A method for extracting and separating phenylethanoid glycoside from cistanche
By combining traditional pulverization with air jet milling as a pretreatment method, along with steam explosion, water extraction, enzymatic hydrolysis, multi-stage membrane filtration, and macroporous resin purification, the problem of low extraction rate and purity of phenylethanoid glycosides in Cistanche deserticola has been solved, achieving efficient extraction and separation and supporting its application in the food and pharmaceutical fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JINAN INST OF FRUIT PRODS CHINA GENERAL SUPPLY & MARKETING COOP
- Filing Date
- 2026-03-11
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies are difficult to efficiently extract and separate phenylethanoid glycosides from Cistanche deserticola, resulting in low extraction rates and low purity. In particular, the presence of impurities such as polysaccharides and tannins leads to low dissolution efficiency and high purification difficulty, which limits its application in the food and pharmaceutical fields.
A two-stage pulverization method combining traditional pulverization and airflow pulverization, along with steam explosion, water extraction, enzymatic hydrolysis, multi-stage membrane filtration, and macroporous resin purification, was employed to disrupt the cell structure of Cistanche deserticola and improve the dissolution rate and purity of phenylethanol glycosides.
The extraction rate of phenylethanol glycosides was increased to 62.13%, and the purity reached 75.6%, which significantly improved the extraction and separation effect of phenylethanol glycosides from Cistanche deserticola and supported its application in the food and pharmaceutical fields.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-extraction technology, and specifically relates to a method for extracting and separating phenylethanol glycosides from Cistanche deserticola. Background Technology
[0002] Cistanche deserticola, the dried fleshy stem with scales of the plant Cistanche deserticola in the Orobanchaceae family, has the effects of tonifying kidney yang, nourishing essence and blood, and moistening the intestines to relieve constipation. Traditionally, it is often used for symptoms such as kidney yang deficiency, deficiency of essence and blood, soreness and weakness of the waist and knees, weakness of muscles and bones, and constipation due to intestinal dryness. Modern pharmacological research has further proved that Cistanche deserticola has significant effects in many aspects such as anti-aging, improving learning and memory ability, and anti-Alzheimer's disease.
[0003] Phenylehnic acid glycosides, as the main active ingredients of Cistanche deserticola, not only exert antioxidant effects by scavenging free radicals and inhibiting lipid peroxidation, but also show significant activity in improving cognitive function, protecting nerve cells, regulating immunity, and anti-fatigue. Their related pharmacological mechanisms have been verified in in vitro cell experiments, animal models, and some clinical studies, providing an important material basis for the development of functional foods, health products, and innovative drugs.
[0004] Currently, research on the extraction and separation of phenylethanoid glycosides from Cistanche deserticola is relatively mature, mainly including water extraction, ultrasonic or microwave-assisted extraction, enzymatic hydrolysis, and supercritical fluid extraction. However, even so, on the one hand, the raw material of Cistanche deserticola is relatively hard and rich in impurities such as polysaccharides, tannins, cellulose, and lignin. The viscous matrix formed by polysaccharides can encapsulate phenylethanoid glycoside molecules, hindering the contact between the extraction solvent and the target component, thus reducing the dissolution efficiency of phenylethanoid glycosides. Furthermore, tannins may combine with phenylethanoid glycosides through hydrogen bonds and hydrophobic interactions to form insoluble complexes, further reducing the extractable amount. On the other hand, phenylethanoid glycosides are polar compounds, requiring the use of highly polar extraction methods. Extraction is performed using solvents, but these solvents also dissolve a large amount of impurities such as polysaccharides and low-molecular-weight sugars, resulting in high viscosity of the extract and a high purification load. If the polarity of the solvent is reduced, the extraction of phenylethanoid glycosides will be incomplete, making it difficult to achieve both "high extraction rate" and "high purity". In addition, the polarity and molecular weight range of polysaccharides and low-polarity phenolic impurities partially overlap with those of phenylethanoid glycosides. This results in similar adsorption-desorption characteristics of polysaccharides and phenylethanoid glycosides during column chromatography, making it difficult to achieve effective separation with a single adsorbent. Ultimately, the extraction rate and purity of phenylethanoid glycosides in Cistanche deserticola remain at a low level, which greatly limits the application and industrialization of phenylethanoid glycosides in the food and pharmaceutical fields. Summary of the Invention
[0005] To address the above technical problems, this invention proposes a method for extracting and separating phenylethanol glycosides from Cistanche deserticola.
[0006] The technical solution of this invention is:
[0007] A method for extracting and isolating phenylethanoid glycosides from Cistanche deserticola, comprising the following steps:
[0008] (1) Cistanche deserticola, dried, first pulverized by a pulverizer, then pulverized by air jet mill to obtain Cistanche deserticola powder;
[0009] (2) The Cistanche deserticola powder in (1) was subjected to steam explosion treatment. The conditions for steam explosion were: steam explosion pressure 0.5~1.5 MPa, steam explosion duration 50~90 s, and material-to-cavity ratio 1:1~3.
[0010] (3) After the steam explosion treatment in (2) is completed, add water to the obtained material, extract at 50~60℃ for 20~60min, centrifuge and filter, repeat 2~3 times, combine the filtrates to obtain water extract;
[0011] (4) Add 0.01% to 0.1% of the water extract by mass to the water extract of (3), and enzymatically hydrolyze at 50 to 60°C for 20 to 50 minutes to obtain the hydrolysate; the compound enzyme preparation is made by mixing pectinase and amylase in a mass ratio of 1:1 to 2.
[0012] (5) The enzymatic hydrolysate in (4) is subjected to multi-stage membrane filtration and then eluted with D-101 macroporous adsorption resin. The eluent is a mixture of 50%~70% ethanol and 0.1% acetic acid aqueous solution in a volume ratio of 6~8:3. The effluent is evaporated, concentrated and then freeze-dried to obtain phenylethanol glycoside powder.
[0013] In the above preparation method provided by the present invention, preferably, in (1) the Cistanche deserticola is dried at a temperature of 40~55°C, more preferably 50°C.
[0014] As a preferred embodiment, the Cistanche deserticola mentioned in (1) is Cistanche tubulosa.
[0015] Preferably, in (1), the primary crushing is to crush the Cistanche deserticola to at least 40 mesh using a crusher.
[0016] As a preferred embodiment, in (1), the airflow pulverization has a pulverization frequency of 30~50 Hz, more preferably 40 Hz, and a pulverizing gas pressure of 0.5~1.0 MPa, more preferably 0.6~0.9 MPa, and most preferably 0.8 MPa.
[0017] As a preferred option, the conditions for the steam explosion treatment described in (2) are: steam explosion pressure 0.8~1.2 MPa, steam explosion duration 60~80 s, and material chamber ratio 1:2.
[0018] More preferably, the conditions for the steam explosion treatment described in (2) are: steam explosion pressure 1.0 MPa, steam explosion duration 70 s, and material chamber ratio 1:2.
[0019] Preferably, the mass-to-volume ratio of the material to water in (3) is 1 g: 10~20 mL.
[0020] As a further preferred embodiment, the mass-to-volume ratio of the material to water in (3) is 1 g: 12~16 mL.
[0021] Preferably, in (4), a compound enzyme preparation is added to the aqueous extract of (3), wherein the compound enzyme preparation comprises 0.03% pectinase and 0.03% amylase based on the mass of the aqueous extract.
[0022] As a preferred embodiment, the specific operation of the multi-stage membrane filtration described in (5) is as follows: membrane filtration is performed sequentially using a 30 kDa spiral wound ultrafiltration membrane, a 200 Da nanofiltration membrane, and a 0.1~1 nm reverse osmosis membrane.
[0023] Preferably, the operating temperature for the multi-stage membrane filtration described in (5) is 50~60°C.
[0024] As a preferred embodiment, in (5) the SSD elution solution is a volume ratio of 60% ethanol to 0.1% acetic acid aqueous solution of 7:3.
[0025] The present invention has the following advantages and effects compared with the prior art:
[0026] (1) This invention improves the crushing method of Cistanche deserticola raw material. It adopts a two-stage crushing method that couples traditional crushing with airflow crushing for pretreatment, which can more thoroughly destroy the lignin, cellulose and other structures in Cistanche deserticola, so that the phenylethanoid glycosides encapsulated in them can be more fully dissolved. Then, Cistanche deserticola is treated in combination with two-stage crushing, steam explosion, water extraction and enzymatic hydrolysis, which breaks the binding of phenylethanoid glycosides by cell structure and polysaccharide molecules from multiple angles. The extraction rate is as high as 62.13%. Compared with the traditional simple one-stage crushing (extraction rate 17.6%) and the crushing pretreatment method that combines one-stage crushing and ultra-fine crushing (extraction rate 45.27%), the extraction rate of phenylethanoid glycosides is increased by 44.53% and 16.86% respectively, and is 8.47% higher than the extraction rate of ultrasonic-assisted extraction process (53.66%).
[0027] (2) In this invention, a multi-stage membrane is first used to pre-purify phenylethanol glycosides, and then a macroporous resin is used for secondary purification. The results show that the purification effects of different types of macroporous resins on phenylethanol glycosides are extremely different. For example, the purity after purification by X-5 macroporous resin is only 59.74%, while the purity after purification by D-101 macroporous resin is the highest, reaching 75.6%. Attached Figure Description
[0028] Figure 1 This is a comparative diagram showing the purification effects of different types of macroporous resins on phenylethanoid glycosides in Cistanche deserticola. Detailed Implementation
[0029] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.
[0030] Example 1
[0031] A method for extracting and separating phenylethanol glycosides from Cistanche deserticola, comprising the following steps:
[0032] (1) Place 300 g of Cistanche tubulosa in a constant temperature hot air drying oven and dry it at 50°C until constant weight. First, use a pulverizer for primary pulverization. The powder passes through a 40-mesh sieve. Then, perform secondary air jet pulverization. During air jet pulverization, the air jet pulverization frequency is 40 Hz and the pulverization gas pressure is 0.8 MPa. After air jet pulverization, Cistanche tubulosa powder is obtained.
[0033] (2) The powder of Cistanche tubulosa in (1) was subjected to steam explosion treatment. The conditions for steam explosion were: steam explosion pressure 1.0 MPa, steam explosion time 70 s, and material-to-cavity ratio 1:2.
[0034] (3) After the steam explosion treatment in (2) is completed, add 15 times the volume of water to the obtained material, extract at 55℃ for 30 min, centrifuge at 4000 r / min for 10 min and filter, repeat 3 times, combine the filtrates to obtain the water extract, the recovery rate of phenylethanol glycosides in the water extract is 58.65% and the purity is 30.69%;
[0035] (4) Add 0.03% pectinase and 0.03% amylase by mass of the aqueous extract to the aqueous extract of (3), and enzymatically hydrolyze at 50℃ for 30 min to obtain the enzymatic hydrolysate. The recovery rate of phenylethanol glycosides in the enzymatic hydrolysate is 60.33%, and the purity is 41.26%.
[0036] (5) The enzymatic hydrolysate in (4) was subjected to multi-stage membrane filtration. Specifically, it was first filtered using a 30 kDa spiral wound ultrafiltration membrane at an operating pressure of 0.8 MPa and a feed temperature of 50°C. Then, it was filtered using a 200 Da nanofiltration membrane at an operating pressure of 1.0 MPa and a feed temperature of 25°C. Finally, it was filtered using a 0.1 nm reverse osmosis membrane at an operating pressure of 2.2 MPa and a feed temperature of 25°C. The collected retentate was eluted using D-101 macroporous adsorption resin. The eluent was a 60% ethanol:0.1% acetic acid aqueous solution with a volume ratio of 7:3. The effluent was evaporated, concentrated, and then freeze-dried to obtain phenylethanol glycoside powder.
[0037] In this invention, the recovery rate of phenylethanol glycosides is calculated according to the following formula:
[0038] Recovery rate of phenylethanol glycosides (%) = C1V1 / C0V0 × 100%;
[0039] Wherein, C1 is the concentration of phenylethanol glycoside solution after treatment (mg / mL), V1 is the volume of phenylethanol glycoside solution after treatment (mL), C0 is the concentration of phenylethanol glycoside in the raw material solution (mg / mL), and V0 is the volume of the raw material solution (mL).
[0040] Extraction rate of phenylethanol glycosides (%) = CV / W × 100%;
[0041] Where C is the concentration calculated in the standard curve (mg / mL), V is the dilution volume of the test solution (mL), and W is the mass of Cistanche deserticola powder (mg).
[0042] The mass of the phenylethanol glycoside powder obtained in this example was 10.2 g, the recovery rate of phenylethanol glycoside was 90.37%, and the purity was 78.94%.
[0043] In this embodiment (5), the effects of different membrane devices on the purity and recovery rate of phenylethanol glycosides are shown in Table 1 below.
[0044] Table 1. Effects of multi-stage membrane purification on the purity and recovery rate of phenylethanoid glycosides Project name Enzymatic hydrolysate Ultrafiltration permeate Nanofiltration retentate Reverse osmosis retentate Phenylethanoid purity (%) 41.26 52.38 60.52 61.71 Phenylethanoid recovery (%) 60.33 69.74 82.96 84.45
[0045] Example 2
[0046] The difference from Example 1 is that the temperature during ultrafiltration in (5) was adjusted, with the feed liquid temperatures being 20°C, 30°C, 40°C, 50°C, and 60°C, respectively. The purity of phenylethanol glycosides permeated by the ultrafiltration membrane at different ultrafiltration temperatures is shown in Table 2.
[0047] Table 2 Purity of phenylethanol glycosides at different ultrafiltration temperatures Temperature (°C) 20 30 40 50 60 Phenylethanoid purity (%) 51.77 52.15 55.96 57.4 60.38
[0048] As can be seen from the data in Table 2, the purity of phenylethanol glycosides generally increases with the increase of ultrafiltration temperature. The purity is relatively high at 50~60℃. However, if the temperature is continuously increased, phenylethanol glycosides are easily lost due to high-temperature decomposition. At the same time, the retention rates of proteins and polysaccharides also decrease.
[0049] Example 3
[0050] Unlike Example 1, in (5), the operating pressure was adjusted when using ultrafiltration membrane filtration. The purity of phenylethanol glycosides permeated by ultrafiltration membrane without operating pressure is shown in Table 3 below.
[0051] Table 3. Purity of phenylethanol glycosides permeated through the ultrafiltration membrane at different ultrafiltration pressures. Pressure (MPa) 0.1 (Example 1) 0.2 0.4 0.6 0.8 1.0 Phenylethanoid purity (%) 60.38 52.8 54.64 58.4 60.38 60.15
[0052] The results in the table show that the operating pressure during ultrafiltration has a significant impact on the purity of phenylethanol glycosides. To a certain extent, the purity of phenylethanol glycosides increases with the increase of ultrafiltration pressure. However, when the operating pressure is 0.8 MPa, further increasing the pressure will actually decrease the purity of phenylethanol glycosides.
[0053] Example 4
[0054] Unlike Example 1, in (5) when D-101 macroporous resin was used to purify phenylethanol glycosides, the ratio of the eluent was adjusted. The effect of different ratios of eluent on the elution effect of phenylethanol glycosides is shown in Table 4 below.
[0055] Table 4. Effect of eluent ratio on the purification efficiency of phenylethanol glycosides Ratio 1：0 9：1 7：3 1：1 3：7 Phenylethanoid purity (%) 75.6 76.27 78.94 70.46 62.61
[0056] The results in Table 4 show that the purity of phenylethanol glycosides obtained by eluting with different ratios of eluent is quite different. As the amount of ethanol added decreases, the purity of phenylethanol glycosides generally shows a trend of first increasing and then decreasing. In particular, when the volume ratio of 60% ethanol to 0.1% acetic acid aqueous solution is 7:3, the purity of phenylethanol glycosides is the highest, and the purification effect is the best at this time.
[0057] Example 5
[0058] Unlike Example 1, in (5) different types of macroporous resins were used to purify phenylethanol glycosides. The purification effects of different types of macroporous resins on phenylethanol glycosides are shown in Table 5 below.
[0059] Table 5. Effect of macroporous resin type on the purification efficiency of phenylethanol glycosides D-101 HPD-100 X-5 DM-301 AB-8 Phenylethanoid purity (%) 75.6 68.33 59.74 65.94 73.27
[0060] The experimental results in Table 5 show that the type of macroporous resin has a significant impact on the purification effect of phenylethanol glycosides. The elution effect of X-5 type resin is the worst, with the purity of phenylethanol glycosides reaching only 59.74%, while the purity of phenylethanol glycosides after elution with D-101 can reach 75.6%.
[0061] Comparative Example 1
[0062] The only difference from Example 1 is that (1) only uses a pulverizer for primary pulverization and no longer performs secondary airflow pulverization. The remaining steps and operations are the same as in Example 1.
[0063] The obtained phenylethanol glycoside powder weighed 6.4 g, the extraction rate of phenylethanol glycoside was 17.6%, the recovery rate after purification was 60.49%, and the purity was 51.2%.
[0064] Comparative Example 2
[0065] The only difference from Example 1 is that (1) uses ultrafine grinding instead of airflow grinding, while the other steps and operations are the same as in Example 1.
[0066] The obtained phenylethanol glycoside powder weighed 8.1 g, with an extraction rate of 45.27%. The recovery rate after purification was 79.4%, and the purity was 63.83%. Compared with Comparative Example 1, the extraction rate of phenylethanol glycoside increased by 27.67%, but compared with the air jet milling in Example 1, the extraction rate of phenylethanol glycoside decreased by 16.86%.
[0067] Comparative Example 3
[0068] The difference from Example 1 is that the steam explosion treatment in (2) is omitted, and in (3), ultrasonic-assisted water extraction is used to obtain the water extract. The specific operation is as follows:
[0069] (2) Add 15 times the volume of water to the Cistanche deserticola powder obtained in (1), extract at 60°C for 30 min with ultrasonic assistance (ultrasonic power of 250 W, ultrasonic duration of 30 min), centrifuge at 4000 r / min for 10 min and filter, repeat 3 times, combine the filtrates to obtain the water extract, and the remaining steps and operations are the same as in Example 1.
[0070] The obtained phenylethanol glycoside powder weighed 4.7 g, and the extraction rate of phenylethanol glycoside was 53.66%.
[0071] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.
Claims
1. A method for extracting and separating phenylethanoid glycosides from Cistanche deserticola, characterized in that, The steps include the following: (1) Cistanche deserticola, dried, first pulverized by a pulverizer, then pulverized by air jet mill to obtain Cistanche deserticola powder; (2) The Cistanche deserticola powder in (1) was subjected to steam explosion treatment. The conditions for steam explosion were: steam explosion pressure 0.5~1.5 MPa, steam explosion duration 50~90 s, and material-to-cavity ratio 1:1~3. (3) After the steam explosion treatment in (2) is completed, add water to the obtained material, extract at 50~60℃ for 20~60 min, centrifuge and filter, repeat 2~3 times, combine the filtrates to obtain water extract; (4) Add 0.01% to 0.1% of the water extract by mass to the water extract of (3), and enzymatically hydrolyze at 50 to 60°C for 20 to 50 minutes to obtain the hydrolysate; the compound enzyme preparation is made by mixing pectinase and amylase in a mass ratio of 1:1 to 2. (5) The enzymatic hydrolysate from (4) is subjected to multi-stage membrane filtration, and then eluted with D-101 macroporous adsorption resin. The eluent is 50%~70% ethanol. The volume ratio of 0.1% acetic acid aqueous solution is 6~8:
3. The effluent is evaporated and concentrated, and then freeze-dried to obtain phenylethanol glycoside powder.
2. The method as described in claim 1, characterized in that, (1) Drying Cistanche deserticola at a temperature of 40~55℃.
3. The method as described in claim 1, characterized in that, The airflow pulverizer described in (1) has a pulverization frequency of 30~50Hz and a pulverizing gas pressure of 0.5~1.0 MPa.
4. The method as described in claim 1, characterized in that, The conditions for steam explosion treatment described in (2) are: steam explosion pressure 0.8~1.2 MPa, steam explosion duration 60~80 s, and material chamber ratio 1:
2.
5. The method as described in claim 1, characterized in that, (3) The mass-volume ratio of the material to water is 1 g: 10~20 mL.
6. The method as described in claim 1, characterized in that, In (4), a compound enzyme preparation is added to the aqueous extract of (3). The compound enzyme preparation includes 0.03% pectinase and 0.03% amylase based on the mass of the aqueous extract.
7. The method as described in claim 1, characterized in that, The specific operation of multi-stage membrane filtration described in (5) is as follows: membrane filtration is performed sequentially using a 30 kDa spiral wound ultrafiltration membrane, a 200 Da nanofiltration membrane, and a 0.1~1 nm reverse osmosis membrane.
8. The method as described in claim 1, characterized in that, The eluent mentioned in (5) is 60% ethanol: The volume ratio of 0.1% acetic acid aqueous solution is 7:3.