Abstract

The present invention discloses a Cistanche extract for improving bone mineral density (BMD). In the extract, the total content of echinacoside and acteoside (verbascoside) is > 25 wt%. The extract is prepared by performing distilled-water extraction of crude powder of Cistanche tubulosa, followed by filtration, centrifugation of the filtrate, concentration, liquid–liquid extraction, concentration with impurity removal, dilution, post-treatment, and then final concentration and drying to obtain the Cistanche extract.

 

 

Cistanche Extract Process

In the Cistanche extract for improving bone mineral density according to the invention, the total content of echinacoside and acteoside is 25–30 wt% (excluding 25 wt%), and the extract is also rich in shikimic acid as well as phenylpropionic-acid-type components and alkaloid-type components, enabling it to stably exert a bone-density-improving effect.

 

Claims

1. Preparation Method

A method for preparing a Cistanche extract for improving bone mineral density, characterized in that it comprises the following steps:

crushing Cistanche tubulosa to obtain crude powder of Cistanche tubulosa;

adding distilled water to the crude powder of Cistanche tubulosa for extraction, filtering, centrifuging the filtrate, and concentrating to obtain a concentrated solution of Cistanche tubulosa;

extracting the concentrated solution of Cistanche tubulosa, concentrating the lower extraction phase to remove impurities, and diluting to obtain a loading solution;

post-treating the loading solution, followed by concentration and drying to obtain the Cistanche extract.

2. Ratio of Material to Water

The method according to Claim 1, wherein the mass-to-volume ratio of the crude powder of Cistanche tubulosa to distilled water is 1 : (15–25).

3. Extraction Temperature

The method according to Claim 1, wherein the distilled-water extraction temperature is 60–100°C.

4. Ratio During Liquid–Liquid Extraction

The method according to Claim 1, wherein during extraction of the concentrated solution, the volume ratio of the concentrated solution of Cistanche tubulosa to extraction solvent is 1 : (1–5).

5. Extraction Solvent

The method according to Claim 4, wherein the extraction solvent comprises at least one of petroleum ether, ethyl acetate, n-hexane, or cyclohexane.

6. Post-Treatment Process

The method according to Claim 1, wherein the post-treatment of the loading solution comprises: passing the loading solution through a macroporous resin, and then eluting sequentially with distilled water and an ethanol solution.

7. Elution Volumes

The method according to Claim 6, wherein the volume ratio of 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. The Extract

A Cistanche extract for improving bone mineral density, characterized in that it is prepared according to any one of Claims 1–7.

9. Active Marker Content

The Cistanche extract according to Claim 8, wherein the total content of echinacoside and acteoside is 25–30 wt%, excluding 25 wt%.

10. Application

Use of the Cistanche extract according to any one of Claims 8–9 in preparing a product for improving bone mineral density, wherein the concentration of the Cistanche extract in the product is 12.5–25 μg/mL.

 

Technical Field

[0001] The present invention relates to the field of medical formulations, and specifically relates to a Cistanche extract for improving bone mineral density, its preparation method, and applications.

Background

[0002] Cistanche refers to the dried fleshy stem with scale leaves of the Orobanchaceae plants Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schenk) Wight. Cistanche has multiple activities such as immune regulation, anti-inflammatory, anti-fatigue, antioxidant, neuroprotective, and reproductive system support.

Phenylethanoid glycosides (PhGs)-with echinacoside and acteoside (verbascoside) being the major PhGs in Cistanche-are important constituents. Echinacoside has been demonstrated to have an effect on improving bone mineral density. However, we found that the bone-density-improving effect of Cistanche extracts is not solely correlated with echinacoside content. Simply controlling echinacoside content cannot ensure that the extract will stably exert a bone-density-improving effect. Therefore, it is crucial to develop a Cistanche extract and a preparation method that can stably deliver the desired bone-density-supporting effect.

[0003] Chinese invention patent application CN102441040A discloses a preparation method for total phenylethanoid glycosides from Cistanche via pulverization, extraction, macroporous resin adsorption, elution, concentration, etc. Under weakly acidic conditions (pH 2–4), the stability of Cistanche phenylethanoid glycosides is significantly improved, and the resulting total glycosides contain echinacoside and acteoside in a combined proportion > 90%. However, it does not verify whether the product can stably improve bone mineral density.

Chinese invention patent application CN105111255A discloses a method for extraction and separation of echinacoside and acteoside from Cistanche, using 75% ethanol as the extraction solvent, AB-8 macroporous resin as the packing material, and 55% ethanol elution, yielding a Cistanche extract with echinacoside > 25%, acteoside > 10%, and total PhGs > 80%. However, it may still not guarantee a stable bone-density-improving effect.

 

Summary of the Invention

[0004] To develop a Cistanche extract and preparation method that can stably support improved bone mineral density, the invention provides:

First Aspect: Preparation Method

A preparation method for a Cistanche extract for improving bone mineral density, comprising:

crushing Cistanche tubulosa to obtain crude powder;

extracting the crude powder with distilled water, filtering, centrifuging, and concentrating to obtain a concentrated solution;

performing extraction; concentrating the lower phase to remove impurities; diluting to obtain a loading solution;

post-treating the loading solution, concentrating, and drying to obtain the Cistanche extract.

[0005] In one embodiment, the crude powder is passed through a 40-mesh sieve.

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

[0007] In one embodiment, the mass-to-volume ratio is 1 : 20.

[0008] In one embodiment, the extraction temperature is 60–100°C.

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

[0010] In one embodiment, extraction is performed 1–2 times, 1 hour each time.

[0011] In one embodiment, filtration is performed through an 80-mesh filter.

[0012] In one embodiment, centrifugation speed is 3000–4000 r/min and time is 3–9 min.

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

[0014] In one embodiment, after centrifugation, the filtrate is concentrated at 70°C to 18%–28% of the initial volume.

[0015] In one embodiment, during extraction of the concentrated solution, the volume ratio of concentrated solution to extraction solvent is 1 : (1–5).

[0016] In one embodiment, the ratio is 1 : 3.

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

[0018] In one embodiment, extraction is performed sequentially using petroleum ether followed by ethyl acetate.

[0019] In one embodiment, "concentration with impurity removal" means concentrating the lower extraction phase to a paste, dissolving it in 300 mL of 50 wt% aqueous ethanol to remove impurities, and adjusting to 1.0–1.5 g crude herb per mL as the loading solution.

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

[0021] In one embodiment, post-treatment comprises passing the loading solution through macroporous resin, and then eluting sequentially with distilled water and an ethanol solution.

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

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

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

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

[0026] In one embodiment, the ethanol solution is 80% (w/w) aqueous ethanol.

Second Aspect: The Extract

[0027] The invention further provides a Cistanche extract prepared by the above method.

[0028] In one embodiment, the total content of echinacoside and acteoside is 25–30 wt%, excluding 25 wt%.

Third Aspect: Use

[0029] The invention further provides use of the Cistanche extract in preparing products for improving bone mineral density, where the extract concentration in the product is 12.5–25 μg/mL.

 

Advantages / Beneficial Effects

[0030] Compared with the prior art, the invention has the following beneficial effects:

The Cistanche extract contains 25–30 wt% total echinacoside and acteoside, and is rich in shikimic acid, phenylpropionic-acid-type components, alkaloids, and fatty acids, enabling it to stably support improvement of bone mineral density.

Within the 12.5–25 μg/mL concentration range, the extract promotes proliferation of MC3T3-E1 cells with excellent proliferative effects.

The extract shows the same dose-related trend in promoting cell proliferation and ALP activity, and can stably promote proliferation and differentiation. In promoting osteoblast mineralization, it shows a better effect than the positive control 17β-estradiol.

The extract stimulates osteoblast-specific transcription factors Runx2 and Osx, enhances osteoblast differentiation, and promotes expression of osteogenic markers OPN and OCN, thereby promoting bone formation. It also increases Bmp2 mRNA expression to enhance osteoblast differentiation and new bone formation by activating the BMP/Smad pathway; furthermore, by regulating Lrp5 mRNA, Lrp6 mRNA, and Wnt3α mRNA, it activates Wnt/β-catenin signaling, affects downstream transcription factor expression, and promotes osteoblast differentiation and bone formation.

The preparation method yields an extract containing phenylethanoid glycosides (echinacoside and acteoside), shikimic acid and phenylpropionic acids, alkaloids, and fatty acids. These components together allow the extract to exhibit consistent dose-dependence in proliferation, differentiation, and mineralization, enabling stable bone-density-supporting effects.

 

Brief Description of the Drawings

[0035] Fig. 1: Effect of extract (Example 1) on MC3T3-E1 cell proliferation
Fig. 2: Effect of extract (Example 2) on MC3T3-E1 cell proliferation
Fig. 3: Effect of extract (Example 3) on MC3T3-E1 cell proliferation
Fig. 4: Effect of extract (Example 1) on ALP activity
Fig. 5: Effect of extract (Example 2) on ALP activity
Fig. 6: Effect of extract (Example 3) on ALP activity
Fig. 7: Photos of Alizarin Red staining of osteoblast mineralization (Example 2)
Fig. 8: Quantitative results of osteoblast mineralization (Example 2)
Fig. 9–12: Effects of Example 2 on Runx2, Osx, Opn, Ocn mRNA expression
Fig. 13: Effect of Example 2 on Bmp2 mRNA expression (BMP/Smad)
Fig. 14–16: Effects of Example 2 on Lrp5, Wnt3α, Lrp6 mRNA expression (Wnt/β-catenin)
Fig. 17–18: Effects of Example 2 on Osx and RUNX2 protein expression

 

Detailed Description / Embodiments

Example 1

[0036] A preparation method comprises:

crushing Cistanche tubulosa, passing through a 40-mesh sieve to obtain 200 g crude powder;

extracting with distilled water at 5× mass (as described), extracting at 75°C for 2 times, 1 h each, concentrating at 70°C to a concentration ratio of 1 : 2.2 to obtain a concentrated solution;

adding absolute ethanol to the concentrated solution for graded ethanol precipitation with final ethanol mass concentrations of 20%, 40%, 60%, 80%, precipitating at 5°C for 24 h, collecting the supernatant after 80 wt% ethanol precipitation;

concentrating and drying at 70°C to obtain the Cistanche extract, with a yield of 20.75%.

Example 2

[0037] A preparation method comprises:

crushing Cistanche tubulosa to obtain 240 g crude powder;

extracting with distilled water, filtering, centrifuging, and concentrating to obtain a concentrated solution;

extracting the concentrated solution; concentrating the lower phase to remove impurities; diluting to obtain a loading solution;

post-treating the loading solution; concentrating and drying at 70°C to obtain the extract, with a yield of 19.69%.

[0038] The crude powder passes through a 40-mesh sieve.
[0039] The mass-to-volume ratio of crude powder to distilled water is 1 : 20.
[0040] Extraction temperature is 80°C.
[0041] Extraction is performed 1–2 times, 1 h each.
[0042] Filtration is through an 80-mesh filter.
[0043] Centrifugation is 3500 r/min for 5 min.
[0044] Concentration after centrifugation: concentrate at 70°C to 20% of initial volume.
[0045] During extraction, the volume ratio of concentrated solution to solvent is 1 : 3.
[0046] Extraction is performed sequentially with petroleum ether and ethyl acetate.
[0047] The lower phase is concentrated to a paste and dissolved in 300 mL of 50 wt% aqueous ethanol to remove impurities.
[0048] Loading solution concentration: 1.0 g crude herb/mL water.
[0049] Post-treatment: pass through macroporous resin, elute with distilled water then ethanol solution.
[0050] Flow rate through resin: 3 mL/min.
[0051] Resin: AB-8, resin volume 0.93 L.
[0052] Distilled water-to-resin volume ratio: 7 : 1. Ethanol-solution-to-resin ratio: 6 : 1.
[0053] Ethanol solution: 80% (w/w) aqueous ethanol.

 

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Example 3

[0053] A preparation method comprises:

crushing Cistanche tubulosa, passing through a 40-mesh sieve to obtain 120 g crude powder;

extracting with 12× mass of 50 wt% aqueous ethanol, extracting at 70°C, 2 times, 1 h each, concentrating at 70°C to a ratio of 1 : 2.5 to obtain a concentrated solution;

adding water to adjust concentration to 0.2 g crude herb/mL water, centrifuging at 3500 r/min for 5 min, taking the supernatant as the loading solution;

passing through macroporous resin; sequentially eluting with distilled water and ethanol solution; collecting the ethanol elution and concentrating/drying at 70°C to obtain the extract with a yield of 24.30%.

[0054] Resin: HPD750, resin volume 0.93 L.
[0055] Distilled water-to-resin ratio: 3 : 1. Ethanol-solution-to-resin ratio: 4 : 1.
[0056] Ethanol solution: 30% (w/w) aqueous ethanol.

 

Performance Testing

1.1 Cell Proliferation Assay

[0057] Osteoblasts are the main functional cells responsible for bone formation, involving proliferation of pre-osteoblasts, production of bone matrix, and final differentiation. The MC3T3-E1 cell line is commonly used to study osteogenic activity. Osteoblast differentiation involves proliferation, differentiation, and mineralization-manifested as increased pre-osteoblast number, maturation into osteoblasts, and formation of new bone matrix.

Alkaline phosphatase (ALP) is an osteoblast phenotype-related protein detectable during cell proliferation and matrix maturation and is a commonly used early marker of osteoblast differentiation.

Therefore, by evaluating MC3T3-E1 proliferation and ALP activity, the osteogenesis-promoting activity of the extracts prepared in Examples 1–3 was compared.

MC3T3-E1 cells were revived and passaged. After stabilization, cells were digested, centrifuged, and collected. Cell density was adjusted so that each 100 μL contained 5×1035 \times 10^35×103 cells and was seeded into a 96-well plate. After overnight culture, the medium was replaced with complete medium containing Example 1–3 extracts at concentrations of 6.25, 12.5, 25, and 50 μg/mL, or containing 0.1 M estradiol (positive control), or medium alone (blank control), and cultured for 48 h. Under light-protected conditions, MTT stock solution (10%–20% of the well volume) was added, incubated for 4 h in a CO₂ incubator, medium removed, and 200 μL DMSO added to dissolve formazan. OD values were measured at 490 nm.

Results: Fig. 1 (Example 1), Fig. 2 (Example 2), Fig. 3 (Example 3).

[0058] Cell proliferation rate (%) = OD(Example 1–3)−OD(Blank)OD(Blank)×100%\frac{OD(\text{Example 1–3}) - OD(\text{Blank})}{OD(\text{Blank})} \times 100\%OD(Blank)OD(Example 1–3)−OD(Blank)​×100%

[0059] As shown in Fig. 1–3, the positive control promoted MC3T3-E1 proliferation. Example 1 promoted proliferation at 6.25, 12.5, and 25 μg/mL; the proliferation rate at 12.5 μg/mL was 1.4× that of the positive control, but without significant difference (P > 0.05). Example 2 promoted proliferation at 6.25, 12.5, 25, and 50 μg/mL; at 12.5, 25, and 50 μg/mL its effects were superior to the positive control (P < 0.01 or P < 0.001), with proliferation rates 2.55×, 3.46×, and 2.59× of the positive control. Example 3 promoted proliferation at 6.25, 12.5, 25, and 50 μg/mL; at 12.5, 25, and 50 μg/mL it was superior to the positive control (P < 0.01 or P < 0.001), with proliferation rates 1.40×, 1.76×, and 0.93× of the positive control. This indicates that Example 2 had the best effect on promoting MC3T3-E1 proliferation.

1.2 ALP Activity Assay

[0060] ALP activity was measured using an ALP assay kit. The stock concentration of p-nitrophenol was 10 mM, and the standard curve concentrations were 0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.2, and 0.25 mM, prepared by gradient dilution with detection buffer.

For each sample well in a 96-well plate, 49 μL detection buffer, 1 μL protein extracted from the treated MC3T3-E1 cells (from section 1.1), and 50 μL chromogenic substrate (added last) were added. The reaction proceeded for 30 min in the incubator protected from light, terminated by adding 100 μL stop solution, and absorbance measured at 405 nm. ALP activity was calculated from the standard curve.

Results: Fig. 4 (Example 1), Fig. 5 (Example 2), Fig. 6 (Example 3).

[0061] ALP activity (U/L) = (relative molar concentration of protein) × 1000 × 100 / 30
(1000 for molar unit conversion; 100 for total reaction volume; 30 for 30 min reaction time)

ALP is an early marker of MC3T3-E1 differentiation. Fig. 4–6 show that the positive control increased ALP activity. Example 1 did not promote ALP activity. Example 2 increased ALP activity at 12.5, 25, and 50 μg/mL; at 25 and 50 μg/mL differences vs. blank were significant (P < 0.01). Example 3 increased ALP activity at 12.5 and 25 μg/mL (P < 0.001), but with increasing concentration, the enhancement decreased. This indicates that only Examples 2 and 3 increased ALP activity and supported differentiation.

[0062] Overall, only Example 2 showed dose-positive correlation in both MC3T3-E1 proliferation and ALP activity at 12.5 and 25 μg/mL. Example 3 showed dose-positive correlation for proliferation but dose-negative correlation for ALP. Comparatively, Example 2 is more suitable for improving bone mineral density.

1.3 Mineralization (Alizarin Red Staining) of Example 2

[0063] Since Example 2 showed the best activity and dose correlation compared with Examples 1 and 3, mineralization experiments were conducted to further evaluate its effect.

[0064] MC3T3-E1 cells were treated after 72 h culture with Example 2 extract at 12.5, 25, and 50 μg/mL, incubated at 37°C for 14 days (based on calcium nodule formation), with medium changes every 2 days. Alizarin Red staining was performed: medium removed, cells fixed with 10% neutral formalin (300 μL/well) for 30 min, rinsed, stained with 0.1% Alizarin Red (300 μL/well) for 30 min (room temperature, protected from light), and washed until no red color remained. Images were taken. Then 10% cetylpyridinium chloride solution (400 μL/well) was added and shaken for 1 h to dissolve calcified nodules. The dissolved solution was transferred to a 96-well plate (100 μL/well) and OD measured at 562 nm to calculate mineralization rate. Photos: Fig. 7; quantitative results: Fig. 8.

[0065] Mineralization rate (%) = OD(Example 2)−OD(Blank)OD(Blank)×100%\frac{OD(\text{Example 2}) - OD(\text{Blank})}{OD(\text{Blank})} \times 100\%OD(Blank)OD(Example 2)−OD(Blank)​×100%

[0066] Fig. 7 shows that calcium nodule numbers increased sequentially with concentration vs. blank. Fig. 8 indicates that Example 2 promoted mineralization in a concentration-dependent manner at 12.5, 25, and 50 μg/mL, with the strongest effect at 50 μg/mL, significantly better than the positive control (P < 0.001). Because mineralized nodules represent the final stage of osteoblast differentiation, these results further confirm the bone-density-supporting effect of Example 2 and demonstrate its stable activity. Example 2 was superior to estradiol, indicating potential value for bone health products.

Gene Expression and Pathway Mechanism (Example 2)

[0067] Example 2 extract (12.5, 25, 50 μg/mL) was applied to MC3T3-E1 cells for 3 days. RNA extraction, quantification, reverse transcription (20 μL system; 800 ng RNA input), and qRT-PCR were conducted to quantify relative expression levels of Runx2 mRNA, Osx mRNA, Opn mRNA, and Ocn mRNA. Results: Fig. 9–12.

[0068] Runx2 and Osx are key downstream regulators in BMP signaling, inducing osteoblast differentiation. Runx2 is important in the early commitment stage, while Osx functions in terminal differentiation. OPN and OCN are important indicators for osteoblast activity and tissue mineralization. Fig. 9–12 show that Example 2 increased relative expression of these markers, supporting enhanced osteoblast differentiation and bone formation.

[0069] Example 2 upregulated Bmp2 mRNA (Fig. 13), indicating activation of the BMP/Smad pathway, which supports osteoblast differentiation and new bone formation.

[0071] Example 2 increased relative expression of Lrp5 mRNA, Lrp6 mRNA, and Wnt3α mRNA (Fig. 14–16), supporting activation of Wnt/β-catenin signaling, which is closely related to osteoblast proliferation, differentiation, and bone formation.

Protein Expression (Example 2)

[0073] Western blot analysis showed that Example 2 increased Osx and RUNX2 protein expression (Fig. 17–18), indicating regulation at both transcriptional and translational levels to promote osteoblast differentiation.

[0075] These activity tests show that Example 2 provided the best proliferation-promoting effect and was the only one with consistent dose correlation for both proliferation and ALP. Example 2 was also superior to estradiol in promoting MC3T3-E1 proliferation and mineralization, with comparable differentiation effects, suggesting optimal bone-density-supporting potential.

 

Chemical Composition Testing

6.1 Phenylethanoid Glycosides Content

[0076] With reference to the "Cistanche" assay method in the Chinese Pharmacopoeia, echinacoside and acteoside contents in Examples 1–3 were measured (results shown in Table 1). Based on echinacoside, acteoside, and total PhGs, the trend was consistent: Example 3 > Example 1 > Example 2, and acteoside in Example 3 was 3.85× and 4.27× that of Examples 1 and 2, respectively. Combining activity and content results indicates that echinacoside and acteoside alone do not directly determine the osteogenic effects.

 

Table 1. Contents of Echinacoside and Acteoside in Cistanche Extracts (%)

Sample (Example)	Echinacoside (%)	Acteoside / Verbascoside (%)	Total (%)
Example 1	28.68	1.63	30.31
Example 2	26.27	1.47	27.74
Example 3	30.39	6.27	36.66

 

6.2 Other Components (UPLC/Q-TOF/MS)

[0078] UPLC/Q-TOF/MS was used to compare chemical components among Examples 1–3. The response values are shown in Table 2.

[0079] Besides echinacoside and acteoside, Example 2 contained additional shikimic acid and phenylpropionic-acid-type components, and also alkaloids. Compared with Examples 1 and 3, Example 2 had 11 phenylpropionic/shikimic-acid-related components with the highest responses, including:

Table 2. UPLC/Q-TOF/MS Response Values of Key Components in Cistanche Extracts (Examples 1–3)

注：原表为"响应值(response value)";我保留为纯数值,不加单位(与图表一致).表内分为两组：Shikimic acid & phenylpropionic acid derivatives(莽草酸及苯丙酸类)与 Alkaloids(生物碱).

A. Shikimic acid & phenylpropionic acid derivatives

Compound	Example 1	Example 2	Example 3
Isodanphenolic acid B (异丹酚酸B)	1,514,590.932	5,985,000.378	5,279,315.212
p-Hydroxyphenylpropionic acid (对羟基苯丙酸)	2,897,237.374	6,144,507.902	1,139,582.918
Shikimic acid (莽草酸)	6,539,932.014	8,883,034.928	7,442,038.152
(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	1,514,590.932	5,985,000.378	5,279,315.212
Benzoylacetone (苯甲酰丙酮)	4,235,952.540	4,526,539.189	4,297,281.060
Epipinoresinol 4′-O-β-D-glucoside (表丁香树脂酚 4′-O-β-D-葡萄糖苷)	2,758,386.748	4,338,477.068	1,182,637.856
Yangmei Changshan glycoside* (杨梅常山苷)	713,259.014	9,202,359.822	841,791.016
Jionoside A1 (焦地黄苯乙醇苷A1)	2,184,856.679	3,736,929.302	3,098,713.743
3,4-Dimethylbenzoic acid (3,4-二甲基苯甲酸)	585,269.7562	1,386,349.28	1,400,433.204
Robustaside D	1,693,753.421	3,272,248.064	4,236,657.164
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	4,489,728.765	1,167,879.841	824,633.4

 

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B. Alkaloids

Compound	Example 1	Example 2	Example 3
Verticine (野百合碱)	1,306,481.28	524,919.97	4,057,704.757
Dihydrocoptisine (氢化可他宁碱)	6,406,217.365	9,533,850.521	4,649,410.535
Thymine (胸腺嘧啶)	2,658,397.084	2,715,160.269	554,956.4602
Gelsemine (钩吻碱)	3,698,263.919	1,554,195.385	5,144,366.562
Yohimbine (育亨宾)	3,929,806.438	1,286,932.427	9,431,924.132

isodanphenolic 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,

epipinoresinol 4′-O-β-D-glucoside,

myricetin-related glycoside (as listed),

jionoside 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.

It also contained 5 alkaloids, including:

verticine,

dihydrocoptisine (as listed),

thymine,

gelsemine-related alkaloid (as listed),

yohimbine.

[0080] Based on comparative results of proliferation/ALP activity, phenylethanoid glycoside assays, and chemical profiling, it can be concluded that phenylethanoid glycosides (echinacoside and acteoside), shikimic/phenylpropionic acids, alkaloids, and terpenoids jointly constitute the specific composition of the Example 2 extract, producing consistent dose-dependence (12.5–25 μg/mL) across proliferation, differentiation, and mineralization, enabling stable bone-density-supporting effects.

[0081] Table 2: response values of components.
[0082] The invention, through the process of Example 2, provides a Cistanche extract with total echinacoside + acteoside > 25% and enriched shikimic/phenylpropionic acids and alkaloids, allowing stable bone-density-supporting effects. Within 12.5–25 μg/mL, it promotes MC3T3-E1 proliferation, differentiation, and mineralization in a dose-enhancing manner with consistent dose correlation, supporting better and more stable bone health outcomes and enabling use in products related to improving bone mineral density.