Extraction method for improving extraction stability of verbascoside in cistanche and application
Through gradient extraction technology and acidic regulation combined with prickly pear extract and tea extract, the problem of large differences in the content of mutton in traditional preparation of Cistanche extracts is solved, and the stability and application stability of the extract are greatly improved.
Patent Information
- Application Number
- CN202510309015.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-06-20
AI Technical Summary
Traditional methods of preparing Cistanche extract lead to large differences in the content of muttonin and are easy to decompose under neutral and alkaline conditions, limiting its wide application in the food field.
Using gradient extraction technology, the pH of the 50% ethanol solution was adjusted to 5-6 using 5mol/L hydrochloric acid solution, and ultrasonic gradient extraction was performed in combination with prickly pear extract and tea extract, followed by centrifugation, filtration, alcohol precipitation and freeze-drying to obtain stable Cistanche extract.
The extraction stability of muttonin in Cistanche is significantly improved, the content fluctuates between batches by less than 2%, and the stability of the chromaticity value when applied to the wine body reaches ≤1.5%, ensuring the stability of product quality.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of extraction of active ingredients from medicinal materials, and particularly relates to an extraction method and application for improving the extraction stability of acteoside in Cistanche deserticola. Background Art
[0002] In the fields of traditional Chinese medicine and food, acteoside in Cistanche deserticola extract, as an important bioactive ingredient, has been widely used in health foods and pharmaceuticals. Research shows that Cistanche deserticola extract has significant anti-tumor, anti-inflammatory, skin-protecting and other effects, and the content of acteoside in it is relatively high, having good development potential.
[0003] However, the traditional method for preparing Cistanche deserticola extract has certain limitations. During the extraction process, acteoside in Cistanche deserticola may be affected by the extraction environment, resulting in significant differences in the acteoside content between batches of Cistanche deserticola extracts obtained. Some research shows that acteoside is prone to decomposition under neutral and alkaline conditions. Although the use of acidic extraction methods can inhibit its decomposition, there are still significant differences in the acteoside content between multiple batches of Cistanche deserticola extracts obtained, and the chromaticity value changes greatly. These factors limit the wide application of Cistanche deserticola extract in the food field. Summary of the Invention
[0004] The purpose of the present invention is to provide an extraction method and application for improving the extraction stability of acteoside in Cistanche deserticola.
[0005] The present invention is achieved through the following technical solutions:
[0006] An extraction method for improving the extraction stability of acteoside in Cistanche deserticola, comprising the following steps:
[0007] Step 1. Raw material screening: Collect Cistanche deserticola medicinal materials, cut them into thin slices, seal them with a sealed bag, and store them frozen at low temperature for later use;
[0008] Step 2. Preparation of extraction solvent: Use 5 mol / L hydrochloric acid solution to adjust the pH of 50% ethanol solution to 5 - 6 as the extraction solvent;
[0009] Step 3. Gradient extraction: Add the thin slices of Cistanche deserticola medicinal materials prepared in Step 1, Rosa roxburghii extract, and tea extract into the extraction solvent prepared in Step 2 according to a mass ratio of 1:(0.2 - 0.5%):(0.2 - 0.5%) for ultrasonic gradient extraction;
[0010] Step 4. Concentration: Centrifuge at a rotational speed of 3000 - 4000 rpm / min for 5 - 10 min, then pass through a ceramic membrane with a pore size of 50 nm, collect the permeate of the ceramic membrane, and concentrate the permeate of the ceramic membrane through a nanofiltration membrane with a pore size of 1000 Da, and collect the retentate of the nanofiltration membrane;
[0011] Step 5. Alcohol precipitation: Use 95% ethanol to adjust the nanofiltration membrane retentate to make the ethanol concentration 80%, and carry out alcohol precipitation for 12 - 15 h;
[0012] Step 6. Drying: The nanofiltration membrane retentate after alcohol precipitation is centrifuged, the supernatant is taken and concentrated to remove ethanol, and then freeze-dried to obtain the cistanche deserticola extract.
[0013] Preferably, in Step 1, the cistanche deserticola medicinal materials are cut into thin slices with a thickness of 2 cm, the vibrating screen vibrates to remove surface impurities and debris, the sealed bag can isolate moisture, the low-temperature storage temperature is -4°C, and the low-temperature storage time is 24 - 26 h.
[0014] Preferably, in Step 3, the polyphenol content in the rosa roxburghii tratt extract is ≥90%, the catechin content in the tea extract is ≥90%, and the mass ratio of the cistanche deserticola medicinal materials, rosa roxburghii tratt extract and tea extract is cistanche deserticola medicinal materials: rosa roxburghii tratt extract: tea extract = 1: 0.3% - 0.5%: 0.3% - 0.5%.
[0015] Preferably, in Step 3, the mass-to-volume ratio of the cistanche deserticola medicinal materials to the extraction solvent = 1: 6 g / mL.
[0016] Preferably, in Step 3, use an ultrasonic extractor to start the first extraction for 20 - 30 min at an ultrasonic power of 120 - 140 W and an extraction temperature of 30 - 40°C, and collect the extract; then start the second extraction for 10 - 20 min at an ultrasonic power of 140 - 160 W and an extraction temperature of 40 - 50°C, collect the extract, and combine the two extracts.
[0017] Preferably, in Step 4, the rotation speed of the centrifuge is 3000 - 4000 rpm / min, the centrifugation time is 5 - 10 min, the pore size of the ceramic membrane is 50 nm, and the pore size of the nanofiltration membrane is 1000 Da.
[0018] Preferably, in Step 6, the rotation speed of the centrifuge is 3000 - 4000 rpm / min, the centrifugation time is 5 - 10 min, and the concentration temperature ≤40°C.
[0019] Preferably, in Step 6, the fluctuation of the acteoside content between batches of the obtained cistanche deserticola extract is less than 2%, and more preferably ≤1.2%.
[0020] The present invention also provides the application of the cistanche deserticola extract obtained by the above method in improving the chromaticity stability of the wine body. Calculated by adding 1 g of cistanche deserticola extract to 500 mL of base wine, the difference in chromaticity values in the wine body is ≤1.5%, and more preferably ≤1.1%.
[0021] The beneficial effects produced by the present invention are:
[0022] 1. Compared with the traditional method for preparing Cistanche deserticola extract, the verbascoside in the Cistanche deserticola extract prepared by the present invention has strong stability and good repeatability between batches, and the RSD value of the verbascoside content between batches of the obtained Cistanche deserticola extract is ≤2%.
[0023] 2. The present invention utilizes gradient extraction technology and adopts a mixed extraction method with roxburghii extract (polyphenol content ≥ 90%) and tea extract (catechin content ≥ 90%) to improve the extraction stability of verbascoside in Cistanche deserticola extract. The extraction process of verbascoside has good extraction stability and is safe and easy to operate.
[0024] 3. When the Cistanche deserticola extract prepared by the present invention is applied to wine, the RSD value of the chromaticity value between batches is ≤1.5%, and the product quality is stable. DETAILED DESCRIPTION
[0025] Example 1
[0026] This embodiment discloses an extraction method for improving the extraction stability of verbascoside in Cistanche deserticola, and the specific steps are as follows:
[0027] (1) Cut the Cistanche deserticola into 2 cm thick slices, vibrate with a vibrating screen to remove surface impurities and debris, and seal in a moisture-proof sealed bag and store in a -4°C environment for 24 hours. Then use 5 mol / L hydrochloric acid solution to adjust the pH of the 50% ethanol solution to 5 as the extraction solvent. Add 1 kg of cryogenically frozen Cistanche deserticola, 2 g of Rosa roxburghii extract (polyphenol content ≥ 90%), and 2 g of tea extract (catechin content ≥ 90%) to 6 L of 50% ethanol solution, use an ultrasonic extractor at an ultrasonic power of 120 W and an extraction temperature of 30°C to start the first extraction for 20 minutes, and collect the extract. Then start the second extraction at an ultrasonic power of 140 W and an extraction temperature of 40°C for 10 minutes, collect the extract, and combine the two extracts to obtain a total of 11 L.
[0028] (2) Use a centrifuge to centrifuge for 5 minutes at a speed of 3000 rpm / min, then pass through a ceramic membrane with a pore size of 50 nm, collect the ceramic membrane permeate, pass the ceramic membrane permeate through a nanofiltration membrane with a pore size of 1000 Da for concentration, and collect the nanofiltration membrane retentate. Use 95% ethanol to adjust the nanofiltration membrane retentate to an ethanol concentration of 80%, and precipitate with alcohol for 12 hours. Finally, centrifuge the nanofiltration membrane retentate after alcohol precipitation at a speed of 3000 rpm / min for 5 minutes, take the centrifugal supernatant, concentrate at a concentration temperature of 37°C to remove ethanol, and then freeze-dry to obtain 56g of Cistanche extract.
[0029] Example 2
[0030] This embodiment discloses an extraction method for improving the extraction stability of acteoside in Cistanche deserticola, and the specific steps are as follows:
[0031] (1) Cut the Cistanche deserticola medicinal materials into thin slices with a thickness of 2 cm, vibrate them with a vibrating sieve to remove surface impurities and debris, and seal them in a moisture-proof sealed bag and store them in a -4°C environment for 25 h. Then use a 5 mol / L hydrochloric acid solution to adjust the pH of a 50% ethanol solution to 5.5 as the extraction solvent. Add 1 kg of cryogenically frozen Cistanche deserticola medicinal materials, 3 g of Rosa roxburghii Tratt extract (polyphenol content ≥ 90%), and 3 g of tea extract (catechin content ≥ 90%) to 6 L of 50% ethanol solution, and start the first extraction for 25 min using an ultrasonic extractor at an ultrasonic power of 130 W and an extraction temperature of 35°C, and collect the extract. Then start the second extraction for 15 min at an ultrasonic power of 150 W and an extraction temperature of 45°C, collect the extract, and combine the two extracts to obtain a total of 11.1 L.
[0032] (2) Centrifuge at a speed of 3500 rpm / min for 7 min using a centrifuge, then pass through a ceramic membrane with a pore size of 50 nm, collect the permeate of the ceramic membrane, and concentrate the permeate of the ceramic membrane through a nanofiltration membrane with a pore size of 1000 Da, and collect the retentate of the nanofiltration membrane. Adjust the retentate of the nanofiltration membrane with 95% ethanol to make its ethanol concentration 80%, and perform alcohol precipitation for 14 h. Finally, centrifuge the retentate of the nanofiltration membrane after alcohol precipitation at a speed of 3500 rpm / min for 7 min, take the centrifugal supernatant, concentrate and remove ethanol at a concentration temperature of 35°C, and then perform freeze-drying to obtain 62 g of Cistanche deserticola extract.
[0033] Example 3
[0034] This embodiment discloses an extraction method for improving the extraction stability of acteoside in Cistanche deserticola, and the specific method steps are as follows:
[0035] (1) Cut the Cistanche deserticola medicinal materials into thin slices with a thickness of 2 cm, vibrate them with a vibrating sieve to remove surface impurities and debris, and seal them in a moisture-proof sealed bag and store them in a -4°C environment for 26 h. Then use a 5 mol / L hydrochloric acid solution to adjust the pH of a 50% ethanol solution to 6 as the extraction solvent. Add 1 kg of cryogenically frozen Cistanche deserticola medicinal materials, 5 g of Rosa roxburghii Tratt extract (polyphenol content ≥ 90%), and 5 g of tea extract (catechin content ≥ 90%) to 6 L of 50% ethanol solution, and start the first extraction for 30 min using an ultrasonic extractor at an ultrasonic power of 140 W and an extraction temperature of 40°C, and collect the extract. Then start the second extraction for 20 min at an ultrasonic power of 160 W and an extraction temperature of 50°C, collect the extract, and combine the two extracts to obtain a total of 11.2 L.
[0036] (2) Centrifuge at 4000 rpm / min for 10 min using a centrifuge, then pass through a ceramic membrane with a pore size of 50 nm, collect the permeate of the ceramic membrane, concentrate the permeate of the ceramic membrane through a nanofiltration membrane with a pore size of 1000 Da, and collect the retentate of the nanofiltration membrane. Adjust the retentate of the nanofiltration membrane with 95% ethanol to make the ethanol concentration 80%, and perform alcohol precipitation for 15 h. Finally, centrifuge the retentate of the nanofiltration membrane after alcohol precipitation at 4000 rpm / min for 10 min, take the centrifugal supernatant, concentrate and remove ethanol at a concentration temperature of 40 °C, and then perform freeze-drying to obtain 60 g of cistanche deserticola extract.
[0037] Comparative Example 1
[0038] The difference between this comparative example and Example 1 is that in Step 1, it was not stored in an environment at -4 °C.
[0039] Comparative Example 2
[0040] The difference between this comparative example and Example 1 is that in Step 2, a 5 mol / L hydrochloric acid solution was not used to adjust the pH of the 50% ethanol solution.
[0041] Comparative Example 3
[0042] The difference between this comparative example and Example 1 is that gradient extraction was not used in Step 3. Specifically, 1 kg of low-temperature frozen cistanche deserticola medicinal materials, 2 g of rosa roxburghii tratt extract (polyphenol content ≥ 90%), and 2 g of tea extract (catechin content ≥ 90%) were added to 12 L of 50% ethanol solution, and ultrasonic extraction was performed using an ultrasonic extractor at an ultrasonic power of 120 W and an extraction temperature of 30 °C for 2 times, 20 min each time, and the extract was collected.
[0043] Comparative Example 4
[0044] The difference between this comparative example and Example 1 is that co-extraction with rosa roxburghii tratt extract (polyphenol content ≥ 90%) and tea extract (catechin content ≥ 90%) was not performed during extraction in Step 3. Specifically, 1 kg of low-temperature frozen cistanche deserticola medicinal materials was added to 6 L of 50% ethanol solution, and ultrasonic extraction was started using an ultrasonic extractor at an ultrasonic power of 120 W and an extraction temperature of 30 °C for 20 min for the first time, and the extract was collected. Then, ultrasonic extraction was started at an ultrasonic power of 140 W and an extraction temperature of 40 °C for 10 min for the second time, and the extract was collected. The two extracts were combined to obtain a total of 11 L.
[0045] Comparative Example 5
[0046] The difference between this comparative example and Example 1 is that the alcohol precipitation step was not used in Step 5.
[0047] Comparative Example 6
[0048] The difference between this comparative example and Example 1 is that only 1 kg of cryogenically frozen Cistanche deserticola medicinal material and 2 g of Rosa roxburghii extract (polyphenol content ≥ 90%) are added to 6 L of 50% ethanol solution and ultrasonic extraction is performed using an ultrasonic extractor, and 2 g of tea extract (catechin content ≥ 90%) is not added.
[0049] Comparative Example 7
[0050] The difference between this comparative example and Example 1 is that only 1 kg of cryogenically frozen Cistanche deserticola medicinal material and 2 g of tea extract (catechin content ≥ 90%) are added to 6 L of 50% ethanol solution and ultrasonic extraction is performed using an ultrasonic extractor, and 2 g of Rosa roxburghii extract (polyphenol content ≥ 90%) is not added.
[0051] Test Example 1 Determination of the stability of verbascoside content between batches of Cistanche deserticola extract
[0052] The stability of the verbascoside content between different batches of the Cistanche deserticola extracts of various embodiments and comparative examples was detected by high performance liquid chromatography.
[0053] ①Reagents and instruments
[0054] Verbascoside standard (purity 97.6%), China Food and Drug Administration;
[0055] UltiMate3000 High Performance Liquid Chromatograph Thermo Fisher Scientific Inc.;
[0056] AB135-S analytical balance Mettler-Toledo;
[0057] ZORBAX Eclipse Plus-C18 chromatographic column (4.6mm×250mm, 5μm) Agilent Technologies Co., Ltd.
[0058] ②Chromatographic method
[0059] Chromatographic conditions: ZORBAX Eclipse Plus-C18 chromatographic column (4.6 mm × 250 mm, 5 μm); detection wavelength 330 nm; flow rate 1.0 mL / min; column temperature 25 °C; injection volume 10 μL; mobile phase: acetonitrile (A) - 0.1% acetic acid solution (B); gradient elution conditions: 0 - 15 min, 7.5% - 9% A; 15 - 17 min, 9% - 12% A; 17 - 25 min, 12% - 13% A; 25 - 40 min, 13% - 15% A; 40 - 60 min, 15% A; 60 - 70 min, 15% - 20% A; 70 - 85 min, 20% A; 85 - 95 min, 20% - 30% A; 95 - 100 min, 30% - 90% A; 100 - 100.1 min, 90% - 7.5% A; 100.1 - 105 min, 7.5% A.
[0060] ③ Calculation method for the stability between batches of Cistanche deserticola extract
[0061] Calculate the RSD value of the content of Cistanche deserticola between batches of Cistanche deserticola extract according to the following formula:
[0062] Relative standard deviation (RSD) = standard deviation (SD) / arithmetic mean (X) of the calculation results
[0063]
[0064] where x i is each measured value; is the mean value of the measured values; n is the number of measured values.
[0065] Table 1 RSD values of acteoside content between batches of Cistanche deserticola extract in different examples and comparative examples (n ≥ 5)
[0066]
[0067]
[0068] As can be seen from Table 1, the RSD values of acteoside content among batches of the cistanche deserticola extract obtained in Examples 1-3 are all less than 2%, indicating strong stability. However, the RSD values of acteoside content among batches of the cistanche deserticola extract obtained in Comparative Examples 1-7 are all higher than 5%, indicating poor stability. This shows that the steps for preparing the cistanche deserticola extract in the present invention are integral, and the lack of any technical step in the present invention will have a great impact on the stability of acteoside content in the cistanche deserticola extract. In addition, the present inventors found during the experiment that the extracts of rosa roxburghii tratt (polyphenol content ≥ 90%) and tea (catechin content ≥ 90%) have strong reducibility and can stabilize the structure of acteoside without being damaged. It can also be seen from Comparative Examples 6-7 that only when the extracts of rosa roxburghii tratt (polyphenol content ≥ 90%) and tea (catechin content ≥ 90%) are used in combination can the RSD values of acteoside content among batches of the obtained cistanche deserticola extract be less than 2%, showing strong stability, and the use of any one of them alone cannot achieve the above effect.
[0069] Test Example 2 Determination of the chromaticity stability of the cistanche deserticola extract among batches after being applied to the wine body
[0070] A colorimeter was used to detect the stability of chromaticity values among different batches of the cistanche deserticola extract in each example and comparative example.
[0071] Preparation of the test samples: 1 g of the cistanche deserticola extract in each example and comparative example was respectively taken and added to 500 mL of 37% vol base liquor. After being fully dissolved, the chromaticity of each sample was detected using a colorimeter, and the RSD values of the chromaticity of the cistanche deserticola extract among batches after being applied to the wine body in each example and comparative example were calculated using the following formula:
[0072] Relative standard deviation (RSD) = standard deviation (SD) / arithmetic mean (X) of the calculation results
[0073]
[0074] where x i is each measured value; is the mean of the measured values; n is the number of measured values.
[0075] Table 2 RSD values of the chromaticity of the cistanche deserticola extract among batches after being applied to the wine body in different examples and comparative examples (n ≥ 5)
[0076]
[0077]
[0078] As can be seen from Table 2, the RSD values of the chromaticity after the cistanche deserticola extracts obtained in Examples 1-3 were applied to the wine body were all less than 1.5%, indicating strong stability. However, the RSD values of the chromaticity after the cistanche deserticola extracts obtained in Comparative Examples 1-7 were applied to the wine body were all higher than 4.5%, indicating poor stability. This shows that the steps for preparing the cistanche deserticola extract in the present invention are integral, and the lack of any technical step in the present invention will have a great impact on the chromaticity stability after the cistanche deserticola extracts are applied to the wine body. It can also be seen from Comparative Examples 6-7 that only the combined use of the rosa roxburghii tratt extract (polyphenol content ≥ 90%) and the tea extract (catechin content ≥ 90%) can make the RSD values of the chromaticity after the obtained cistanche deserticola extracts are applied to the wine body less than 1.5%, indicating strong stability, and the use of any one of them alone cannot achieve the above effect.
[0079] Based on the above analysis results of the data, the cistanche deserticola extract prepared in the examples of the present invention has the characteristics of good stability of the acteoside content between batches and small fluctuations in the chromaticity value when applied to the wine body. To sum up, the above examples are only illustrative examples of the preferred embodiments of the present invention and do not cover all the scopes of the invention embodiments. Those of ordinary skill in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is subject to the scope defined by the claims.
Claims
1. A method for improving the extraction stability of verbascoside in Cistanche deserticola, characterized in that: The following steps are involved: Step 1: Raw material selection: collect Cistanche deserticola medicinal materials, cut into thin slices, seal in a sealed bag, and store at low temperature for later use; Step 2: Extraction solvent preparation: Use 5 mol / L hydrochloric acid solution to adjust the pH of 50% ethanol solution to 5-6 as the extraction solvent; Step 3, gradient extraction: adding the Cistanche deserticola slices, Rosa roxburghii extract and tea extract prepared in step 1 to the extraction solvent prepared in step 2 in a mass ratio of 1: (0.2-0.5%): (0.2-0.5%) for ultrasonic gradient extraction; Step 4, concentration: centrifuge at a speed of 3000-4000 rpm / min for 5-10 minutes, then pass through a ceramic membrane with a pore size of 50 nm, collect the ceramic membrane permeate, pass the ceramic membrane permeate through a nanofiltration membrane with a pore size of 1000 Da for concentration, and collect the nanofiltration membrane retentate; Step 5, alcohol precipitation: Use 95% ethanol to adjust the nanofiltration membrane retentate to an ethanol concentration of 80%, and precipitate with alcohol for 12-15 hours; Step 6, drying: centrifuging the nanofiltration membrane retentate after alcohol precipitation, concentrating the supernatant to remove ethanol, and then freeze-drying to obtain the Cistanche deserticola extract.
2. The method according to claim 1, characterized in that In the step 1, the Cistanche deserticola medicinal material is cut into 2 cm thick slices, and the surface impurities and debris are removed by vibration screen. The sealed bag can isolate moisture. The low-temperature storage temperature is -4°C and the low-temperature storage time is 24-26 hours.
3. The method according to claim 1, characterized in that In the step 3, the polyphenol content in the roxburghii extract is ≥90%, the catechin content in the tea extract is ≥90%, and the mass ratio of the Cistanche deserticola medicinal material, the roxburghii extract and the tea extract is Cistanche deserticola medicinal material: roxburghii extract: tea extract = 1: 0.3%-0.5%: 0.3%-0.5%.
4. The method according to claim 1, characterized in that: In the step 3, the mass volume ratio of Cistanche deserticola medicinal material to the extraction solvent is 1:6 g / mL.
5. The method according to claim 1, characterized in that: In the step three, an ultrasonic extractor is used to start the first extraction for 20-30 minutes at an ultrasonic power of 120-140W and an extraction temperature of 30-40°C, and the extract is collected; then a second extraction is started for 10-20 minutes at an ultrasonic power of 140-160W and an extraction temperature of 40-50°C, and the extract is collected and the two extracts are combined.
6. The method according to claim 1, characterized in that In the step 4, the rotation speed of the centrifuge is 3000-4000 rpm / min, the centrifugation time is 5-10 min, the pore size of the ceramic membrane is 50 nm, and the pore size of the nanofiltration membrane is 1000 Da.
7. The method according to claim 1, characterized in that In step six, the rotation speed of the centrifuge is 3000-4000 rpm / min, the centrifugation time is 5-10 min, and the concentration temperature is ≤40°C.
8. The method according to claim 1, characterized in that: In the step 6, the fluctuation of the verbascoside content between the obtained Cistanche deserticola extract batches is less than 2%.
9. Use of the Cistanche deserticola extract prepared by the method of any one of claims 1 to 8 in improving the chromaticity stability of wine, characterized in that: When 1g of Cistanche deserticola extract is added to 500mL of base wine, the color value difference in the wine is ≤1.5%.