Preparation process of maitaiweng saponin E

Abstract

The application discloses a production and preparation process of agrostemma githago saponin E and belongs to the field of natural product chemistry. The process efficiently prepares agrostemma githago saponin E by taking agrostemma githago as raw material, and realizes directional enrichment and purification of the target component through a ladder type process of 'raw material degreasing pretreatment, 70% water alcohol reflux extraction, sequential solvent distribution of n-hexane-ethyl acetate-n-butanol, macroporous resin (HP-20 / XAD-7) enrichment, silica gel column gradient chromatography, preparation type C18-HPLC refining, and trehalose / cyclodextrin co-freeze drying stabilization', process parameters are controllable, the product purity is greater than or equal to 95%, and the storage stability is significantly improved (degradation rate is less than 0.6% when stored in dark at-20 DEG C for 12 months), and the operation process meets the requirements of industrial production, and has good industrial applicability.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and preparation technology, and in particular to a production and preparation process for maltodextrin E. Background Technology

[0002] Agrostemmoside E is a triterpenoid glycoside saponin derived from Agrostemma githago, with the molecular formula C. 84 H 128 O 45 It has a molecular weight of 1856.77, which is large and has a complex structure (as shown in Formula I).

[0003]

[0004] Ophiopogonin E possesses various biological activities, including anti-inflammatory, antioxidant, and antitumor effects, and has significant development value in the pharmaceutical and health product fields. However, its natural content is extremely low, it exhibits chemical instability, and its crude extract contains numerous impurities (such as other saponins, tannins, and polyphenols), limiting its industrial application. Existing processes often result in low purity, contain residual impurities, or require multi-step chromatographic separation, making efficient large-scale production difficult.

[0005] Therefore, there is a need for an efficient, scalable, and repeatable process to produce maltodextrin E with pharmaceutical-grade purity while minimizing degradation and maximizing yield. Summary of the Invention

[0006] This invention provides a process for producing maltodextrin E, which includes the following steps:

[0007] (1) Raw material pretreatment: Select the above-ground parts (stems, leaves, flowers) or seeds of *Ophiopogon japonicus* as raw materials, dry them and then crush them to a particle size of <1 mm (to increase the solvent contact area), and defatt them with petroleum ether or n-hexane at room temperature for 2-3 hours. This step can remove more than 90% of fat-soluble impurities (such as phytosterols and fatty acids) in advance, avoid impurities from co-dissolving with the target saponins during subsequent extraction, and reduce the difficulty of purification.

[0008] (2) Water-alcohol reflux extraction: Mix the defatted plant powder with a 60-80% ethanol aqueous solution (volume percentage, ethanol accounts for 60-80%) at a material-to-liquid ratio of 1:(8-10) and reflux extract at 50-60℃ for 2-4 hours; preferably a 70% ethanol aqueous solution (to balance saponin solubility and impurity inhibition: too high an ethanol concentration will easily dissolve resin impurities, while too low an ethanol concentration will reduce the saponin dissolution rate). Gentle reflux at 50-60℃ can reduce saponin hydrolysis, and the extraction efficiency is 3-5 times higher than that of the existing room temperature soaking.

[0009] (3) Solvent partitioning and enrichment: After dissolving the crude extract in water, it is partitioned sequentially with n-hexane, ethyl acetate and n-butanol: the n-hexane phase removes residual lipid-soluble impurities, the ethyl acetate phase removes moderately polar impurities (such as flavonoids and oligosaccharides), and the n-butanol phase directionally retains saponin components; this step can initially enrich the target components and the impurity removal rate is more than 60%.

[0010] (4) Macroporous resin purification: The n-butanol phase is loaded into HP-20 or XAD-7 type macroporous resin (both types of resins have large adsorption capacity and high desorption rate for saponins). First, water is used to wash away water-soluble impurities (such as monosaccharides and inorganic salts), and then the saponins are eluted with 70% methanol aqueous solution (volume percentage, methanol accounts for 70%). The concentration of 70% methanol was determined by optimization - if it is lower than 60%, the saponins will not be completely eluted, and if it is higher than 80%, polar impurities will be easily introduced. This step can improve the purity of saponins to 60-70%.

[0011] (5) Silica gel column chromatography: The macroporous resin elution fraction was dissolved in a 1:1 methanol-water mixture (to ensure sample solubility), loaded onto a silica gel column, and eluted with a chloroform-methanol-water gradient (70:30:5→60:40:10). Gradient elution can effectively separate saponin homologues with similar structures (such as the separation degree between sclerosing saponin E and sclerosing saponin D is improved to more than 1.5). The target fraction was collected by TLC or HPLC monitoring, and the purity was further improved to 85-90%.

[0012] (6) Preparative HPLC purification: The target fraction was loaded onto a C18 stationary phase preparative HPLC system, using water-acetonitrile containing 0.05% TFA as the eluent (TFA can improve the peak shape of saponins and reduce tailing), and eluted for 30-45 minutes at a 30-60% acetonitrile concentration gradient; the sample was then purified by LC-MS / MS (monitoring the characteristic molecular ion peak of Ophiopogon japonicus saponin E) and... 1 H / 13 After C NMR identification and concentration of the target fractions, sterculia saponin E with a purity ≥95% was obtained.

[0013] (7) Stabilization treatment (optional): Mix the refined maltose saponin E with 5-10% trehalose or cyclodextrin (trehalose / cyclodextrin can form hydrogen bonds to protect saponin molecules and inhibit hydrolysis and oxidation), and freeze-dry (low-temperature freeze-drying avoids saponin degradation caused by high temperature); the freeze-dried product is stored at -20℃ under light-protected conditions, and the shelf life is extended to more than 12 months.

[0014] Optionally, in some embodiments, the extraction solvent is a 70% aqueous ethanol solution.

[0015] Optionally, in some embodiments, the macroporous resin is of type HP-20.

[0016] Optionally, in some embodiments, the silica gel column elution gradient is 70:30:5 (chloroform-methanol-water) for 1 hour, then switched to 60:40:10 for continued elution.

[0017] Optionally, in some implementations, the stabilizing excipient is 8% trehalose.

[0018] The production process of malt extract saponin E in this invention has the following beneficial effects:

[0019] (1) High extraction efficiency: Through “defatting pretreatment + optimization of water and alcohol extraction parameters”, the dissolution rate of maltose saponin E is increased by 3-5 times, and the extraction time is shortened from 8-12 hours to 2-4 hours, which greatly reduces energy consumption;

[0020] (2) High product purity: The product purity is ≥95% by using a step-by-step purification method of "solvent partitioning-dual column chromatography-preparative HPLC", which meets the requirements of pharmaceutical grade raw materials;

[0021] (3) High yield: The yield of Ophiopogon japonicus saponin E reaches 0.01-0.05% of the mass of dried Ophiopogon japonicus raw material;

[0022] (4) By “excipient protection + freeze drying + low temperature and light protection storage”, the product has a shelf life of more than 12 months, which solves the problem of easy degradation of maltose saponin E;

[0023] (5) Good industrial applicability: All process parameters are controllable (such as temperature, solvent concentration, elution gradient), the batch-to-batch product purity and yield deviation is ≤5%, and the solvents used (ethanol, chloroform, methanol, etc.) are easy to recover, which meets the requirements of green industrial production. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the embodiments of the present invention are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. All conventional modifications and adjustments made based on the technical solutions of the present invention shall fall within the scope of protection of the present invention.

[0025] Example 1

[0026] 1. Raw material preparation

[0027] Select fresh above-ground parts of the barley grass (stems + leaves + flowers), wash them, dry them in a forced-air dryer at 45℃ until the moisture content is <8%, and crush them to a particle size of 0.8mm (pass through a 20-mesh sieve) for later use.

[0028] 2. Specific steps

[0029] (1) Take 1.0 kg of pulverized aerial part powder, add 3.0 L of petroleum ether (boiling range 60-90℃), stir and degrease at room temperature (25℃) for 2.5 hours, filter, collect the filter residue (petroleum ether is recovered and reused), and vacuum dry the filter residue at 50℃ until there is no petroleum ether residue.

[0030] (2) Add the defatted filter residue to 10.0L of 70% ethanol aqueous solution (volume percentage, ethanol accounts for 70%), place it in a 20L round bottom flask, and reflux extract in a constant temperature water bath at 55℃ for 3 hours, stirring once every 30 minutes during the extraction. After extraction, filter while hot (filter cloth pore size 0.22μm), collect the filtrate, and extract the filter residue again with 5.0L of 70% ethanol aqueous solution (2 hours). Combine the two filtrates and concentrate under reduced pressure at 60℃ and -0.08MPa until there is no ethanol odor, to obtain 85.2g of crude extract.

[0031] (3) Dissolve the crude extract in 200 mL of deionized water, transfer it to a 1 L separatory funnel, add 200 mL of n-hexane (shake for 10 minutes, let stand for 30 minutes to separate the layers), and discard the n-hexane phase; add 200 mL of ethyl acetate to the remaining aqueous phase, shake and separate the layers in the same way, and discard the ethyl acetate phase; finally add 200 mL of n-butanol, shake and separate the layers, collect the n-butanol phase, concentrate the n-butanol phase to dryness under reduced pressure, and obtain 12.6 g of enriched product.

[0032] (4) Dissolve the enriched product in 50 mL of deionized water and load it onto a pre-activated HP-20 macroporous resin column (column specifications: diameter 5 cm, height 60 cm, resin packing volume 100 mL); first, elute with water at a flow rate of 2 BV / h (BV is column volume) for 5 BV to remove sugar impurities; then elute with 70% methanol aqueous solution (volume percentage, methanol accounts for 70%) at a flow rate of 2 BV / h for 4 BV, collect the methanol eluent, concentrate to dryness, and obtain 4.8 g of crude saponin.

[0033] (5) Dissolve the crude saponin in 10 mL of a 1:1 methanol-water mixture (volume ratio) and load it onto a silica gel column (column specifications: diameter 3 cm, height 40 cm, silica gel particle size 200-300 mesh, packing weight 50 g); first use chloroform-methanol-water = 70:30:5 (volume ratio) as the eluent and elute at a flow rate of 1 BV / h for 1 hour; then switch to chloroform-methanol-water = 60:40:10 and continue eluting at a flow rate of 1 BV / h, collecting one fraction every 10 mL; monitor by TLC (developing solvent: chloroform-methanol-water = 65:35:8), combine the fractions with Rf values ​​of 0.4-0.5, concentrate to dryness, and obtain 1.5 g of the pre-purified product.

[0034] (6) HPLC: The product before purification was dissolved in 5 mL of methanol and loaded onto a preparative reversed-phase HPLC system (instrument: Agilent 1260, column: Zorbax SB-C18, 250 mm × 10 mm, 5 μm); elution conditions: mobile phase A was 0.05% TFA aqueous solution, mobile phase B was acetonitrile, gradient elution (0-10 min: 30% B; 10-30 min: 30%-60% B; 30-40 min: 60% B), flow rate 3 mL / min, column temperature 30 ℃, detection wavelength 203 nm; the fraction corresponding to the chromatographic peak with retention time of 18.5-19.5 min was collected, concentrated to dryness, and 0.32 g of pure senna saponin E was obtained.

[0035] (7) Mix 0.32g of pure product with 0.0256g of 8% trehalose (mass ratio), add 5mL of deionized water to dissolve, and place in a freeze dryer (pre-freeze at -50℃ for 2 hours, vacuum dry at -30℃ for 12 hours) to obtain 0.34g of freeze-dried product.

[0036] 3. Result Verification

[0037] Purity: As determined by analytical HPLC (under the same preparative chromatographic conditions), the purity is 97.2%.

[0038] Yield: Based on dried aerial parts, the yield was 0.032%;

[0039] Stability: After 12 months of storage at -20℃ in the dark, the purity of the lyophilized product was still 96.8% as determined by HPLC, and the degradation rate was <0.5%.

[0040] Example 2

[0041] 1. Raw material preparation

[0042] Take 1.0 kg of wheatgrass seeds, dry them at 40℃ with forced air until the moisture content is <7%, and crush them to a particle size of 0.5 mm (pass through a 30-mesh sieve).

[0043] 2. Specific steps

[0044] (1) Raw material pretreatment: Degrease with 3.0L n-hexane at room temperature for 3 hours, filter and dry the filter residue at 50℃ under vacuum;

[0045] (2) Water-ethanol reflux extraction: Add 10.0L of 70% ethanol aqueous solution (volume percentage, ethanol accounts for 70%), reflux extract at 55℃ for 3.5 hours, repeat the extraction once, combine the filtrates and concentrate to obtain 92.5g of crude extract;

[0046] Steps (3)-(6) are the same as in Example 1 (HP-20 resin, silica gel column gradient elution, and preparative HPLC conditions are consistent).

[0047] (7) Stabilization treatment: Add 8% trehalose and freeze dry.

[0048] 3. Result Verification

[0049] Purity: 96.5%;

[0050] Yield: 0.048% (the saponin content in the seeds is higher than that in the aerial parts);

[0051] Stability: After 12 months of storage, the purity is 96.1% and the degradation rate is <0.5%.

[0052] Example 3

[0053] 1. Raw material preparation

[0054] Same as Example 1 (above-ground portion 1.0 kg).

[0055] 2. Specific steps

[0056] (1) Raw material pretreatment: petroleum ether degreasing for 2 hours;

[0057] (2) Water-ethanol reflux extraction: 10.0L of 65% ethanol aqueous solution (volume percentage, ethanol accounts for 65%) was used for reflux extraction at 60℃ for 2.5 hours. The extraction was repeated once, and the crude extract was concentrated to obtain 81.3g.

[0058] (3) Solvent partitioning enrichment: Same as in Example 1, 11.8 g of enriched product was obtained;

[0059] (4) Macroporous resin purification: XAD-7 type resin was used, and eluted with 70% water and methanol to obtain 4.5g of crude saponin;

[0060] (5)-(6) are the same as in Example 1, and 0.28g of pure product is obtained by preparative HPLC;

[0061] (7) Stabilization treatment: Add 10% cyclodextrin (by mass) and freeze dry.

[0062] 3. Result Verification

[0063] Purity: 95.8%;

[0064] Yield: 0.028%;

[0065] Stability: After 12 months of storage, the purity is 95.3% and the degradation rate is <0.6%.

[0066] Example 4

[0067] 1. Raw material preparation and steps

[0068] Same as Example 1, but step (7) stabilization treatment was omitted. 0.32g of pure product obtained by preparative HPLC was directly vacuum dried at 40°C for 4 hours to obtain 0.32g of dried product.

[0069] 2. Result Verification

[0070] Initial purity: 97.0%;

[0071] Stability: After 3 months of storage at 4℃ in the dark, the purity dropped to 92.1% (degradation rate of 5.1%), and after 6 months it dropped to 88.3%, proving that stabilization treatment can significantly improve stability.

[0072] Example 5

[0073] 1. Raw material preparation

[0074] 10.0 kg of the above-ground parts of the wheatgrass were crushed to a particle size of <1 mm.

[0075] 2. Specific steps

[0076] (1) Raw material pretreatment: Degrease with 30L petroleum ether at room temperature for 2.5 hours (industrial grade petroleum ether, which can be recycled and reused).

[0077] (2) Water-ethanol reflux extraction: 100L industrial extraction tank was used, 100L of 70% ethanol aqueous solution (volume percentage, ethanol accounts for 70%) was added, and the mixture was refluxed at 55℃ for 3 hours. The extraction was repeated once. After plate and frame filtration, the filtrate was concentrated by an industrial-grade vacuum concentrator to obtain 845g of crude extract.

[0078] (3) Solvent partitioning enrichment: Dispersed with 2000 mL of deionized water, and partitioned sequentially with 2000 mL of n-hexane, ethyl acetate, and n-butanol (industrial grade), and concentrated to obtain 128 g of enriched product;

[0079] (4) Macroporous resin purification: An industrial-grade HP-20 resin column (diameter 20cm, height 100cm, resin packing volume 30L) was used. After washing with water for 5 BV, the column was washed with 70% water and methanol for 4 BV. The crude saponin was concentrated to obtain 49g.

[0080] (5) Silica gel column chromatography: Industrial grade silica gel column (diameter 10cm, height 80cm, silica gel packing amount 500g), gradient elution and merging of target fractions to obtain 15.2g;

[0081] (6) Preparative HPLC purification: Industrial-grade preparative HPLC (column specifications: 500mm×20mm, C18) was used, and 3.3g of pure product was obtained after gradient elution;

[0082] (7) Stabilization treatment: 3.5g of product was obtained by freeze-drying 8% trehalose.

[0083] 3. Result Verification

[0084] Purity: 97.0%;

[0085] Yield: 0.033% (basically consistent with the yield at a 1kg scale, proving that the process can be scaled up).

[0086] Batch stability: In three consecutive batch scale-up experiments, the yield fluctuated by ±0.002% and the purity fluctuated by ±0.3%, which meets the requirements for industrialization.

Claims

1. A process for the production of Makino Saponin E, characterized in that, The method comprises the following steps: (a) Pretreatment of raw material: taking the aerial part or seed of Gentiana manshurica Kitag., drying and crushing, and then defatting at room temperature with a defatting solvent, which is petroleum ether or n-hexane; (b) Water-alcohol extraction: mixing the defatted plant powder of step (a) with 60-80% ethanol aqueous solution at a solid-liquid ratio of 1:(8-10), and refluxing at 50-60°C for 2-4 hours, and then filtering and concentrating under reduced pressure to obtain a crude extract; (c) Solvent partitioning and enrichment: dissolving the crude extract of step (b) in water, and then sequentially performing solvent partitioning with n-hexane, ethyl acetate and n-butanol, and retaining the n-butanol phase; (d) Macroporous resin chromatography purification: loading the n-butanol phase of step (c) to a macroporous resin, first eluting with water to remove sugar impurities, and then eluting with 70% water-methanol to obtain saponin components, and collecting and concentrating the eluate; the macroporous resin is HP-20 type macroporous resin or XAD-7 type macroporous resin; (e) Silica gel column chromatography purification: dissolving the concentrated fraction of step (d) in a methanol-water mixture, and then loading to a silica gel column, and eluting with a chloroform-methanol-water gradient elution system, i.e. sequentially eluting with chloroform / methanol / water mixed solutions with a volume ratio of 70:30:5 and 60:40:10, and then monitoring and collecting fractions containing target components by TLC or HPLC; (f) Preparative HPLC final separation: The target fraction collected in step (e) was loaded onto a preparative reverse phase HPLC with C18 stationary phase, gradient elution was performed using water-acetonitrile containing 0.05% trifluoroacetic acid as eluent, acetonitrile concentration from 30% to 60%, the molecular ion peak m / z and 1 H / 13 C NMR identified saponin E, the corresponding fractions were combined and concentrated to obtain saponin E product.

2. The process according to claim 1, characterized in that, In step (a), the particle size of the crushed raw material is less than 1 mm, and the defatting time is 2-3 hours.

3. The process of claim 1, wherein, In step (b), the concentration of ethanol in the ethanol aqueous solution is 70%.

4. The process of claim 1, wherein, In step (e), the volume ratio of the methanol-water mixture is 1:

1.

5. The process of claim 1, wherein, In step (f), the elution time of the preparative HPLC is 30-45 minutes.

6. The process of claim 1, wherein, Further comprising step (g) stabilization treatment: mixing the Gentiana manshurica saponin E product obtained in step (f) with 5-10% trehalose or cyclodextrin, and then jointly performing freeze-drying treatment.

7. The process of claim 6, wherein, The storage condition of the product after freeze-drying treatment in step (g) is -20°C, and the product is stored in the dark.

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