Aralia mandshurica-sourced new skeleton saponin compound and preparation method thereof

Abstract

The invention relates to the technical field of separation and purification of active ingredients of traditional Chinese medicines, and discloses a saponin compound derived from aralia elata and a preparation method thereof. The saponin compound has a structure as shown in a formula (I). The preparation method comprises the following steps: carrying out hydrous ethanol extraction on aralia mandshurica root bark, enriching through macroporous adsorption resin, carrying out directional enrichment on acidic saponin through anion exchange resin, carrying out coarse separation and preparative liquid phase refining by adopting reversed-phase C18 chromatography, and carrying out window collection in combination with online detection to obtain a target saponin compound. The method can improve the separation selectivity of the target component, reduce the co-elution phenomenon and improve the purity and batch stability of the final product, and is suitable for preparation of the aralia mandshurica sourced saponin compound.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology of active ingredients in traditional Chinese medicine, and more specifically to a novel skeleton saponin compound derived from *Aralia elata* and its preparation method. Background Technology

[0002] *Aralia elata*, the dried root, root bark, or stem bark of the plant *Aralia elata* (family Araliaceae), is a natural medicinal plant resource. Current research indicates that *Aralia elata* contains saponins, flavonoids, polysaccharides, and other secondary metabolites, among which saponins have attracted attention due to their high research value. In particular, triterpenoid saponins, due to their diverse structural types and complex sugar chain linkages, have become a key focus in the study of the chemical composition of *Aralia elata*.

[0003] In existing technologies, the acquisition of saponins from *Aralia elata* typically involves extraction with ethanol or aqueous ethanol, followed by separation and purification using macroporous adsorption resins, silica gel column chromatography, or reversed-phase chromatography. While this approach can achieve preliminary enrichment of total saponins from *Aralia elata*, it still suffers from insufficient selectivity for certain target saponins with similar polarity, similar glycan structures, or containing acidic glycosides. In practice, target components often co-elute with other saponins, glycoside impurities, and co-extracted impurities, resulting in insufficient peak resolution, a wide collection window, unstable final product purity, and poor reproducibility.

[0004] Furthermore, existing methods for separating saponins from *Aralia elata* mostly focus on obtaining total saponin fractions or conventional monomeric components. For acidic saponins with specific structural characteristics, especially target saponins containing glucuronic acid residues, there is a lack of targeted enrichment and fine purification processes designed based on their molecular structure. Relying solely on conventional macroporous resin separation or single-stage reversed-phase chromatography often fails to simultaneously ensure the purity, yield, and batch stability of the target product, thus hindering the stable preparation and subsequent research applications of these saponin compounds.

[0005] Therefore, it is necessary to provide a saponin compound derived from *Aralia elata* and its preparation method, especially a preparation method that can target the structural characteristics of the target saponin, improve separation efficiency, improve the purity of the final product and enhance reproducibility, so as to meet the actual needs of the separation and purification of target components of natural products. Summary of the Invention

[0006] The purpose of this invention is to provide a novel skeletal saponin compound derived from *Aralia elata* and its preparation method, in order to solve the problems of difficulty in the directional enrichment of target components, easy co-elution with similar polar saponins and glycoside impurities, unstable purity of the final product, and poor reproducibility in the existing *Aralia elata* saponin separation and purification process.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In one aspect, the present invention provides a saponin compound having the structure shown in formula (I).

[0009] Preferably, the molecular formula of the saponin compound is C41H66O15.

[0010] Preferably, the saponin compound has a [M-H2O]^- ion peak at m / z 779.4193±0.5 in high-resolution mass spectrometry.

[0011] Preferably, the saponin compound has the following characteristic chemical shift signals in the ^13 CNMR spectrum: δ108.33±0.5, δ109.86±0.5, δ69.83±0.5 and δ85.48±0.5.

[0012] Preferably, the saponin compound is named aralia elata saponin C.

[0013] Another aspect of the present invention provides a method for preparing the above-mentioned saponin compound, comprising the following steps:

[0014] S1. After crushing the root bark of *Aralia elata*, extract it by heating and reflux with 60%–80% ethanol (by volume) 2–4 times. Combine the extracts and concentrate under reduced pressure to obtain the ethanol extract.

[0015] S2. Dilute the alcohol extract with water to an alcohol content of 5% to 15%, load the sample onto a macroporous adsorption resin column, and perform gradient elution with water and ethanol with a volume fraction of 10% to 90%. Collect the eluted fraction with a volume fraction of 50% to 80% ethanol to obtain the saponin-enriched fraction.

[0016] S3. Dissolve the saponin-enriched fraction in an aqueous alcohol solution, load the sample onto an anion exchange resin column, first elute neutral impurities with water or an alcohol aqueous solution with a volume fraction of 5% to 20%, then elute with a volatile ammonium salt aqueous solution, and collect the acidic saponin-enriched fraction.

[0017] S4. After desalting the acidic saponin-enriched fraction, the sample is loaded onto a reversed-phase C18 column and the first-step reversed-phase separation is performed using an acetonitrile-water system or a methanol-water system. The fraction containing the main peak of the target saponin is collected.

[0018] S5. The fraction containing the main peak of the target saponin is subjected to preparative liquid phase purification by gradient elution with an acetonitrile-water system containing 0.01% to 0.05% acid. The fraction corresponding to the target peak is collected, concentrated and dried to obtain the saponin compound.

[0019] Preferably, the macroporous adsorption resin in step S2 is selected from one or at least two of MCI-GEL resin, AB-8 resin and HPD-100 resin.

[0020] Preferably, the anion exchange resin in step S3 is a weakly basic anion exchange resin, and the volatile ammonium salt aqueous solution is an ammonium bicarbonate solution or an ammonium acetate solution with a concentration of 0.05 mol / L to 0.50 mol / L.

[0021] Preferably, the acid in step S5 is selected from formic acid, acetic acid, or trifluoroacetic acid, with formic acid being the most preferred.

[0022] Preferably, in step S5, high performance liquid chromatography or liquid chromatography-mass spectrometry is used to detect the fraction online, and the fraction is collected in a window according to the target peak retention time and the target ion peak signal.

[0023] Preferably, the liquid phase purity of the obtained saponin compound is not less than 95%.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] The preparation method of this invention is based on the structural characteristics of the target saponin molecule, which contains glucuronic acid residues and has acidic response characteristics. After the initial enrichment with macroporous resin, anion exchange resin is further introduced for directional enrichment, so that the target saponin can be effectively separated from neutral saponins, glycoside impurities and other co-extractable impurities, thereby reducing the burden of subsequent purification.

[0026] This invention adopts a stepwise purification route of "anion exchange directional enrichment + reversed-phase C18 crude separation + preparative liquid phase purification", which organically combines different separation mechanisms. It first achieves the enrichment of acidic saponin fractions and then achieves further purification of the target main peak. This helps to reduce the co-elution of similar polar components and improve the separation degree, purity and yield of the target product.

[0027] This invention employs online detection in the purification stage, combined with target peak retention time and target ion peak signal for window collection. Compared with conventional collection methods that rely on experience, this improves the accuracy of fraction collection, enhances batch-to-batch repeatability and final product quality stability, and is more conducive to subsequent process scale-up and stable preparation. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 The saponin compounds of the present invention have the structure of formula (I);

[0030] Figure 2 The chart shows the 13C-NMR and 1H-NMR data of the saponin compounds of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Equivalent substitutions or conventional modifications made by those skilled in the art based on the description herein without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

[0032] The preferred raw material used in this invention is the root bark of *Aralia elata*. The root bark can be washed, air-dried, or dried at low temperature before being pulverized for later use. The particle size can be adjusted according to the extraction efficiency and subsequent filtration conditions, generally to meet the requirements of ethanol reflux extraction and subsequent concentration and loading operations. The ethanol used can be a solution of appropriate concentration prepared from analytical grade or industrial grade ethanol; the water used is preferably purified water or deionized water. The macroporous adsorption resin, anion exchange resin, reversed-phase C18 packing material, and preparative liquid chromatography column can all be conventionally available products in the art, as long as they can achieve the purpose of saponin enrichment, purification, and separation described in this invention.

[0033] Example 1: Preparation of saponin compounds from *Aralia elata*

[0034] 2.0 kg of *Aralia elata* root bark was crushed and placed in an extraction container. 7 L of 70% ethanol was added, and the mixture was heated under reflux for 2 h. After filtration, the extraction was repeated twice under the same conditions, and the three extracts were combined. The combined extract was concentrated under reduced pressure to obtain approximately 400 mL of ethanol extract. 200 mL of this extract was diluted to 2.5 L with 10% ethanol, filtered, and loaded onto a pre-equilibrated MCI-GEL macroporous adsorption resin column. Gradient elution was then performed using an ethanol-water system, with the elution order being 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol. Each eluent was collected separately, and after solvent recovery, the sample was dried under low temperature and vacuum to obtain a powder. Thin-layer chromatography was used to focus on the elution fractions containing concentrated saponins, from 50% ethanol to 70% ethanol, with the 70% ethanol fraction (Fr.1) being the target for subsequent fine separation.

[0035] Take 4.00 g of the 70% ethanol Fr.1 fraction, dissolve it in 20% methanol aqueous solution, filter, and load the sample into a weakly basic anion exchange resin column pre-equilibrated with pure water and 10% methanol aqueous solution. First, elute with pure water to remove inorganic salts and some water-soluble impurities, then elute with 10% methanol aqueous solution to remove neutral small molecules and some neutral saponin impurities; subsequently, elute with 0.10 mol / L ammonium bicarbonate aqueous solution, collecting the eluent containing acidic saponins. Concentrate this eluent under reduced pressure to remove most of the volatile salts, then reconstitute with pure water, and desalt using lyophilization or reduced pressure to obtain the acidic saponin-enriched fraction.

[0036] The acidic saponin-enriched fraction was dissolved in an acetonitrile-water mixture, filtered, and then wet-loaded onto a reversed-phase C18 column for the first step of reversed-phase separation. The mobile phase was an acetonitrile-water system, preferably containing 0.01% formic acid in the aqueous phase. Elution was performed using gradient elution at acetonitrile:water ratios of 38:62, 40:60, 43:57, 47:53, and 50:50 (volume ratio). The elution peaks were collected in fractions based on the chromatogram, and the fractions containing the main peak of the target saponin were combined.

[0037] The fraction containing the target saponin main peak was concentrated again and then purified by preparative liquid chromatography (HPLC). The preferred mobile phase for preparative HPLC was an acetonitrile-water system containing 0.01%–0.05% formic acid, and elution could be performed using isocratic or small gradient elution methods depending on the retention behavior of the target peak. During purification, the target peak was detected online using high-performance liquid chromatography (HPLC), and the target ion peak signal was monitored using liquid chromatography-mass spectrometry (LC-MS). Fractions corresponding to the target peak were collected within a specific time window; when the retention time of the target peak was stable and the response of the target ion peak was significantly higher than that of adjacent impurity peaks, the fraction within the corresponding time window was collected. The collected target fraction was concentrated under reduced pressure to remove the organic solvent, and then freeze-dried to obtain a white powdery target saponin compound.

[0038] The purity of the obtained target product can be determined by high performance liquid chromatography, and it can be further used for mass spectrometry and nuclear magnetic resonance structure confirmation.

[0039] In a preferred embodiment, the graded purification process of "macroporous adsorption resin pre-separation - anion exchange directional enrichment - reverse-phase C18 crude separation - preparative liquid phase purification" can make the separation between the target component and neutral saponins, glycosides, and similar polar components more complete, thereby improving the purity and batch stability of the final product.

[0040] Another specific implementation of the preparation method in Example 2

[0041] 1.0 kg of *Aralia elata* root bark was crushed and extracted with ethanol under reflux in the same manner as in Example 1, except that 65% ethanol was used as the extraction solvent, and the extraction time was 1.5 h each time, for a total of 3 extractions. The extracts were combined and concentrated under reduced pressure to obtain an ethanol extract. The extract was diluted to an alcohol concentration of about 10% and loaded into an AB-8 macroporous adsorption resin column. Gradient elution was performed using 10%, 30%, 50%, 70%, and 90% ethanol aqueous solutions. The 50%–70% ethanol eluent fraction was collected, concentrated, and then the saponin-enriched fraction was obtained.

[0042] The saponin-enriched fractions were dissolved in a 15% methanol aqueous solution and loaded onto a weakly basic anion exchange resin column. The column was eluted first with pure water and then with a 15% methanol aqueous solution, followed by elution with a 0.20 mol / L ammonium acetate aqueous solution. The acidic saponin-enriched fractions were collected. After concentration and desalting of the eluent, a first-step reversed-phase coarse separation was performed using a methanol-water system, collecting the fractions showing the concentrated target peaks. Subsequently, preparative liquid chromatography purification was performed using an acetonitrile-water system containing 0.02% formic acid, combined with online detection for window collection, to obtain the target saponin compounds.

[0043] This embodiment demonstrates that changing the type of macroporous adsorption resin, the type of anion exchange elution salt, and the composition of the organic phase in the subsequent reversed-phase system does not alter the basic technical concept of this invention, which involves the directional enrichment of acidic saponins followed by two-stage reversed-phase purification. As long as the effective enrichment, purification, and separation of the target saponins can still be achieved, they can all be included within the scope of protection of this invention.

[0044] Example 3: Structural confirmation of the target saponin compound

[0045] The target product obtained in Example 1 or Example 2 was subjected to high-resolution mass spectrometry, nuclear magnetic resonance (NMR), and two-dimensional spectroscopy analysis. The target product was a white powder, which turned purple in ethanol-sulfurized sulfuric acid. High-resolution mass spectrometry showed a [M-H2O]^- ion peak at m / z 779.4193, with the molecular formula C41H66O15. ^1H NMR showed that the compound contained 7 methyl signals, and the sugar proton signals were mainly distributed in the δ 3.5–6.5 range; ^13C NMR showed two sugar terminal carbon signals at δ 108.33 and δ 109.86, and characteristic carbon signals at δ 69.83 and δ 85.48 were also observed. Combining HSQC, HMBC, and ^1H-^1HCOSY two-dimensional correlation spectroscopy analysis, it can be determined that the sugar chain is linked to the C-3 position of the aglycone. The aglycone part has the skeleton characteristics of the araliane II described in the previous case, that is, compared with ursolic acid type saponins, it does not form double bonds at the C-12 and C-13 positions, and has hydroxyl groups at the C-16 and C-20 positions. Finally, the obtained compound can be confirmed as araliane II-3-O-[α-L-furanarabinose(1”→4')-β-D-glucuronic acid], and named araliane saponin C.

[0046] Comparative Example 1: Separation method without anion exchange-directed enrichment

[0047] 4.00 g of 70% ethanol Fr.1 fraction from the same source and batch as in Example 1 was taken and directly loaded onto a reversed-phase C18 column using an acetonitrile-water system for gradient elution without anion exchange resin enrichment. The fractions were collected according to conventional liquid chromatography and then purified again using preparative liquid chromatography. Comparison revealed that without anion exchange-directed enrichment, the sample entering the reversed-phase system contained higher levels of neutral saponins and other co-extracted impurities, with more impurity peaks adjacent to the target peak, resulting in a wider fraction merging range. This made it more likely that the target peak and adjacent impurity peaks would partially overlap in the subsequent preparative liquid chromatography, increasing the difficulty of controlling the collection window and leading to relatively poorer retention behavior of the target peak and stability of the final product purity during repeated separations.

[0048] Comparative Example 2 uses a refinement method that does not employ online window collection control.

[0049] In Example 1, the fraction containing the target saponin main peak obtained from the first step of reverse-phase separation was used for preparative liquid phase purification. However, collection was based solely on empirical observation of the main peak's appearance using conventional UV detection, without window control incorporating the target ion peak signal. Comparison revealed that when sample batch composition fluctuated slightly, relying solely on empirical collection easily resulted in the target peak's fore-and-aft or tail portion entering the collected fraction along with impurities, increasing the purity fluctuation of the final product. Using the online detection described in this invention, combined with target peak retention time and target ion peak signal for window collection, is more beneficial for improving the consistency of collection between different batches and the stability of the final product quality.

[0050] In this invention, the type of macroporous adsorption resin, the type of anion exchange resin, the concentration of the volatile ammonium salt solution, the specific ratio of the acetonitrile-water or methanol-water system during reverse-phase separation, the type and concentration of acid during preparative liquid-phase purification, the online detection method, and the specific collection window can all be adaptively adjusted according to the source of raw materials, equipment conditions, and the retention behavior of the target peak. As long as the core remains the same—firstly, the acidic saponins in the extract of *Aralia elata* buds are directionally enriched, and then the target saponins are purified in two stages by reverse-phase purification and collected and controlled by an online window—it should be considered an equivalent embodiment of this invention.

Claims

1. A novel skeletal saponin compound derived from *Aralia elata*, characterized in that, The saponin compound has the structure shown in formula (I).

2. The saponin compound according to claim 1, characterized in that, The molecular formula of the saponin compound is C41H66O15.

3. The saponin compound according to claim 1 or 2, characterized in that, The saponin compound has a [M-H2O]^- ion peak at m / z 779.4193±0.5 in high-resolution mass spectrometry.

4. The saponin compound according to any one of claims 1 to 3, characterized in that, The saponin compounds exhibit the following characteristic chemical shift signals in the ^13 CNMR spectrum: δ108.33±0.5, δ109.86±0.5, δ69.83±0.5 and δ85.48±0.

5.

5. The saponin compound according to any one of claims 1 to 4, characterized in that, The saponin compound was named Aralia elata saponin C.

6. A method for preparing the saponin compound according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. After crushing the root bark of *Aralia elata*, extract it by heating and reflux with 60%–80% ethanol (by volume) 2–4 times. Combine the extracts and concentrate under reduced pressure to obtain the ethanol extract. S2. Dilute the alcohol extract with water to an alcohol content of 5% to 15%, load the sample onto a macroporous adsorption resin column, and perform gradient elution with water and ethanol with a volume fraction of 10% to 90%. Collect the eluted fraction with a volume fraction of 50% to 80% ethanol to obtain the saponin-enriched fraction. S3. Dissolve the saponin-enriched fraction in an aqueous alcohol solution, load the sample onto an anion exchange resin column, first elute neutral impurities with water or an alcohol aqueous solution with a volume fraction of 5% to 20%, then elute with a volatile ammonium salt aqueous solution, and collect the acidic saponin-enriched fraction. S4. After desalting the acidic saponin-enriched fraction, the sample is loaded onto a reversed-phase C18 column and the first-step reversed-phase separation is performed using an acetonitrile-water system or a methanol-water system. The fraction containing the main peak of the target saponin is collected. S5. The fraction containing the main peak of the target saponin is subjected to preparative liquid phase purification by gradient elution with an acetonitrile-water system containing 0.01% to 0.05% acid. The fraction corresponding to the target peak is collected, concentrated and dried to obtain the saponin compound.

7. The preparation method according to claim 6, characterized in that, The macroporous adsorption resin mentioned in step S2 is selected from one or at least two of MCI-GEL resin, AB-8 resin and HPD-100 resin.

8. The preparation method according to claim 6, characterized in that, The anion exchange resin mentioned in step S3 is a weakly basic anion exchange resin, and the volatile ammonium salt aqueous solution is an ammonium bicarbonate solution or an ammonium acetate solution with a concentration of 0.05 mol / L to 0.50 mol / L.

9. The preparation method according to claim 6, characterized in that, In step S5, high performance liquid chromatography or liquid chromatography-mass spectrometry is used to detect the fraction online, and the fraction is collected in a window according to the target peak retention time and the target ion peak signal.

10. The preparation method according to any one of claims 6 to 9, characterized in that, The liquid phase purity of the obtained saponin compound is not less than 95%.

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