Preparation process of high-content ligustrazine osmanthus standard active substance
High-content privetin was extracted from osmanthus flowers using a purely physical processing method. The extraction was carried out by heating pure water, followed by membrane separation and purification with macroporous resin. This method solved the problems of cumbersome preparation process and high cost in the existing technology, and realized efficient and environmentally friendly preparation and large-scale production of privetin.
Patent Information
- Application Number
- CN202311134856.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-04
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2043-09-04
AI Technical Summary
Existing technologies are difficult to efficiently prepare high-content privetin glycosides from osmanthus standard active ingredients, and the preparation process is cumbersome, costly, and difficult to achieve large-scale production.
High-purity privetin was extracted from osmanthus using a purely physical processing method. Modern membrane equipment and centrifugation technology, including pure water heating extraction, filtration, membrane separation, and concentration, were used in combination with macroporous resin and gel chromatography purification to prepare high-purity privetin.
The preparation of high-content privetin has been achieved, reducing production costs, improving the environmental friendliness and safety of the preparation process, and facilitating large-scale production.
Smart Images
Figure CN117247421B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of natural drug component extraction technology, specifically to a preparation process of a high-content privetin standard active ingredient from osmanthus. Background Technology
[0002] Osmanthus fragrans (Thunb.) Lour., also known as mountain osmanthus or rock osmanthus, grows among rocky hills and belongs to the genus Osmanthus in the family Oleaceae. There are 31 species of Osmanthus worldwide, 26 of which are endemic in China, making China the distribution center of this genus and the earliest country in the world to cultivate and use osmanthus. Osmanthus is one of my country's ten traditional famous flowers, with a cultivation history of over 2500 years. There are numerous varieties of osmanthus, approximately 154 in my country, divided into four cultivar groups: Four Seasons Osmanthus, Silver Osmanthus, Golden Osmanthus, and Red Osmanthus. According to the *Chinese Materia Medica*, osmanthus, also known as sweet osmanthus flower, is the flower of Osmanthus fragrans. It is harvested when it blooms in September and October, impurities are removed, it is dried in the shade, and stored in an airtight container. Osmanthus is rich in over 100 active ingredients, with representative functional components including polyphenols, flavonoids, and terpenes. Osmanthus has a pungent and warm nature, and enters the lung, spleen and kidney meridians. It has the effects of warming the lungs and resolving phlegm, dispelling cold and relieving pain. It is mainly used to treat cold pain in the stomach and abdomen, abdominal pain due to cold hernia, amenorrhea and dysmenorrhea.
[0003] Privet glycoside is a secoiridoid derivative containing a tyrosol unit and a unique open-ring secoiridoid structural unit. It exhibits both hydrophilic and lipophilic properties, and is soluble in hot water and certain proportions of organic solvents such as ethanol, water, methanol, and acetone. Its chemical structure is very similar to oleuropein (oleuropein contains a hydroxytyrosol structural unit, and the rest is the same as privet glycoside), and it possesses high medicinal value.
[0004] Tang Min et al. obtained an extract from Osmanthus fragrans using a 95% ethanol-water flash extraction method, followed by sequential extraction with petroleum ether, ethyl acetate, and n-butanol. From the ethyl acetate extract, privet glycoside was purified using silica gel chromatography, polyamide column chromatography, and dextran gel chromatography (West China Pharmaceutical Journal, 2009, 24(1), 010-013). This method for purifying and preparing privet glycoside is cumbersome, impractical, and unsuitable for large-scale preparation.
[0005] Tong Jun et al. used HPLC-ESI-QTOF-MS liquid chromatography-mass spectrometry to qualitatively analyze Osmanthus fragrans and reported that it contains privetin (Acta Pharmaceutica Sinica 2023, 58(3), 750-759). Literature search showed that there were no reports on the content of privetin in Osmanthus fragrans. Privetin standard products are expensive on the market, about 8,000-10,000 yuan / 10mg, and it is difficult and costly to prepare high-purity privetin.
[0006] Therefore, it is necessary and promising to develop new methods for the efficient preparation of high-content privetin standard active substances using osmanthus as raw material. Summary of the Invention
[0007] The purpose of this invention is to provide a preparation process for high-content privetin Osmanthus standard active ingredients, using Osmanthus as raw material and employing a purely physical processing technology to efficiently prepare high-content privetin Osmanthus standard active ingredients PHEG60 and PHEG70.
[0008] Therefore, the present invention adopts the following technical solution:
[0009] This invention provides a preparation process for a high-content standard active ingredient of privetin from osmanthus, comprising the following steps:
[0010] (1) Take dried osmanthus flowers, add a certain amount of pure water, heat and stir for a certain time, filter, and collect filtrate 1 and residue 1 respectively; add a certain amount of pure water to the residue 1, heat and stir for a certain time, filter, and collect filtrate 2.
[0011] (2) Combine filtrate 1 and filtrate 2, filter again to remove fine particles, collect the filtrate, and separate the components in the filtrate;
[0012] The separation method is as follows: the filtrate is eluted through a macroporous resin gradient or collected by an ultrafiltration membrane, and the solvent used in the separation process is pure water or ethanol-water solvent.
[0013] (3) The eluent or ultrafiltration membrane retentate collected in step (2) is concentrated by membrane separation, wherein the membrane separation type includes nanofiltration membrane or RO membrane. The obtained concentrate is further concentrated under reduced pressure or freeze-dried to obtain an extract, wherein the extract is the standard active ingredient of osmanthus, total phenylethanol glycosides (PHEG).
[0014] The purity of privet glycoside in the standard active ingredient PHEG60 of osmanthus is ≥50%, and the total phenylethanoid glycoside content is ≥60%.
[0015] Preferably, it is any one or more of the following: silver osmanthus, golden osmanthus, and red osmanthus.
[0016] Preferably, the amount of pure water added is 10-50 mL per 1 g of osmanthus flowers;
[0017] More preferably, the amount of pure water added is 15-30 mL per 1 g of osmanthus flowers.
[0018] Preferably, the temperature of the two pure water extractions in step (1) is 70-100℃;
[0019] More preferably, the temperature of the two pure water extractions in step (1) is 85-95℃;
[0020] More preferably, the extraction temperature in step (1) is 90-95℃ for both extractions.
[0021] Preferably, the first stirring time in step (1) is 30-180 min;
[0022] More preferably, the first stirring time in step (1) is 60-90 min;
[0023] And / or, the second stirring time in step (1) is 15-60 min;
[0024] More preferably, the second stirring time in step (1) is 30-45 min.
[0025] Preferably, the filtration method in step (1) or step (2) is screen filtration, centrifugal filtration or membrane filtration.
[0026] More preferably, the filter mesh size is 100-200 mesh; and / or, the centrifugal filtration includes disc centrifugation, horizontal centrifugation and tubular centrifugation; and / or, the membrane filtration is ceramic membrane filtration.
[0027] Preferably, the macroporous resin in step (2) includes any one or more of the following types: D101, HP-20, AB-8, XAD1600, LSA-21, and DM-301.
[0028] Preferably, the concentration of ethanol in the ethanol-water solution in step (2) is 0-95%, and the gradient elution process first uses a low-concentration ethanol-water solution and then uses a high-concentration ethanol-water solution for elution, and the gradient elution of the ethanol-water solution is repeated 1-5 times.
[0029] More preferably, the concentration gradient of ethanol in the aqueous ethanol solution can be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
[0030] For example, the solution is first eluted with 30%-40% ethanol and water for 2-3 column volumes, and then eluted with 60%-70% ethanol and water for 1-2 column volumes.
[0031] Preferably, the ultrafiltration membrane separation method in step (2) is as follows: the filtrate is filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons, and the filtrate is then filtered through an ultrafiltration membrane with a molecular weight cutoff of 300 Daltons, and the ultrafiltration membrane retentate is collected.
[0032] Preferably, the RO membrane concentration method in step (3) is further as follows: the RO membrane is concentrated to a solid content of 20-30%, frozen overnight at 0-4°C, the frozen liquid is centrifuged and filtered, and the centrifuged precipitate is vacuum dried or freeze-dried to obtain the extract of Osmanthus standard active ingredient phenylethanol total glycosides PHEG70, wherein the purity of privet glycoside is ≥60% and the phenylethanol total glycosides are ≥70%.
[0033] Preferably, the method further includes a purification step, wherein the purification method is as follows: the total phenylethyl glycosides (PHEG) of the Osmanthus standard extract is dissolved in a 95% ethanol aqueous solution, passed through a gel chromatography column, and eluted with 95% ethanol water. The eluent rich in privet glycosides is collected, and the eluent is concentrated under reduced pressure to obtain the extract; wherein the extract is high-purity privet glycosides.
[0034] More preferably, the gel chromatography column is a Sepheedex LH-20 gel chromatography column.
[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0036] The preparation process provided by this invention uses osmanthus as raw material. my country has numerous varieties of osmanthus, making the raw material readily available. This preparation process can obtain high-content privetin-based standard active ingredients from osmanthus. HPLC analysis shows that the total phenylethanoid glycosides (PHEG60, including verbascoside, isoverascoside, 10-hydroxyprivetin, and privetin) are ≥60%, with privetin content ≥50%; PHEG70 shows privetin content ≥60%, and total phenylethanoid glycosides ≥70%. Further purification of the total phenylethanoid glycosides obtained using this preparation process yields high-purity privetin. This preparation process primarily utilizes pure water extraction, with the only organic solvent being edible alcohol (ethanol). It comprehensively utilizes modern membrane equipment such as ceramic membranes, nanofiltration membranes, and ultrafiltration membranes, as well as purely physical processes such as centrifugation and freeze-drying. Therefore, this preparation process is more environmentally friendly, safer, significantly reduces production costs, is easily scalable, and has excellent application prospects. Attached Figure Description
[0037] Figure 1 This is the HPLC spectrum of PHEG60, the standard active ingredient of Osmanthus fragrans in Example 1 of this invention, at a wavelength of 280 nm.
[0038] Figure 2 This is the HPLC spectrum of GH-B (privet glycoside) in Example 4 of the present invention at a wavelength of 280 nm;
[0039] Figure 3 This is the hydrogen spectrum of GH-B (privet glycoside) in Example 4 of the present invention;
[0040] Figure 4This is the carbon spectrum of GH-B (privet glycoside) in Example 4 of the present invention;
[0041] Figure 5 This is the ESI-MS mass spectra of GH-B (privet glycoside) in Example 4 of the present invention. Detailed Implementation
[0042] The specific implementation methods of the technical solution described herein will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for the purpose of describing the technical solution in detail, and not for limiting the technical solution. Based on the embodiments in this description, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this description.
[0043] Example 1
[0044] A preparation process for a high-content privetin standard active ingredient from osmanthus flowers includes the following steps:
[0045] (1) Take 1500g of dried Osmanthus fragrans flowers, add 22.5L of pure water, heat to 95℃, stir and extract for 60min, filter through a 200-mesh filter, and collect filtrate 1 and residue 1 respectively; add 15L of pure water to the residue 1, heat to 95℃, stir and extract for 30min, filter through a 200-mesh filter, and collect filtrate 2.
[0046] (2) Combine filtrate 1 and filtrate 2, filter by centrifuge, and directly load the supernatant into an AB-8 macroporous resin column (column bed volume 7.5L). Elute with 30L of 30% ethanol aqueous solution, and then continue eluting with 10L of 90% ethanol aqueous solution. Collect the 90% ethanol aqueous eluent.
[0047] The 90% ethanol-water eluent collected in step (2) was concentrated and evaporated under reduced pressure to obtain extract A, which is the standard active ingredient of Osmanthus PHEG60-1.
[0048] Example 2
[0049] A preparation process for a high-content privetin standard active ingredient from osmanthus flowers includes the following steps:
[0050] (1) Take 20kg of dried Osmanthus fragrans flowers, add 300L of pure water to a 500L extraction tank, heat to 90℃, stir and extract for 60min, filter through a 200-mesh filter, and collect filtrate 1; add 200L of pure water to the filter residue again, heat to 90℃, stir and extract for 30min, filter through a 200-mesh filter, and collect filtrate 2.
[0051] (2) Combine filtrate 1 and filtrate 2, filter them twice through a ceramic membrane, and pass the ceramic membrane filtrate directly through a D101 macroporous resin column (column bed volume 150L) at a loading flow rate of 300L / h. After loading, add 75L of pure water for elution, then continue elution with 450L of 30% ethanol aqueous solution, and then add 200L of 60% ethanol aqueous solution for elution. Collect the 60% ethanol aqueous eluent.
[0052] (3) The 60% ethanol-water eluent collected in step (2) is concentrated by nanofiltration membrane, and the 60% ethanol-water permeate is recovered. The nanofiltration membrane retentate is 20L, which is concentrated and dried under reduced pressure to obtain extract B. The extract is the standard active ingredient of Osmanthus PHEG60-2.
[0053] Example 3
[0054] A preparation process for a high-content privetin standard active ingredient from osmanthus flowers includes the following steps:
[0055] (1) Take 20kg of dried Osmanthus fragrans flowers, add 300L of pure water, heat to 95℃, stir and extract for 60min, filter through a 200-mesh filter and collect filtrate 1; add 200L of pure water to the residue again, heat to 90℃, stir and extract for 30min, filter through a 200-mesh filter and collect filtrate 2.
[0056] (2) Combine filtrate 1 and filtrate 2, filter through a ceramic membrane, then pass the ceramic membrane filtrate through a polyethersulfone ultrafiltration membrane (molecular weight cutoff of 10,000 Daltons), and then pass the 10,000 Dalton ultrafiltration membrane filtrate through a 300 Dalton ultrafiltration membrane (molecular weight cutoff of 300 Daltons). Collect the 300 Dalton ultrafiltration membrane retentate.
[0057] (3) The ultrafiltration membrane retentate collected in step (2) is concentrated by RO membrane until the solid content Brix is 20%. The concentrate is placed in a cold storage overnight, centrifuged and filtered, and the centrifuged precipitate is freeze-dried to obtain extract C, wherein extract C is the standard active ingredient of Osmanthus PHEG70.
[0058] Example 4
[0059] A preparation process for a high-content privetin standard active ingredient from osmanthus flowers includes the following steps:
[0060] (1) Take 20kg of dried Osmanthus fragrans flowers, add 400L of pure water, heat to 95℃, stir and extract for 60min, filter with a 200-mesh filter, and collect filtrate 1; add 200L of pure water to the residue again, heat to 80℃, stir and extract for 30min, filter with a 200-mesh filter, and collect filtrate 2.
[0061] (2) Combine filtrate 1 and filtrate 2, filter them with a tubular centrifuge, load the filtrate directly onto an HP-20 macroporous resin column (column bed volume 200L), elute with 400L of 40% ethanol aqueous solution, and then elute with 300L of 70% ethanol aqueous solution. Collect the eluent of 70% ethanol aqueous solution.
[0062] (3) The eluent collected in step (2) is concentrated by nanofiltration membrane, and the ethanol aqueous solution is recovered. The nanofiltration membrane retentate is 30L, which is concentrated under reduced pressure and vacuum to obtain extract D. The extract is the standard active ingredient of Osmanthus PHEG60-3.
[0063] (4) Take 10g of extract D, dissolve it in 40ml of 95% ethanol aqueous solution, pass it through a Sepheedex LH-20 gel chromatography column, and elute with 95% ethanol water. Detect the eluent by HPLC. After combining the fractions, concentrate under reduced pressure to obtain high-purity compound E (labeled as GH-B).
[0064] Test examples: chemical structure identification and purity analysis
[0065] (I) Detection and analysis by proton, carbon, and ESI-MS mass spectrometry
[0066] The GH-B prepared in Example 4 was analyzed by proton NMR, carbon NMR, and ESI-MS mass spectrometry. The results are as follows: Figure 3-5 As shown.
[0067]
[0068] The structure of ligustroside is shown in Formula I. Its molecular weight is 524 Daltons and its CAS number is 35897-92-8.
[0069] right Figure 3-4 The 1H NMR, 1C NMR, and ESI-MS mass spectrometry analyses showed that the structure was completely consistent with Formula I. This indicates that GH-B prepared in Example 4 of this invention is ligustroside.
[0070] (II) Content Analysis (HPLC)
[0071] Test conditions:
[0072] A Waters e2695 HPLC system was used with a 2998PDA Detector.
[0073] The chromatographic column is an Atlantis. TM T3, 5µm, 4.6x250mm; detection wavelengths: 250nm, 280nm; flow rate: 1ml / min; column temperature: 30±2℃. The mobile phase gradient is shown in Table 1 below.
[0074]
[0075] Table 1. Mobile Phase Gradient Table
[0076] The extracts AE prepared in Examples 1-4 were analyzed by HPLC, and the results are as follows: Figure 1 , Figure 2 As shown in Table 2.
[0077] As shown in Table 2, the total phenylethanoid glycosides content of PHEG60 prepared in Examples 1, 2 and 4 is higher than 60%, and the content of the main indicator component, ligustrazine, is higher than 50%; the total phenylethanoid glycosides content of PHEG70 prepared in Example 3 is higher than 70%, and the content of the main indicator component, ligustrazine, is higher than 60%.
[0078] Figure 1 This is the chromatogram of PHEG60-1, the standard active ingredient of Osmanthus fragrans prepared in Example 1, at a wavelength of 280 nm. Figure 2 This is the chromatogram of the high-purity compound GH-B obtained in Example 4 at a wavelength of 280 nm. A comparison shows that... Figure 4 The low number of impurity peaks indicates that high-purity privet glycosides were isolated through purification steps.
[0079] Table 2. Content of privetin and other phenylethanoid glycosides in the standard active ingredients.
[0080]
[0081] The preparation process of this invention can be used to prepare a series of osmanthus standard active substances with high content of privetin, and to separate high purity privetin.
[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A preparation process for a high-content privetin-containing standard active ingredient from osmanthus, characterized in that, Includes the following steps: (1) Take dried osmanthus flowers, add a certain amount of pure water, heat and stir for a certain time, filter, and collect filtrate 1 and residue 1 respectively; add a certain amount of pure water to the residue 1, heat and stir for a certain time, filter, and collect filtrate 2. (2) Combine filtrate 1 and filtrate 2, filter again, collect the filtrate, and separate the components in the filtrate; the separation method is: pass the filtrate through a macroporous resin gradient elution and collect the eluent; The macroporous resin is selected from any one or more of the following models: D101, HP-20, AB-8, XAD1600, LSA-21, and DM-301. The solvent used for gradient elution of the macroporous resin is an aqueous ethanol solution with a concentration of 0-95%. In the gradient elution process, a low-concentration aqueous ethanol solution is used for elution first, followed by a high-concentration aqueous ethanol solution. The number of gradient elutions with the aqueous ethanol solution is 1-5 times. (3) The eluent collected in step (2) is concentrated by membrane separation to obtain the standard active ingredient of osmanthus, total phenylethanol glycosides, wherein the membrane separation type is selected from nanofiltration membrane; Furthermore, the purification process includes the following steps: the total phenylethyl glycosides of the osmanthus standard active ingredient are dissolved in a 95% ethanol aqueous solution, passed through a gel chromatography column, and eluted with 95% ethanol water. The eluent rich in privet glycosides is collected, and the solution is concentrated under reduced pressure to obtain privet glycosides. The gel chromatography column was a Sepheedex LH-20 gel chromatography column.
2. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 1, characterized in that, The osmanthus mentioned is any one or more of silver osmanthus, golden osmanthus, and red osmanthus.
3. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 1, characterized in that, The amount of pure water added is 15-50 mL per 1 g of osmanthus flowers.
4. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 1, characterized in that, The extraction temperature in step (1) is 70-100℃ for both extractions.
5. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 1, characterized in that, The first extraction time in step (1) is 30-180 min; and / or the second extraction time is 15-60 min.
6. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 1, characterized in that, The filtration method in step (1) or step (2) includes one or more of the following: filter screen, centrifugal filtration, or membrane filtration.
7. The preparation process of the high-content privetin-osmanthus standard active ingredient according to claim 6, characterized in that, The filter screen has a mesh size of 100-200; and / or, the centrifugal filtration includes disc centrifugation, horizontal centrifugation, and tubular centrifugation; and / or, the membrane filtration is ceramic membrane filtration.