Rapid extraction method of leucopelargonidin from hippophae rhamnoides l. leaves

Abstract

The application discloses a kind of quick extraction methods of Hippocastanin in sea-buckthorn leaf, belong to plant active ingredient extraction technical field, the extraction method, including the following steps: defatted sea-buckthorn leaf powder is mixed with ethanol aqueous solution, extraction, and extract liquid is obtained, first vacuum concentration is reduced, and concentrated extract is obtained, the concentrated extract is mixed with distilled water, and separation first extract liquid;The first extract liquid is mixed with calcium oxide, after reaction, adjust pH, filter, and reaction liquid is obtained;Using macroporous cation resin, the reaction liquid is adsorbed and treated, second vacuum concentration is reduced, and concentrated reaction liquid is obtained;The concentrated reaction liquid is mixed with methanol, and precipitate is obtained after standing, and first extract is obtained;The first extract is heated and concentrated, crystallization, and hippocastanin is obtained.The extraction efficiency of the extraction method is high, the purification step is simple, and the purity of hippocastanin extracted is high.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, and particularly relates to a rapid extraction method of strychnine from sea buckthorn leaves. Background Technology

[0002] Sea buckthorn ( *Hippophae rhamloides* L. is a plant belonging to the genus *Hippophae* in the family Elaeagnaceae. It is a thorny, nitrogen-fixing, deciduous, dioecious shrub. Sea buckthorn leaves are rich in phenols, steroids, triterpenes, polysaccharides, vitamins, amino acids, and inositol, possessing antioxidant, anti-cancer, blood sugar and lipid-lowering, and metabolic regulating functions. It can serve as a good source of bioactive substances and functional foods. *Hippophae rhamloides* L. (molecular formula: ...) Figure 2 (As shown) is a characteristic active ingredient in sea buckthorn leaves, with outstanding medicinal effects. It has been proven to have adjuvant effects in cancer treatment, promoting cell proliferation, adjuvant treatment of diabetes, antibacterial effects, and reducing stomach damage, attracting widespread attention from scholars in the pharmaceutical field. However, due to the low distribution and content of malachite alcohol in plant raw materials, and the large number of isomers in the extract, the extraction of high-purity malachite alcohol is difficult and research is limited.

[0003] The existing extraction technology for malachite alcohol from sea buckthorn leaves is patent CN202111645344.4 filed by Guilin Rhine Biotechnology Co., Ltd. Its extraction process is as follows: (1) Take sea buckthorn leaves and pulverize them into ultrafine powder, add pure water and stir evenly, extract at room temperature, filter the extracted sea buckthorn liquid, centrifuge the filtrate in stages, and press the filter residue dry to obtain the extract; (2) The extract is passed through a macroporous adsorption resin to adsorb impurities, and the effluent and washing liquid are collected; (3) The effluent and washing liquid from step (2) are purified through an organic solvent nanofiltration membrane to obtain a decolorized and retained membrane solution; the organic solvent nanofiltration membrane includes a first-stage organic solvent nanofiltration membrane and a second-stage organic solvent nanofiltration membrane, the first-stage organic solvent nanofiltration membrane being an organic solvent nanofiltration membrane with a molecular weight of 800-1500 Daltons, and the second-stage organic solvent nanofiltration membrane being an organic solvent nanofiltration membrane with a molecular weight of 100-250 Daltons; this step mainly uses... (4) The decolorized retentate is fed into an anion exchange resin, then eluted with pure water, and the effluent and washing liquid are collected. Then it is fed into a cation exchange resin, and after that, it is eluted with pure water, and the effluent and washing liquid are collected. Anions and cations can be separated from the extract by adsorption, precipitation or ion exchange using anion and cation exchange resins. (5) The effluent and washing liquid collected after passing through the cation exchange resin in step (4) are passed through a reverse osmosis membrane to obtain a retentate. This removes dissolved salts, colloids, microorganisms and organic matter from the extract. (6) The retentate is concentrated into an extract, then dissolved in an organic solvent, stirred evenly, acid is added to adjust the pH to acidic, cooled to crystallize, and dried to obtain the white malachite alcohol product. In this method, since white malachite alcohol is highly polar and easily soluble in water, pure water is chosen as the extraction solvent. However, the use of pure water during extraction also causes a large amount of other water-soluble impurities to dissolve, including water-soluble pigments, proteins, sugars, etc., making it difficult to remove impurities in the subsequent process. In addition, during macroporous resin elution, highly polar substances are eluted first with the aqueous solution, while less polar substances are more strongly adsorbed by the macroporous resin and are eluted later. This step mainly utilizes the macroporous resin to remove impurities with slightly lower polarity. However, in step (1) of this method, pure water, which is highly polar, is chosen as the extraction solvent. The crude extract contains relatively few polar substances, so the amount of impurities that can be removed by the macroporous adsorption resin in this step is small, resulting in low impurity removal efficiency. This method also utilizes ultrafine grinding technology, macroporous resin purification, organic solvent nanofiltration membrane purification, anion and cation exchange resin purification, and reverse osmosis for impurity removal. Its preparation process is complex, involves many types of equipment, has high energy consumption, and has a large extraction cost.

[0004] In addition, Tao Cui of Beijing Forestry University wrote "Preparation and In Vitro Hypoglycemic Activity Study of Seabuckthorn Leaf White Pestilence Alcohol" which describes the extraction and purification of seabuckthorn leaf white pestilence alcohol, including the following steps: (1) Prepare crude white pestilence alcohol extract using the optimal extraction process. Weigh 100g of dried seabuckthorn leaf powder with a particle size of 20 mesh, add 100mL of 50% ethanol, and extract by ultrasonication three times, 40min each time. Combine the filtrates, remove the solvent by vacuum distillation at 60℃, and concentrate into an extract. (2) Pass the extract through a dextran gel column to adsorb impurities. Collect the eluent, evaporate the solvent under vacuum, and weigh it. The purity of white pestilence alcohol in the final seabuckthorn leaf purified product is 72.7%. This method uses dextran gel for purification, but Sephadex G-10 gel powder is expensive, and the dextran gel column has a slow column speed and low processing efficiency. This purification method has simple steps and does not involve crystallization, resulting in a low purity of the white pestilence alcohol extract. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a rapid extraction method for baicalein from sea buckthorn leaves, which features high extraction efficiency, simple purification steps, and high purity of the extracted baicalein.

[0006] To achieve the above objectives, this invention provides a rapid extraction method for spirulinaol from sea buckthorn leaves, comprising the following steps:

[0007] Defatted sea buckthorn leaf powder was mixed with an ethanol aqueous solution and extracted to obtain an extract. The extract was concentrated under vacuum for the first time to obtain a concentrated extract. The concentrated extract was mixed with distilled water to separate the initial extract.

[0008] The initial extract was mixed with calcium oxide, and after the reaction, the pH was adjusted and the mixture was filtered to obtain the reaction solution.

[0009] The reaction solution was adsorbed using a macroporous cation exchange resin and then concentrated under vacuum to obtain a concentrated reaction solution.

[0010] The concentrated reaction solution was mixed with methanol and allowed to stand to precipitate, yielding the initial extract.

[0011] The initial extract was heated, concentrated, and crystallized to obtain basilol.

[0012] Preferably, the defatted sea buckthorn leaf powder is prepared by mixing sea buckthorn leaf powder with petroleum ether, reflux treatment, and drying. Using petroleum ether for defatting can remove the waxy layer on the surface of sea buckthorn leaves, which can affect the dissolution of active ingredients. Hot reflux treatment with petroleum ether can remove the waxy layer of sea buckthorn leaves and increase the dissolution of malachite alcohol.

[0013] Preferably, the ethanol-water solution is an 80% (v / v) ethanol-water solution, and the mixing ratio of the defatted sea buckthorn leaf powder to the 80% (v / v) ethanol-water solution is 100g:1500mL. The extraction is performed three times, the extraction temperature is 20-28℃, and the extraction time for each extraction is 2 hours. The 80% (v / v) ethanol-water solution is used as the extraction solvent because malachite alcohol is highly water-soluble. Extraction with an 80% (v / v) ethanol-water solution reduces the dissolution of water-soluble impurities. After concentration, dissolving the extract in distilled water reduces low-polarity impurities in the extract. This process controls both water-soluble and polar impurities in the extract, simplifying subsequent purification operations.

[0014] Preferably, the vacuum degree of the first vacuum concentration is 0.07 MPa, the temperature is 55°C, and the time is 20-30 min; the ratio of the concentrated extract to distilled water is 1 g: 10 mL.

[0015] Preferably, the ratio of the initial extract to calcium oxide is 100 mL: 0.6 g, the reaction time is 15–30 min, and the pH is adjusted to 7–8. During the extraction process, calcium oxide (CaO) is added. CaO reacts with water to generate Ca(OH)₂. Ca(OH)₂ reacts with proteins, acidic inorganic substances, and acidic organic substances in the initial extract to form flocculent precipitates, thereby removing water-soluble impurities from the filtrate.

[0016] Preferably, the vacuum degree of the second vacuum concentration is 0.07 MPa, the temperature is 55°C, and the time is 20-30 min; the volume of the concentrated reaction solution is 15-20% of the total volume of the reaction solution.

[0017] Preferably, the volume ratio of the concentrated reaction solution to methanol is 1:5, and the settling time is 2 hours.

[0018] Preferably, the concentrated reaction solution is mixed with methanol, and the precipitation process is repeated three times. The precipitates obtained from the three processes are combined to obtain the initial extract. The concentrated reaction solution is mixed with methanol because malachite alcohol has low solubility in cold methanol. This property can be used to precipitate malachite alcohol and other compounds with low solubility in cold methanol from the filtrate.

[0019] Preferably, the primary extract is concentrated by heating at 90-100°C for 15-20 minutes after adjusting the pH to 5-6 with a 1M HCl aqueous solution; the primary extract is concentrated to 1 / 3 of its original volume. The primary extract is a flocculent precipitate containing some polysaccharide compounds in addition to malachite alcohol and monosaccharides. These compounds are insoluble in methanol under heating conditions. Acid hydrolysis is performed by adjusting the pH to 5-6 with a 1M HCl solution to dissolve the insoluble polysaccharides. Heating the solution allows the acid-hydrolyzed primary extract to fully dissolve in methanol.

[0020] Preferably, the crystallization temperature is 4°C, and the crystallization time is 2-3 days. After crystallization, crystals are obtained, and the crystals are washed twice with methanol to obtain strychnine. Strychnine has low solubility in cold methanol, but it can dissolve in methanol when heated, and can crystallize upon cooling. This property is used to evaporate, concentrate, cool, and crystallize strychnine to obtain a crystalline substance with high purity.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] This invention provides a rapid extraction method for leucosterol from sea buckthorn leaves. The purity of the leucosterol sample prepared by this invention can reach 94.15%. By selecting a suitable solvent, the dissolution of other water-soluble impurities is reduced during the extraction of leucosterol, thus reducing subsequent purification operations. A large number of impurities in the extract are removed by CaO precipitation, and the low solubility of leucosterol in methanol solution is utilized for further precipitation and separation, eliminating cumbersome purification steps such as macroporous resin purification and organic solvent nanofiltration membrane purification. The extraction method described in this invention involves simple equipment with low energy consumption and utilizes the solubility properties of leucosterol multiple times, improving the extraction efficiency of the leucosterol sample and obtaining a high yield and high purity product in one step. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The X-ray diffraction pattern is shown for the malachite alcohol sample prepared in Example 1.

[0025] Figure 2 The molecular formula is cypermethrin;

[0026] Figure 3 Infrared spectra of the malachite alcohol sample and the malachite alcohol reference standard prepared in Example 1 (the sample is the malachite alcohol sample prepared in Example 1);

[0027] Figure 4 The image shows the HPLC-QQQ-MS secondary mass spectrum of the white malachite alcohol sample prepared in Example 1.

[0028] Figure 5 The HPLC-QQQ-MS total ion chromatogram of the white malachite alcohol sample prepared in Example 1 is shown below.

[0029] Figure 6 The image shows the sample product of basilol prepared in Example 1, where A and B are crystal morphology images of basilol sample, and C is a morphology image of basilol sample.

[0030] Figure 7 This is a diagram illustrating the extraction process of white pea wood alcohol in this invention;

[0031] Figure 8 The above is the 1H NMR spectrum of the supernatant corresponding to the initial extract obtained in Example 1;

[0032] Figure 9 The first extract prepared in Example 1 has a hydrogen nuclear magnetic resonance spectrum.

[0033] Figure 10 The 1H NMR spectrum of baicalein reference standard;

[0034] Figure 11 The carbon NMR spectrum of the white malachite alcohol sample prepared in Example 1;

[0035] Figure 12 This is the carbon NMR spectrum of the reference standard, basilol. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] The reference standard of white cypress alcohol used in the embodiments of this invention was purchased from EFA Biotechnology (Chengdu) Co., Ltd.; the sea buckthorn leaf powder was obtained from the leaves of Chinese sea buckthorn collected from Lexiu Town, Hezuo City, Gannan Tibetan Autonomous Prefecture, Gansu Province (102°92'E, 34°98'N, altitude 2960m), which were dried and then pulverized through a 40-mesh sieve using a high-speed multi-functional pulverizer.

[0042] Example 1

[0043] Weigh out sea buckthorn leaf powder, mix sea buckthorn leaf powder with petroleum ether at a ratio of 1g:20mL, heat and reflux at 65℃ for 2h, fully volatilize the petroleum ether and dry to obtain defatted sea buckthorn leaf powder.

[0044] Weigh 100g of defatted sea buckthorn leaf powder and mix it with 1500mL of 80% ethanol aqueous solution. Extract the mixture three times at 25℃ for 2 hours each time. Combine the extracts from the three extractions and concentrate them under vacuum at 0.07MPa and 55℃ for 25 minutes to obtain 51.4g of concentrated extract. Mix the concentrated extract with distilled water at a ratio of 1g:10mL, stir to dissolve, let it stand to precipitate, and then filter to obtain 498mL of initial extract and 25g of insoluble extract.

[0045] The initial extract was mixed with distilled water at a volume ratio of 1:1 to obtain a diluted solution. CaO was added to the diluted solution at a ratio of 100 mL: 0.6 g, and the mixture was stirred for 15 min. The pH was adjusted to 7 using a 1 M HCl aqueous solution. After standing and precipitation, the mixture was filtered to obtain 450 mL of reaction solution.

[0046] The reaction solution was adsorbed onto a weakly acidic macroporous cation exchange resin (D152, particle size 0.315–1.25 mm) at a flow rate of 0.5 BV / h. 435 mL of the filtrate was collected and concentrated for 25 min under vacuum conditions of 0.07 MPa and 55 °C to obtain a concentrated reaction solution. The volume of the concentrated reaction solution was 20% of the total volume of the reaction solution.

[0047] The concentrated reaction solution was mixed with methanol at a volume ratio of 1:5. After mixing, flocculent precipitate gradually appeared in the mixture. After standing for 2 hours, the precipitate was retained. This step was repeated 3 times, and the precipitates were combined to obtain the initial extract.

[0048] The pH of the initial extract was adjusted to 5.5 using 1M HCl aqueous solution, and then concentrated at 95℃ for 20 min to one-third of the original volume, yielding 26 mL of acid hydrolysate. This hydrolysate was then cooled at 4℃ for 3 days to crystallize, yielding crystals. The crystals were washed twice with methanol, resulting in a total of 0.41 g of malachite alcohol sample (e.g., ...). Figure 6 (As shown).

[0049] Example 2

[0050] Weigh out sea buckthorn leaf powder, mix sea buckthorn leaf powder with petroleum ether at a ratio of 1g:20mL, heat and reflux at 60℃ for 2h, fully volatilize the petroleum ether and dry to obtain defatted sea buckthorn leaf powder.

[0051] Weigh 100g of defatted sea buckthorn leaf powder and mix it with 1500mL of 80% ethanol aqueous solution. Extract the extract using ultrasound at 250W and 40kHz for 30min to obtain the extract. Concentrate the extract under vacuum at 0.07MPa and 55℃ for 20min to obtain the concentrated extract. Mix the concentrated extract with distilled water at a ratio of 1g:10mL, stir to dissolve, allow to stand and precipitate, and then filter to obtain the initial extract.

[0052] The initial extract was mixed with distilled water at a volume ratio of 1:1 to obtain a diluted solution. CaO was added to the diluted solution at a ratio of 100 mL: 0.6 g, and the mixture was stirred for 15 min. The pH was adjusted to 7.2, and the mixture was allowed to stand and precipitate before filtration to obtain the reaction solution.

[0053] The reaction solution was adsorbed onto a weakly acidic macroporous cation exchange resin (D152, particle size 0.315–1.25 mm) at a flow rate of 0.5 BV / h. The filtrate was collected and concentrated under vacuum for 30 min at a vacuum of 0.07 MPa and a temperature of 55 °C to obtain a concentrated reaction solution. The volume of the concentrated reaction solution was 18% of the total volume of the reaction solution.

[0054] The concentrated reaction solution was mixed with methanol at a volume ratio of 1:5. After mixing, flocculent precipitate gradually appeared in the mixture. After standing for 2 hours, the precipitate was retained. This step was repeated 3 times, and the precipitates were combined to obtain the initial extract.

[0055] The pH of the initial extract was adjusted to 5 using 1M HCl aqueous solution, and then concentrated at 95℃ for 15 min to 1 / 3 of the original volume to obtain an acid hydrolysate. The acid hydrolysate was placed at 4℃ and cooled for 2 days to crystallize, and the crystals were washed twice with methanol to obtain the white malachite alcohol sample.

[0056] Example 3

[0057] Weigh out sea buckthorn leaf powder, mix sea buckthorn leaf powder with petroleum ether at a ratio of 1g:20mL, heat and reflux at 70℃ for 2h, fully volatilize the petroleum ether and dry to obtain defatted sea buckthorn leaf powder.

[0058] Weigh 100g of defatted sea buckthorn leaf powder and mix it with 1500mL of 80% ethanol aqueous solution. Extract the mixture three times at 25℃ for 2 hours each time. Combine the extracts from the three extractions to obtain the extract. Concentrate the extract under vacuum at 0.07MPa and 55℃ for 30 minutes to obtain a concentrated extract. Mix the concentrated extract with distilled water at a ratio of 1g:10mL, stir to dissolve, allow to stand and precipitate, and then filter to obtain the initial extract.

[0059] The initial extract was mixed with distilled water at a volume ratio of 1:1 to obtain a diluted solution. CaO was added to the diluted solution at a ratio of 100 mL: 0.6 g. The mixture was stirred and reacted for 30 min. The pH was adjusted to 7.5. After standing and settling, the mixture was filtered to obtain the reaction solution.

[0060] The reaction solution was adsorbed onto a weakly acidic macroporous cation exchange resin (D152, particle size 0.315–1.25 mm) at a flow rate of 0.5 BV / h. The filtrate was collected and concentrated under vacuum for 20 min at a vacuum degree of 0.07 MPa and a temperature of 55 °C to obtain a concentrated reaction solution. The volume of the concentrated reaction solution was 13% of the total volume of the reaction solution.

[0061] The concentrated reaction solution was mixed with methanol at a volume ratio of 1:5. After mixing, flocculent precipitate gradually appeared in the mixture. After standing for 2 hours, the precipitate was retained. This step was repeated 3 times, and the precipitates were combined to obtain the initial extract.

[0062] The pH of the initial extract was adjusted to 6 using 1M HCl aqueous solution, and then concentrated at 100℃ for 15 min to 1 / 3 of the original volume to obtain an acid hydrolysate. The acid hydrolysate was placed at 4℃ and cooled to crystallize for 3 days to obtain crystals. The crystals were washed twice with methanol to obtain the white malachite alcohol sample.

[0063] Comparative Example 1

[0064] The only difference from Example 1 is that the 80% ethanol aqueous solution mixed with defatted sea buckthorn leaf powder is replaced with a 50% ethanol aqueous solution.

[0065] Comparative Example 2

[0066] The only difference from Example 1 is that the methanol mixed with the concentrated reaction solution is replaced with a 90% volume-fraction aqueous methanol solution.

[0067] Comparative Example 3

[0068] The only difference from Example 1 is that the step of "adjusting the pH of the initial extract to 5.5 with 1M HCl aqueous solution, heating and concentrating at 95°C for 20 minutes to 1 / 3 of the original volume to obtain acid hydrolysate" is not included. Instead, the initial extract is heated at 90°C for 5 minutes, the insoluble flocculent matter is filtered through a filter paper funnel, and then heated and concentrated at 95°C for 20 minutes to 1 / 2 of the original volume to obtain concentrated initial extract. The extract is then cooled and crystallized at 4°C for 3 days to obtain crystals, which are washed twice with methanol to obtain the white malachite alcohol sample.

[0069] Comparative Example 4

[0070] The only difference from Example 1 is that the step of "weighing sea buckthorn leaf powder, mixing sea buckthorn leaf powder with petroleum ether at a ratio of 1g:20mL, heating and refluxing at 65°C for 2h, fully evaporating the petroleum ether and drying to obtain defatted sea buckthorn leaf powder" is not included. Instead, sea buckthorn leaf powder (not defatted) is directly mixed with 1500mL of 80% ethanol aqueous solution, and the remaining steps are the same as those described in Example 1 to prepare the white peacock alcohol sample.

[0071] Comparative Example 5

[0072] The only difference from Example 1 is that the step of "mixing the concentrated reaction solution with methanol at a volume ratio of 1:5, after mixing, flocculent precipitate gradually appears in the mixture, after standing for 2 hours, retaining the precipitate, repeating this step 3 times, and combining the precipitates to obtain the initial extract" is not included. Instead, the step of "mixing the concentrated reaction solution with methanol at a volume ratio of 1:5, after mixing, flocculent precipitate gradually appears in the mixture, after standing for 2 hours, retaining the precipitate to obtain the initial extract" is included.

[0073] Experimental Example 1

[0074] Nuclear magnetic resonance detection of primary extract and its supernatant

[0075] To further clarify the distribution of baicalein during the extraction process, the primary extract and baicalein reference standard in Example 1 were subjected to nuclear magnetic resonance detection and hydrogen spectrum analysis.

[0076] The results are as follows Figure 8 , 9 As shown in Figure 10, the hydrogen spectrum signal of cymenol in the supernatant corresponding to the initial extract in Example 1 is relatively weak. Figure 8 The primary extract contained a strong response to the characteristic hydrogen spectrum of cymenol. Figure 9 ), and its characteristic 1H spectrum is the same as that of the reference standard for cymbidium faberi (Calvatia spp.). Figure 10 This proves that basilol is distributed in the primary extract.

[0077] Nuclear magnetic resonance detection of the white malachite alcohol sample prepared in Example 1

[0078] The white malachite alcohol sample and white malachite alcohol reference standard prepared in Example 1 were subjected to nuclear magnetic resonance detection and carbon spectrum analysis.

[0079] The results are as follows Figure 11 and 12 As shown, the characteristic carbon spectrum of the basilol sample prepared in Example 1 is the same as that of the basilol reference standard, proving that the sample prepared in Example 1 is indeed a basilol sample.

[0080] Experiment Example 2

[0081] X-ray diffraction analysis of basilol samples

[0082] The crystal structure of the basilol sample prepared in Example 1 was determined by X-ray diffraction.

[0083] The results are as follows Figure 1 As shown, it can be proven that the white malachite alcohol sample prepared in Example 1 is a crystalline material, and the angles of its strongest diffraction peaks are 16.32°, 20.90°, 27.90°, 36.18°, 17.08°, and 38.56°, respectively.

[0084] Experimental Example 3

[0085] Detection of basilol samples by nuclear magnetic resonance spectroscopy

[0086] The white malachite alcohol sample prepared in Example 1 was detected by nuclear magnetic resonance spectroscopy.

[0087] The results are as follows: NMR data of the baicalein sample prepared in Example 1:

[0088] 1 H-NMR (D2O, 500MHz) δ: 3.33 (1H, dd, J=3.0, 9.6Hz, H-2), 3.38 (3H, s, H-OMe), 3.54 (2H, m, H-3 , 4), 3.67 (1H, dd, J=9.7, 3.2Hz, H-5), 3.99 (1H, t, J=3.7Hz, H-6), 4.20 (1H, t, J=3.7Hz, H-1);

[0089] 13 C-NMR (D2O, 125MHz) δ: 56.88 (Me), 67.13 (C-1), 80.15 (C-2), 72.82 (C-3), 71.91 (C-4), 70.34 (C-5), 71.35 (C-6).

[0090] Experiment Example 4

[0091] Infrared spectroscopy analysis of white pea alcohol samples

[0092] The white pea alcohol sample prepared in Example 1 was analyzed by infrared spectroscopy.

[0093] The results are as follows Figure 3 As shown, the characteristic peaks of the baicalein sample prepared in Example 1 are consistent with those of the baicalein reference standard, with the main peaks appearing at 3291, 2910, 1439, 1325, 1139, 1042, 910, and 756 cm⁻¹. -1 Nearby. 3291cm -1 The broad peak at 2910 cm⁻¹ is the peak of hydroxyl stretching vibration; -1 These are stretching vibration peaks of hydrocarbons (CH); 1439, 1325, 1139, and 1042 cm⁻¹. -1 Both peaks belong to the CO vibration of the ring, at 910 and 756 cm⁻¹. -1 These are vibrational peaks of CH and CC.

[0094] Experimental Example 5

[0095] HPLC-QQQ-MS Detection of White Peony Alcohol Samples

[0096] High performance liquid chromatography coupled with mass spectrometry (HPLC) was used, with a triple quadrupole mass spectrometer, to detect the basilol sample prepared in Example 1.

[0097] The results are as follows Figure 4 and 5 As shown, the malachite alcohol sample prepared in Example 1 has an m / z of 217.0 [M+Na] in positive ion mode. + The quasi-molecular ion peak has a relative molecular mass of 194.0, consistent with the reference standard of basilol.

[0098] Experimental Example 6

[0099] The purity of the white malachite alcohol samples prepared in Examples 1-3 and Comparative Examples 1-5 was calculated using the external standard quantification method.

[0100] Table 1. Purity of the malachite alcohol samples prepared in Examples 1-3 and Comparative Examples 1-5

[0101] Group Purity (%) of white malachite alcohol sample Example 1 94.15 Example 2 90.20 Example 3 93.44 Comparative Example 1 88.97 Comparative Example 2 86.14 Comparative Example 3 70.01 Comparative Example 4 89.35 Comparative Example 5 78.89

[0102] The results are shown in Table 1. The purity of the white malachite alcohol sample prepared in Example 1 of this invention can reach 94.15%. The purity of the white malachite alcohol samples prepared by the preparation method of this invention is all higher than 90%. In Comparative Example 1, the purity of the extracted white malachite alcohol was affected after changing the concentration of the extraction solvent. In Comparative Example 2, the purity of the white malachite alcohol sample was reduced after adjusting the methanol concentration. In Comparative Example 3, the step of acid hydrolysis of the initial extract was omitted, resulting in the failure of polysaccharide impurities in the sea buckthorn leaf powder to dissolve, and a large amount of precipitated substances significantly affected the crystallization process of white malachite alcohol. In Comparative Example 4, the sea buckthorn leaf powder was not defatted, resulting in limited dissolution of the effective active ingredients in the sea buckthorn leaf powder, which limited the precipitation of white malachite alcohol and reduced the purity of the prepared white malachite alcohol sample. In Comparative Example 5, the step of repeatedly treating the concentrated reaction solution and methanol mixture to precipitate flocculent matter three times was omitted, resulting in less white malachite alcohol precipitating from the concentrated solution and affecting the yield of the white malachite alcohol sample.

[0103] The extraction method described in this invention is simple to operate. A suitable solvent is selected to reduce impurities in the extract, minimizing purification steps and achieving high yield and purity in a single step. Analysis shows that the concentration of malachite alcohol in sea buckthorn leaves is 5.244 mg / g. The extraction rate in Example 1 of this invention is 4.00 mg / g, which calculates to a yield of 4 / 5.244*100%, or 76.28%, indicating a high yield. By selecting a suitable solvent, the dissolution of other water-soluble impurities is reduced during malachite alcohol extraction, thus minimizing subsequent purification steps. CaO precipitation is used to remove a large amount of impurities from the extract, and the low solubility of malachite alcohol in cold methanol solution is utilized for further precipitation and separation, eliminating cumbersome purification steps such as macroporous resin purification and organic solvent nanofiltration membrane purification. The extraction method described in this invention involves simple equipment with low energy consumption and utilizes the solubility properties of malachite alcohol multiple times, improving the extraction efficiency of malachite alcohol samples.

[0104] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rapid extraction method for spirulinaol from sea buckthorn leaves, characterized in that, Includes the following steps: Defatted sea buckthorn leaf powder was mixed with an ethanol aqueous solution and extracted to obtain an extract. The extract was concentrated under vacuum for the first time to obtain a concentrated extract. The concentrated extract was mixed with distilled water to separate the initial extract. The ethanol-water solution is an ethanol-water solution with a volume fraction of 80%; The initial extract was mixed with calcium oxide, and after the reaction, the pH was adjusted and the mixture was filtered to obtain the reaction solution. The reaction solution was adsorbed using a macroporous cation exchange resin and then concentrated under vacuum to obtain a concentrated reaction solution. The concentrated reaction solution was mixed with methanol, allowed to stand and precipitate, and the process was repeated 3 times. The precipitates obtained from the 3 times were combined to obtain the initial extract. The initial extract was adjusted to pH 5-6 with 1M HCl aqueous solution, then concentrated by heating at 90-100℃ for 15-20 min, and crystallized to obtain basilol.

2. The rapid extraction method according to claim 1, characterized in that, The defatted sea buckthorn leaf powder is prepared by mixing sea buckthorn leaf powder with petroleum ether, reflux treatment, and drying.

3. The rapid extraction method according to claim 1, characterized in that, The mixing ratio of defatted sea buckthorn leaf powder to 80% ethanol aqueous solution is 100g:1500mL. The extraction is performed 3 times, the extraction temperature is 20-28℃, and the extraction time for each extraction is 2h.

4. The rapid extraction method according to claim 1, characterized in that, The vacuum degree of the first vacuum concentration is 0.07 MPa, the temperature is 55℃, and the time is 20-30 min; the ratio of the concentrated extract to distilled water is 1 g: 10 mL.

5. The rapid extraction method according to claim 1, characterized in that, The ratio of the initial extract to calcium oxide is 100 mL: 0.6 g, the reaction time is 15–30 min, and the pH value is adjusted to 7–8.

6. The rapid extraction method according to claim 1, characterized in that, The vacuum degree of the second vacuum concentration is 0.07 MPa, the temperature is 55°C, and the time is 20-30 min; the volume of the concentrated reaction solution is 15-20% of the total volume of the reaction solution.

7. The rapid extraction method according to claim 1, characterized in that, The volume ratio of the concentrated reaction solution to methanol is 1:5, and the settling time is 2 hours.

8. The rapid extraction method according to claim 1, characterized in that, The initial extract is heated and concentrated to 1 / 3 of its original volume.

9. The rapid extraction method according to claim 1, characterized in that, The crystallization temperature is 4°C, and the crystallization time is 2-3 days. After crystallization, crystals are obtained, and the crystals are washed twice with methanol to obtain basilol.

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