsorption resin, elution, and concentration. The stability of Cistanche deserticola phenylethanoid glycosides is significantly improved under weakly acidic conditions (pH 2-4). The total content of echinacoside and verbascoside in the obtained total phenylethanoid glycosides is greater than 90%, but it has not been verified whether it can stably improve bone density. Chinese invention patent application CN105111255A discloses a method for extracting and separating echinacoside and verbascoside from Cistanche deserticola, using 75% ethanol as the extraction solvent, AB-8 type macroporous resin packing material, and elution with 55% ethanol. This method yields a Cistanche deserticola extract with an echinacoside content of over 25%, a verbascoside content of over 10%, and a total phenylethanoid glycoside content of over 80%. However, it does not necessarily guarantee a stable effect on improving bone density. Summary of the Invention

[0004] In order to develop a Cistanche deserticola extract that can stably improve bone density and its preparation method, the first aspect of the present invention provides a method for preparing a Cistanche deserticola extract that improves bone density, comprising the following steps: Cistanche tubulosa was pulverized to obtain coarse powder of Cistanche tubulosa. Crude powder of Cistanche tubulosa was extracted with distilled water, filtered, and the filtrate was centrifuged and concentrated to obtain concentrated Cistanche tubulosa extract. Extract the concentrated extract of Cistanche tubulosa, concentrate the lower extract to remove impurities, and dilute to obtain the sample solution; The sample solution was post-processed, concentrated, and dried to obtain Cistanche deserticola extract.

[0005] In one embodiment, the coarse powder of Cistanche tubulosa is passed through a 40-mesh sieve.

[0006] In one embodiment, the mass-to-volume ratio of the crude Cistanche tubulosa powder to distilled water is 1:(15-25).

[0007] In one embodiment, the mass-to-volume ratio of the crude Cistanche tubulosa powder to distilled water is 1:20.

[0008] In one embodiment, the temperature for distilled water extraction is 60-100℃.

[0009] In one embodiment, the temperature for distilling water extraction is 80°C.

[0010] In one implementation, the distilled water extraction is performed 1-2 times, each time for 1 hour.

[0011] In one implementation, the filtration is through an 80-mesh filter.

[0012] In one embodiment, the centrifugation rate is 3000-4000 r / min and the centrifugation time is 3-9 min.

[0013] In one embodiment, the centrifugation rate is 3500 r / min and the centrifugation time is 5 min.

[0014] In one embodiment, the concentration after centrifugation is achieved by concentrating the filtrate at 70°C to 18%-28% of its initial volume.

[0015] In one embodiment, during the extraction of the concentrated Cistanche tubulosa extract, the volume ratio of the concentrated Cistanche tubulosa extract to the extractant is 1:(1-5).

[0016] In one embodiment, during the extraction of the concentrated Cistanche tubulosa extract, the volume ratio of the concentrated Cistanche tubulosa extract to the extractant is 1:3.

[0017] In one embodiment, the extractant includes at least one of petroleum ether, ethyl acetate, n-hexane, or cyclohexane.

[0018] In one embodiment, the extraction is performed by sequential extraction with petroleum ether and ethyl acetate.

[0019] In one embodiment, the lower layer extract is concentrated to remove impurities by concentrating the lower layer extract to a paste state and dissolving it in 300 mL of 50 wt% ethanol aqueous solution to remove impurities, adjusting the concentration to 1.0-1.5 g medicinal material / mL as the loading solution.

[0020] In one embodiment, the concentration of the loading solution is 1.0-1.5 g crude drug / mL water.

[0021] As one implementation method, the post-treatment process of the loading solution is as follows: the loading solution is passed through a macroporous resin, and then eluted sequentially with distilled water and ethanol solution.

[0022] In one embodiment, the flow rate of the loading solution through the macroporous resin is 1-5 mL / min.

[0023] In one embodiment, the macroporous resin is AB-8 macroporous resin, and the volume of the macroporous resin is 0.93L.

[0024] In one embodiment, the volume ratio of distilled water to macroporous resin is (5-10):1, and the volume ratio of ethanol solution to macroporous resin is (3-9):1.

[0025] In one embodiment, the volume ratio of distilled water to macroporous resin is 7:1, and the volume ratio of ethanol solution to macroporous resin is 6:1.

[0026] In one embodiment, the ethanol solution is an aqueous ethanol solution with a mass concentration of 80%.

[0027] A second aspect of the present invention provides a Cistanche deserticola extract for improving bone density, wherein the Cistanche deserticola extract is prepared by the preparation method described above.

[0028] In one embodiment, the total content of echinacoside and verbascoside in the Cistanche deserticola extract is 25-30 wt%, excluding 25 wt%.

[0029] A third aspect of the present invention provides an application of a Cistanche deserticola extract for improving bone density, used in the preparation of a product for improving bone density, wherein the concentration of the Cistanche deserticola extract in the product for improving bone density is 12.5-25 μg / mL.

[0030] Compared with the prior art, the present invention has the following beneficial effects: (1) The total content of echinacoside and verbascoside in the Cistanche deserticola extract for improving bone density described in this invention is 25-30 wt%, and it is rich in shikimic acid, phenylpropionic acid, alkaloids and fatty acids, which enable it to stably play the role of improving bone density.

[0031] (2) The Cistanche deserticola extract for improving bone density described in this invention promotes the proliferation of MC3T3-E1 cells in the concentration range of 12.5-25 μg / mL, and has excellent cell proliferation promoting effect.

[0032] (3) The Cistanche deserticola extract for improving bone density described in this invention exhibits the same dose-related effect in promoting cell proliferation and ALP activity, and can stably exert the effect of promoting cell proliferation and differentiation; in promoting osteoblast mineralization, it has a better effect than the positive drug 17β-1 estradiol.

[0033] (4) The Cistanche deserticola extract for improving bone density described in this invention can stimulate osteoblast-specific transcription factors Runx2 and Osx, thereby enhancing osteoblast differentiation and promoting the expression of osteoblast-specific markers OPN and OCN, thus promoting bone formation. It can also increase the relative expression level of Bmp2 mRNA, thereby enhancing osteoblast differentiation and promoting new bone formation by activating the BMP / Smad pathway. Furthermore, it can activate the Wnt / β-catenin pathway signal transduction by regulating Lrp5 mRNA, Lrp6 mRNA, and Wnt3α mRNA, thereby affecting the expression of downstream transcription factors and promoting osteoblast differentiation and bone formation.

[0034] (5) The preparation method of the Cistanche deserticola extract for improving bone density described in this invention yields a Cistanche deserticola extract containing phenylethanol glycosides (echinacoside, verbascoside), shikimic acid and phenylpropionic acid, alkaloids, and fatty acids, which makes its effects on promoting cell proliferation, differentiation, and mineralization show the same dose-dependent relationship, and can stably exert the effect of improving bone density. Attached Figure Description

[0035] Figure 1 The figure shows the test results of the Cistanche deserticola extract prepared in Example 1 on the proliferation of MC3T3-E1 cells; Figure 2 The figure shows the test results of the Cistanche deserticola extract prepared in Example 2 on the proliferation of MC3T3-E1 cells; Figure 3 The figure shows the test results of the Cistanche deserticola extract prepared in Example 3 on the proliferation of MC3T3-E1 cells; Figure 4 The graph shows the test results of ALP activity of the Cistanche deserticola extract prepared in Example 1. Figure 5 The graph shows the test results of ALP activity of the Cistanche deserticola extract prepared in Example 2. Figure 6 The graph shows the test results of ALP activity of the Cistanche deserticola extract prepared in Example 3. Figure 7 Photograph of osteoblast mineralization alizarin red staining with Cistanche deserticola extract prepared in Example 2; Figure 8 The graph shows the test results of the Cistanche deserticola extract prepared in Example 2 on osteoblast mineralization. Figure 9 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on the expression of Runx2 mRNA gene in osteoblast differentiation; Figure 10 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on the expression of Osx mRNA gene in osteoblast differentiation. Figure 11 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on the expression of Opn mRNA gene in osteoblast differentiation. Figure 12 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on the expression of Ocn mRNA gene in osteoblast differentiation. Figure 13 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of Bmp2 mRNA gene in the BMP / Smad pathway; Figure 14 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of Lrp5 mRNA gene in the Wnt / β-catenin pathway; Figure 15 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of Wnt3α mRNA gene in the Wnt / β-catenin pathway; Figure 16 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of Lrp6 mRNA gene in the Wnt / β-catenin pathway; Figure 17 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of Osx protein related to osteoblast differentiation; Figure 18 The figure shows the effect of the Cistanche deserticola extract prepared in Example 2 on upregulating the expression of RUNX2 protein, which is related to osteoblast differentiation. Detailed Implementation

[0036] Example 1 A method for preparing a Cistanche deserticola extract that improves bone density includes the following steps: Cistanche tubulosa was pulverized and passed through a 40-mesh sieve to obtain 200g of coarse powder. Extract the crude powder of Cistanche tubulosa with 5 times its weight of distilled water, extract twice at 75℃ for 1 hour each time, and concentrate the extract at 70℃ with a concentration ratio of 1:2.2 to obtain concentrated Cistanche tubulosa extract; Anhydrous ethanol was added to the concentrated extract of Cistanche tubulosa for fractional alcohol precipitation. The final mass concentrations of ethanol were 20%, 40%, 60% and 80%, respectively. The precipitation was carried out at 5℃ for 24 h, and the supernatant after 80wt% alcohol precipitation was collected. The extract was concentrated and dried at 70℃ to obtain Cistanche deserticola extract with a yield of 20.75%.

[0037] Example 2 A method for preparing a Cistanche deserticola extract that improves bone density includes the following steps: Cistanche tubulosa was pulverized to obtain 240g of coarse powder. Crude powder of Cistanche tubulosa was extracted with distilled water, filtered, and the filtrate was centrifuged and concentrated to obtain concentrated Cistanche tubulosa extract. Extract the concentrated extract of Cistanche tubulosa, concentrate the lower extract to remove impurities, and dilute to obtain the sample solution; The sample solution was post-treated and concentrated at 70℃ and dried to obtain Cistanche deserticola extract. The yield was 19.69%. The coarse powder of Cistanche tubulosa was passed through a 40-mesh sieve.

[0038] The mass-to-volume ratio of the crude powder of Cistanche tubulosa to distilled water is 1:20.

[0039] The temperature for distilled water extraction is 80℃.

[0040] The distilled water extraction is performed 1-2 times, each time for 1 hour.

[0041] The filtration process involves passing the material through an 80-mesh filter.

[0042] The centrifugation rate was 3500 r / min, and the centrifugation time was 5 min.

[0043] The centrifuged concentrate is obtained by concentrating the filtrate at 70°C to 20% of its initial volume.

[0044] During the extraction of the concentrated Cistanche tubulosa extract, the volume ratio of the concentrated Cistanche tubulosa extract to the extractant is 1:3.

[0045] The extraction process involves sequential extraction with petroleum ether and ethyl acetate.

[0046] The lower layer extract is concentrated and purified by concentrating it to a paste-like consistency and then dissolving and purifying it with 300 mL of 50 wt% ethanol aqueous solution.

[0047] The concentration of the loading solution was 1.0 g crude drug / mL water.

[0048] The post-treatment process of the loading solution is as follows: the loading solution is passed through a macroporous resin and then eluted with distilled water and ethanol solution in sequence.

[0049] The flow rate of the loading solution through the macroporous resin was 3 mL / min.

[0050] The macroporous resin is AB-8 macroporous resin, and the volume of the macroporous resin is 0.93L.

[0051] The volume ratio of distilled water to macroporous resin is 7:1, and the volume ratio of ethanol solution to macroporous resin is 6:1.

[0052] The ethanol solution is an aqueous solution of ethanol with a mass concentration of 80%.

[0053] Example 3 A method for preparing a Cistanche deserticola extract that improves bone density includes the following steps: Cistanche tubulosa was pulverized and passed through a 40-mesh sieve to obtain 120g of coarse powder. Crude powder of Cistanche tubulosa was extracted with 12 times its weight of 50wt% ethanol aqueous solution. The extraction was carried out twice at 70℃ for 1 hour each time. The extract was concentrated at 70℃ with a concentration ratio of 1:2.5 to obtain concentrated Cistanche tubulosa extract. Add water to adjust the concentration of the concentrated Cistanche tubulosa extract to 0.2 g crude drug / mL water, centrifuge at 3500 r / min for 5 min, and take the supernatant to obtain the sample solution; The sample solution was passed through a macroporous adsorption resin. After loading, it was eluted sequentially with distilled water and ethanol solution. The ethanol eluent was collected, concentrated, and dried at 70°C to obtain the Cistanche deserticola extract. The yield was 24.30%.

[0054] The macroporous resin is HPD750 macroporous resin, and the volume of the macroporous resin is 0.93L.

[0055] The volume ratio of distilled water to macroporous resin is 3:1, and the volume ratio of ethanol solution to macroporous resin is 4:1.

[0056] The ethanol solution is an aqueous solution of ethanol with a mass concentration of 30%.

[0057] Performance testing Osteoblasts are the main functional cells in bone formation, and their formation mainly includes the proliferation of pre-osteoblasts, the production of bone matrix, and the final differentiation of osteoblasts. The MC3T3-E1 cell line is a commonly used cell model for studying osteogenic activity. Osteoblast differentiation mainly includes three processes: proliferation, differentiation, and mineralization, specifically manifested as an increase in the number of pre-osteoblasts, the differentiation of pre-osteoblasts into mature osteoblasts, and the formation of new bone matrix. Alkaline phosphatase (ALP), as a specific protein associated with osteoblast phenotype, can be detected during cell proliferation and matrix maturation and is a commonly used early marker of osteoblast differentiation. Therefore, the osteogenic activity of the Cistanche deserticola extract prepared in Examples 1-3 was compared by detecting its effects on the proliferation of MC3T3-E1 cells and ALP activity. 1.1 Cell proliferation experiment MC3T3-E1 cells were resuscitated and passaged. After the cells were stabilized, they were digested and collected by centrifugation. The cell density was adjusted to 5 x 10⁶ cells per 100 μL of cell suspension.3 Cells were seeded into 96-well plates. After overnight culture, the culture medium was replaced with complete medium containing 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL of Cistanche deserticola extract prepared in Examples 1-3, or 0.1 M estradiol (positive control) or culture medium (blank control), and cultured for another 48 h. MTT stock solution (10%-20% of the culture medium volume in each well) was added to each well under dark conditions, and the plates were incubated in a CO2 incubator for 4 h. After incubation, the culture medium was removed, and 200 μL of DMSO was added to dissolve formazan. The optical density (OD) of the wells was measured at 490 nm using a microplate reader. The test results of Example 1 are shown below. Figure 1 The test results for Example 2 are shown below. Figure 2 The test results for Example 3 are shown below. Figure 3 .

[0058] Cell proliferation rate (%) = (OD (Examples 1-3) - OD (blank control)) / OD (blank control) × 100%.

[0059] Depend on Figure 1-3 It was found that the positive control promoted the proliferation of MC3T3-E1 cells. Example 1 promoted the proliferation of MC3T3-E1 cells at concentrations of 6.25, 12.5, and 25 μg / mL, with the proliferation rate at 12.5 μg / mL being 1.4 times that of the positive control, but the difference was not statistically significant (P > 0.05). Example 2 promoted the proliferation of MC3T3-E1 cells at concentrations of 6.25, 12.5, 25, and 50 μg / mL, with the proliferation-promoting effect at concentrations of 12.5, 25, and 50 μg / mL being superior to the positive control (P < 0.01 or P < 0.001), with proliferation rates 2.55, 3.46, and 2.59 times that of the positive control, respectively. Example 3 promoted the proliferation of MC3T3-E1 cells at concentrations of 6.25, 12.5, 25, and 50 μg / mL, with the proliferation rate at concentrations of 12.5, 25, and 50 μg / mL being significantly higher than that of the positive control (P < 0.01 or P < 0.001). At a concentration of μg / mL, the proliferation-promoting effect was superior to the positive control (P<0.01 or P<0.001), with proliferation rates 1.40, 1.76, and 0.93 times that of the positive control, respectively. This indicates that Example 2 is more effective in promoting the proliferation of MC3T3-E1 cells.

[0060] 1.2 ALP Activity Assay ALP activity was detected using an ALP detection kit. The stock solution concentration of β-nitrophenol was 10 mM, and the standard curve concentration gradient was: 0 mM, 0.02 mM, 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM, 0.2 mM, and 0.25 mM, diluted serially with detection buffer. 49 μL of detection buffer, 1 μL of protein extracted from MC3T3-E1 cells as described in section 1.1, and 50 μL of chromogenic substrate (added last) were added sequentially to the wells of a 96-well plate. The plate was incubated in the dark for 30 min, and the reaction was terminated by adding 100 μL of stop solution. The absorbance was measured at 405 nm using a microplate reader. The molar concentration of β-nitrophenol corresponding to each protein group was calculated from the standard curve, and ALP activity was calculated. The test results of Example 1 are shown below. Figure 4 The test results for Example 2 are shown below. Figure 5 The test results for Example 3 are shown below. Figure 6 .

[0061] ALP activity (U / L) = relative molar concentration of protein × 1000 × 100 ÷ 30 (1000 represents the conversion of moles, 100 represents the total volume of the reaction system, and 30 represents the reaction time of 30 min.) ALP is an early marker of MC3T3-E1 cell differentiation. Figure 4-6 It was found that the positive control promoted ALP activity in MC3T3-E1 cells. Example 1 had no effect on promoting ALP activity; Example 2 promoted ALP activity at concentrations of 12.5, 25, and 50 μg / mL, with the 25 and 50 μg / mL groups showing significant differences compared to the blank control (P < 0.01); Example 3 promoted ALP activity at concentrations of 12.5 and 25 μg / mL (P < 0.001), but the effect of increasing ALP activity decreased with increasing sample concentration. This indicates that only Examples 2 and 3 can promote increased ALP activity in MC3T3-E1 cells, which is beneficial for osteoblast differentiation.

[0062] In summary, only Example 2 showed a positive correlation between dose and the effects of concentrations of 12.5 and 25 μg / mL on promoting MC3T3-E1 cell proliferation and ALP activity. Example 3 showed a positive correlation between dose and the effects of concentrations of 12.5 and 25 μg / mL on promoting cell proliferation activity, but a negative correlation between dose and the effects on ALP activity. Comparatively, the Cistanche deserticola extract of Example 2 is more suitable for improving bone mineral density.

[0063] 1.3 Effect of Cistanche deserticola extract in Example 2 on alizarin red staining of osteoblast mineralization Based on the clear evidence that Example 2 has the best activity and better dose-relativity compared to Example 1 and Example 3, the effect of Example 2 was further investigated through osteoblast mineralization experiments.

[0064] MC3T3-E1 cells were cultured for 72 h and then treated with drugs (12.5, 25, and 50 μg / mL), and incubated at 37 ℃ for 14 days (depending on the formation of calcium nodules), with the medium changed every 2 days. After incubation, alizarin red staining was performed. The cell culture medium in each well was removed, and 300 μL of 10% (v / v) neutral formalin was added to each well for fixation for 30 min. The cells were then rinsed three times with ultrapure water, and stained with 300 μL of 0.1% (w / v) alizarin red staining solution for 30 min (at room temperature, protected from light). The cells were then washed with ultrapure water until no red color remained. Images were taken under a microscope for archiving, and whole-well images were also taken with a camera. After staining, 400 μL of 10% (w / v) hexadecylpyridine solution was added to each well, and the plate was incubated on a shaker for 1 h until the calcium nodules were completely dissolved. Then, 100 μL of the solution was aspirated into a 96-well plate, and the absorbance was measured at 562 nm using a microplate reader to calculate the mineralization rate. See the image for the actual test sample. Figure 7 The test results are shown below. Figure 8 .

[0065] Mineralization rate (%) = (OD (Example 3) - OD (blank control)) / OD (blank control) × 100%.

[0066] Figure 7 Alizarin Red staining results showed that, compared with the blank control, the number of calcified nodules in cells increased sequentially at concentrations of 12.5, 25, and 50 μg / mL in Example 2. Figure 8 Quantitative results showed that Example 2 promoted osteogenic mineralization in a concentration-dependent manner at concentrations of 12.5, 25, and 50 μg / mL, with the most significant effect at 50 μg / mL, showing a highly significant difference compared to the positive control (P < 0.001). Calcified nodules represent the final stage of osteoblast differentiation, and this result further confirms the bone mineral density-enhancing effect of Example 2, indicating that it can exert a stable effect on increasing bone mineral density. Furthermore, Example 2 showed better efficacy than the positive control estradiol, suggesting its application value in products for improving bone mineral density.

[0067] The effect of Cistanche deserticola extract in Example 2 on upregulating the expression of osteoblast differentiation-related genes induced MC3T3-E1 osteoblast differentiation. After 72 h of cell culture, Cistanche deserticola extract from Example 2 (12.5, 25, 50 μg / mL) was added, and the cells were incubated at 37 ℃ for 3 days, with the medium changed every 2 days (depending on the expression time of different genes). After the drug administration, RNA was extracted according to the kit method, and the RNA sample concentration was detected. The total volume of the reverse transcription PCR system was 20 μL (RNA, RNase-free ddH2O, and 2 × Hifair SuperMix). The RNA loading volume was 800 ng. The volume to be added was calculated based on the RNA concentration, and then RNase-free ddH2O was added to make up to 10 μL. 10 μL of 2 × Hifair SuperMix was added to prepare the reverse transcription reaction mixture, which was immediately placed in a thermal cycler for reverse transcription (program: 25 ℃, 5 min; 42 ℃, 30 min; 85 ℃, 5 min; 4 ℃, permanent). After completion, the samples were stored at -80°C for later use. The relative expression levels of Runx2 mRNA, Osx mRNA, Opn mRNA, and Ocn mRNA were quantified using real-time quantitative polymerase chain reaction. The test results are shown below. Figure 9-12 .

[0068] Runx2 mRNA and Osx mRNA are downstream regulatory signals in the BMP signaling pathway, both of which can induce osteoblast differentiation. Runx2 mRNA plays an important role in the initial directed differentiation stage of progenitor cells, while Osx mRNA plays a role in the terminal stage of this process. OPN mRNA and OCN mRNA are important indicators of osteoblast activity and tissue mineralization. Figure 9-12 The results showed that the Cistanche deserticola extract of Example 2 could increase the relative expression levels of Runx2 mRNA, Osx mRNA, Opn mRNA, and Ocn mRNA, indicating that it stimulated osteoblast-specific transcription factors Runx2 mRNA and Osx mRNA, enhanced osteoblast differentiation, and promoted the expression of osteoblast-specific markers OPN mRNA and OCN mRNA, thereby promoting bone formation.

[0069] Example 2 demonstrates the effect of Cistanche deserticola extract on upregulating the expression of key genes in the BMP / Smad pathway. The experimental method was the same as in Example 2, and the relative expression level of Bmp2 mRNA was detected using qRT-PCR. The test results are shown below. Figure 13 .

[0070] The BMP pathway not only regulates osteoblast differentiation but also significantly impacts new bone formation. BMP, a member of the TGF-β family, is a cysteine-containing acidic glycoprotein; BMP2 is one of its crucial subtypes. BMP2 influences the proliferation and differentiation of mesenchymal stem cells, promoting their transformation into mature osteoblasts and further facilitating their differentiation into osteocytes. It is currently recognized as the most potent growth factor for promoting allogeneic osteogenic growth in vitro. During osteogenic differentiation, BMP2 stimulates osteoblast-specific transcription factors such as Runx2 mRNA and Osx mRNA, thereby enhancing osteoblast differentiation. Figure 13 The results showed that the Cistanche deserticola extract of Example 2 could increase the relative expression level of Bmp2 mRNA, indicating that it enhances osteoblast differentiation and promotes new bone formation by activating the BMP / Smad pathway.

[0071] Example 2 demonstrates the effect of Cistanche deserticola extract on upregulating the expression of key genes in the Wnt / β-catenin pathway. The experimental method was the same as in Example 2, and the relative expression levels of Lrp5 mRNA, Wnt3α mRNA, and Lrp6 mRNA were detected using qRT-PCR. The test results are shown below. Figure 14-16 .

[0072] The Wnt / β-catenin pathway is closely related to osteoblast proliferation, differentiation, bone formation, and the treatment of osteoporosis. It positively regulates bone mass by stimulating an increase in osteoblast number and reducing the apoptosis rate of osteoblasts and osteocytes. When Wnt is active, extracellular Wnt ligand proteins bind to the seven-transmembrane receptors of coiled protein on the cell surface and LRP5 mRNA and LRP6 mRNA, thereby activating signal transduction. Wnt3a mRNA can activate β-catenin expression, and the level of β-catenin determines the expression of downstream transcription factors. For example, the Wnt / β-catenin pathway promotes osteogenic formation through the RUNX2 gene. Figure 14-16 The results showed that the Cistanche deserticola extract of Example 2 could increase the relative expression levels of Lrp5 mRNA, Lrp6 mRNA, and Wnt3α mRNA, indicating that it can promote osteoblast differentiation and bone formation by regulating Lrp5 mRNA, Lrp6 mRNA, and Wnt3α mRNA to activate Wnt / β-catenin pathway signaling and affect the expression of downstream transcription factors.

[0073] The effect of Cistanche deserticola extract in Example 2 on the upregulation of key proteins related to osteoblast differentiation was investigated using Western blotting (WB) to detect the expression of Osx and RUNX2 proteins. Total protein was extracted from each group of cells, and the protein content was measured. The protein concentration was diluted to a uniform level, and the mixture was vortexed at a ratio of V protein solution: V loading buffer = 4:1, and heated at 100°C for 5 min. Samples were added (35 μg of phosphorylated protein and 20 μg of other proteins), and markers were added to both sides of the wells. Electrophoresis was performed at a constant voltage of 80 V for 45 min, followed by 60 min at 120 V. The PVDF membrane was activated in anhydrous methanol for 90 s. Protein transfer was performed using the "filter paper-gel-PVDF membrane-filter paper" procedure. The electrophoresis tank was then filled with pre-cooled transfer buffer, and the entire transfer apparatus was placed in an ice-water bath for 90 min at a constant voltage of 100 V. PVDF membranes were blocked in 5% BSA solution for 60 min, then placed in primary antibody solutions of corresponding dilutions for each protein and incubated overnight at 4 °C. Protein bands incubated with primary antibody were washed with TBST, followed by incubation at room temperature for 2 h with the corresponding secondary antibody solutions. The bands were washed again with TBST. The bands were then immersed in ECL chemiluminescence solution for 30 s and images of each protein band were acquired using a chemiluminescence imager. Protein expression levels were quantified using ImageJ software based on grayscale values. The test results are shown below. Figure 17-18 .

[0074] Figure 17-18 The results showed that the Cistanche deserticola extract of Example 2 could increase the relative expression levels of Osx and RUNX2 proteins, indicating that it can regulate Osx and RUNX2 at both the transcriptional and post-translational levels, thereby promoting osteoblast differentiation.

[0075] The above activity experiments showed that the Cistanche deserticola extract of Example 2 had the best effect on promoting MC3T3-E1 cell proliferation, and only the Cistanche deserticola extract of Example 2 showed the same dose-related effect in promoting cell proliferation and ALP activity, possessing the best effect in promoting cell proliferation and differentiation. Furthermore, the Cistanche deserticola extract of Example 2 was superior to estradiol in promoting MC3T3-E1 cell proliferation and mineralization, and its effect in promoting MC3T3-E1 cell differentiation was comparable to estradiol, suggesting that it has the best effect in improving bone mineral density.

[0076] Chemical Composition Test 6.1 Determination of Phenylehnic Acid Glycoside Content: Referring to the method under the "Determination of Content in Cistanche deserticola" section of the Chinese Pharmacopoeia, the contents of echinacoside and verbascoside in Examples 1-3 were detected. The test results are shown in Table 1. From the total amounts of echinacoside, verbascoside, and phenylethanoid glycosides, the same trend was observed: Example 3 > Example 1 > Example 2. Furthermore, the verbascoside content in Example 3 was 3.85 times and 4.27 times that in Examples 1 and 2, respectively. Based on the combined results of the cell proliferation and ALP activity experiments and content determination, it can be concluded that echinacoside and verbascoside cannot directly determine the effect of Cistanche deserticola extract in promoting osteoblast proliferation and differentiation.

[0077] Table 1

[0078] 6.2 Other chemical composition analysis: The chemical composition of the Cistanche deserticola extracts of Examples 1-3 were compared by UPLC / Q-TOF / MS. The test results of the response values ​​of each component are shown in Table 2.

[0079] As shown in Table 2, besides echinacoside and verbascoside, Example 2 also contains other shikimic acid and phenylpropionic acid components, as well as alkaloid components. Compared with Examples 1 and 3, Example 2 has 11 other shikimic acid and phenylpropionic acid components with the highest response: isostansinic acid B, p-hydroxyphenylpropionic acid, (2R)-2-[(E)-3-[3-[(1R)-1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-4-yl]prop-2-enoyl]oxy-3-(3,4-dihydroxyphenyl)propanoic acid, benzoylacetone, epieugenol 4'-O-β-D-glucoside, myricetin, pyrethroid phenylethanol glycoside A1, 3,4-dimethylbenzoic acid, Robustaside D, 7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2S,3R,4S,5R)-3,4,5-trihydroxytetrahydropyran-2-yl]oxymethyl]tetrahydropyran-2-yl]oxychromen-2-one; 5 alkaloids: limonene, hydrocortisone, thymine, gelseminine, yohimbine.

[0080] Based on the comprehensive comparison of cell proliferation promotion and ALP activity, as well as the results of phenylethanoid glycoside content determination and chemical composition analysis, it can be seen that phenylethanoid glycosides (echinacoside, verbascoside), shikimic acid, phenylpropionic acid, alkaloids, and terpenoids together constitute the specific composition of the Cistanche deserticola extract in Example 2, which makes it exhibit the same dose-dependent relationship (12.5-25 μg / mL) in promoting cell proliferation, differentiation, and mineralization, and can stably exert the effect of increasing bone density.

[0081] Table 2 Response values ​​of each component

[0082] This invention, through the process described in Example 2, yields a Cistanche deserticola extract with a total content of echinacoside and verbascoside >25%, rich in shikimic acid, phenylpropionic acid, and alkaloids, enabling it to stably improve bone mineral density. Within a concentration range of 12.5-25 μg / mL, its effects on promoting MC3T3-E1 cell proliferation, differentiation, and mineralization all increase with increasing dosage, maintaining a similar dose-dependent relationship among the three effects. This demonstrates a better and more stable effect in preventing osteoporosis and improving bone mineral density, making it suitable for use in products related to improving bone mineral density.

Claims

1. A method for preparing an extract of Cistanche to improve bone density, characterized in that, The method comprises the following steps: The tuberose Cistanche is crushed to obtain tuberose Cistanche coarse powder; The tuberose Cistanche coarse powder is extracted with distilled water, filtered, centrifuged, concentrated to obtain tuberose Cistanche concentrated solution; The tuberose Cistanche concentrated solution is extracted, the lower layer extract is concentrated and impurities are removed, and the sample solution is obtained by dilution; The sample solution is post-processed, concentrated and dried to obtain Cistanche extract.

2. The preparation method of the Cistanche extract for improving bone density according to claim 1, characterized in that, The mass-volume ratio of the tuberose Cistanche coarse powder to distilled water is 1: (15-25).

3. The preparation method of the Cistanche extract for improving bone density according to claim 1, characterized in that, The temperature of the distilled water extraction is 60-100℃.

4. The preparation method of the Cistanche extract for improving bone density according to claim 1, characterized in that, When the tuberose Cistanche concentrated solution is extracted, the volume ratio of the tuberose Cistanche concentrated solution to the extractant is 1: (1-5).

5. The method for preparing the Cistanche deserticola extract for improving bone density according to claim 4, characterized in that, The extractant comprises at least one of petroleum ether, ethyl acetate, n-hexane or cyclohexane.

6. The preparation method of the Cistanche extract for improving bone density according to claim 1, characterized in that, The post-processing process of the sample solution is as follows: the sample solution is passed through a macroporous resin, and then eluted with distilled water and an ethanol solution in sequence.

7. The preparation method of the C. ferum extract for improving bone density according to claim 6, wherein, The volume ratio of the distilled water to the macroporous resin is (5-10): 1, and the volume ratio of the ethanol solution to the macroporous resin is (3-9):

1.

8. A Cistanche extract for increasing bone density, characterized in that, The Cistanche extract is prepared by the preparation method of any one of claims 1-7.

9. The extract of C. monnieri of claim 8, wherein, The total content of echinacoside and verbascoside in the Cistanche extract is 25-30wt%, not including 25wt%.

10. Use of an extract of Cistanche according to any one of claims 8-9 for increasing bone density, characterized in that, The Cistanche extract is applied to prepare a product for improving bone density, and the concentration of the Cistanche extract in the product for improving bone density is 12.5-25μg / mL.

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