Separation and identification of steroidal saponins from allium macrostemon bunge and its application

Abstract

The present application mainly relates to the separation and identification and application of active ingredients in traditional Chinese medicine, and specifically discloses a separation and purification of a steroidal saponin compound in Allii Macrostemonis Bulbus. According to the physical and chemical properties and high-resolution electrospray mass spectrometry (HR-ESI-MS), one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy (1D- / 2D-NMR) and other spectral technologies, the structure of the compound is identified, the structure is analyzed and the molecular formula is determined, and finally named as Allii Macrostemonis Bulbus saponin U. The pharmacological activity of Allii Macrostemonis Bulbus saponin U is studied through in vitro cell experiments, and it is found that Allii Macrostemonis Bulbus saponin U significantly improves the cell viability of mouse hippocampal neurons after glutamate treatment, has good neuroprotective activity, and can be used for the preparation and application of anti-nerve injury drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the isolation, purification and preparation of a steroidal saponin from Allium macrostemon, and the determination of its neuroprotective activity. Background Technology

[0002] Hippocampal neuronal damage and death are associated with various neurological diseases, including neurodegenerative disorders such as depression, Alzheimer's disease, and anxiety. Currently, drugs used clinically to improve nerve damage still suffer from problems such as long onset of action, persistent side effects, and limited therapeutic efficacy. Therefore, the search for effective drugs with fewer side effects and neuroprotective effects is particularly urgent. Allium macrosae (Allium macrosae) or Allium chinense (Allium chinense G. Don) are dried bulbs of plants in the Liliaceae family and have the effects of regulating qi, relieving chest tightness, promoting yang, and dispersing stagnation. In recent years, in-depth research on the effective components of Allium macrosae has revealed that steroidal saponins are an important pharmacodynamic basis for its efficacy, leading to increased research on steroidal saponin components in Allium macrosae. To date, according to literature reports on the chemical composition of Allium macrosae, no research reports have been found on the compound Allium macrosae saponin U and its neuroprotective effects. Summary of the Invention

[0003] This invention innovatively discovers and successfully isolates allium saponin U (spirostane-5,25(27)-dien-12β-O-Ac-3-O-β-D-glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→3)]-O-β-D-glucopyranosyl(1→4)-O-β-D-galactopyranosid, {spirostane-5,25(27)-dien-12β-O-Ac-3-O-β-D-glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→3)]-O-β-D-glucopyranosyl(1→4)-O-β-D-galactopyranosid} or macrostemonoside U) from Allium macrostemonoside samples.

[0004] The compound Allium saponin U in this invention has been preliminarily shown to have neuroprotective effects.

[0005] This invention provides a compound from Allium macrostemon and a method for isolating the compound from Allium macrostemon.

[0006] This invention first provides a compound, named Allium macrostemon saponin U, with the molecular formula: C 44 H 70 O 18 The structural formula is as follows:

[0007] The method for separating the compound described in this invention specifically comprises the following steps:

[0008] Step 1: Crush the dried Allium macrostemon sample and pass it through a 40-mesh sieve. Dry the Allium macrostemon powder. Soak the Allium macrostemon powder in petroleum ether (60-90℃) at a solid-liquid ratio of 1:5 for 1 hour. Extract with ultrasound at 100W for 30 minutes at room temperature. Filter and repeat 6 times. Soak the residue of the medicinal material after petroleum ether extraction in 80% ethanol solution at a solid-liquid ratio of 1:5 for 1 hour. Extract with ultrasound at 100W for 30 minutes. Filter and repeat 6 times. Combine the filtrates, concentrate under reduced pressure at 35℃, and freeze-dry to obtain Allium macrostemon ethanol extract.

[0009] Step 2: Dissolve the ethanol extract of Allium macrostemon from Step 1 in 2 times the volume of distilled water, and then extract it sequentially with petroleum ether, dichloromethane and n-butanol in a gradient manner, extracting four times with each solvent. Combine the extracts, concentrate the n-butanol fraction under reduced pressure at 35°C, and then freeze-dry to obtain the n-butanol extract of Allium macrostemon.

[0010] Step 3: The n-butanol extract from Step 2 was subjected to column chromatography on a 200-300 mesh silica gel column (Qingdao Ocean Chemical Co., Ltd.). Gradient elution was performed using dichloromethane-methanol-water in a volume ratio of 5:2:1 to 13:8:2 to obtain Fr.3.1 to 3.12.

[0011] Step 4: Fr.3.5 from Step 3 was subjected to ODS (YMC Corporation, Japan) column chromatography with a gradient elution of methanol-water in a ratio of 40:60 to 90:10 to obtain 5 fractions, namely Fr.3.5.1 to 3.5.5.

[0012] Step 5: Fr.3.5.5 from Step 4 was subjected to ODS (YMC Corporation, Japan) column chromatography with a gradient elution of methanol-water in a ratio of 60:40 to 90:10 to obtain the compound Allium macrostemon saponin U.

[0013] The present invention further provides the use of the compound in the preparation of anti-neuronal injury drugs, specifically, in the preparation of drugs for the prevention, treatment or improvement of neuronal injury.

[0014] As a preferred embodiment of the present invention, the neuronal damage-related diseases include depression, Alzheimer's disease, anxiety disorder, etc.

[0015] The advantages of this invention over the prior art are as follows: This invention isolates a steroidal saponin compound, Allium saponin U, from Allium macrostemon. This compound has good protective activity against Glu-induced HT22 cell damage. This invention lays the foundation for the development and utilization of drugs against neuronal damage. Attached Figure Description

[0016] Appendix Figure 1 This is the HR-ESI-MS spectrum of Allium macrostemon saponin U of the present invention;

[0017] Appendix Figure 2 The present invention relates to Allium macrostemon saponin U. 1 H-NMR (600MHz, Pyridine-d5) spectrum;

[0018] Appendix Figure 3 The saponin U of Allium macrostemon of this invention 13 C10-NMR (150MHz, Pyridine-d5) spectrum;

[0019] Appendix Figure 4 This is the HMQC spectrum of Allium macrostemon saponin U of the present invention;

[0020] Appendix Figure 5 This is the HMBC spectrum of Allium macrostemon saponin U of the present invention;

[0021] Appendix Figure 6 This is the COSY spectrum of Allium macrostemon saponin U of the present invention;

[0022] Appendix Figure 7 This is the NOESY spectrum of Allium macrostemon saponin U of the present invention. Detailed Implementation

[0023] I will now further illustrate the separation and purification method of the present invention with specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] Example 1: The isolation and preparation of the compound Allium macrostemon saponin U of the present invention specifically includes:

[0025] The structural formula of the compound Allium macrostemon saponin U in this embodiment is as follows:

[0026]

[0027] The method for isolating and preparing this compound specifically includes the following steps:

[0028] Step 1: After pulverizing the dried Allium macrostemon sample, pass it through a 40-mesh sieve. Dry the Allium macrostemon powder. Soak the Allium macrostemon powder in petroleum ether (60-90℃) at a solid-liquid ratio of 1:5 for 1 hour. Extract with ultrasound at 100W for 30 minutes at room temperature. Filter and repeat 6 times. Soak the residue of the medicinal material after petroleum ether extraction in 80% ethanol solution at a solid-liquid ratio of 1:5 for 1 hour. Extract with ultrasound at 100W for 30 minutes. Filter and repeat 6 times. Combine the filtrates and concentrate under reduced pressure at 35℃ until there is no alcohol odor. Freeze-dry to obtain Allium macrostemon ethanol extract.

[0029] Step 2: Dissolve the ethanol extract of Allium macrostemon from Step 1 in 2 times the volume of distilled water, and then extract it sequentially with petroleum ether, dichloromethane and n-butanol in a gradient manner, extracting four times with each solvent. Combine the extracts, concentrate the n-butanol fraction under reduced pressure at 35°C, and then freeze-dry to obtain the n-butanol extract of Allium macrostemon.

[0030] Step 3: The n-butanol extract from Step 2 was subjected to column chromatography on a 200-300 mesh silica gel column (Qingdao Ocean Chemical Co., Ltd.). Gradient elution was performed using dichloromethane-methanol-water in a volume ratio of 5:2:1 to 13:8:2 to obtain Fr.3.1 to 3.12.

[0031] Step 4: Fr.3.5 from Step 3 was subjected to ODS column chromatography with a gradient elution of methanol-water in a ratio of 20:80 to 70:30 to obtain 5 fractions, namely Fr.3.5.1 to 3.5.5.

[0032] Step 5: Fr.3.5.5 from Step 4 was subjected to ODS column chromatography with a gradient elution of methanol-water in a ratio of 60:40 to 90:10 to obtain the compound Allium macrostemon saponin U.

[0033] 2. Structural identification of the obtained compounds, specifically including:

[0034] Step 1: The obtained compound is a white amorphous powder. The Molish reaction with Liebermann-Burchard is positive, but the reaction with Ehrlich reagent is negative. After heating with 10% concentrated H2SO4-EtOH on a thin-layer silica gel plate, it turns purple-red. After standing for a period of time, it eventually turns yellow-green, suggesting that the compound may be a spirostane alcohol type saponin.

[0035] Step 2: HR-ESI-MS of the positive ion yielded the m / z value as: 1125.5114 [M+Na]+ (theoretical value is 1125.5094). Figure 1 Based on its 1H and 13C spectra, its molecular formula was determined to be C. 53 H 82 O 24; 1H-NMR (δppm, 600MHz, Pyridine-d5) data of this compound (Table 1, Figure 2 and Figure 3 The high-field region indicates the presence of characteristic signals from two tertiary methyl protons at δH 0.83 (3H,s,Me-18), 0.70 (3H,d,J=5.64Hz,Me-19), and one secondary methyl proton at δH 1.15 (3H,d,J=6.90Hz,Me-21). The 13C-NMR data (δppm, 150MHz, Pyridine-d5) of this compound (Table 1) indicate the presence of 53 carbon signals, of which 29 carbon signals are assigned to the sapogenin and 24 carbon signals are assigned to the four sugar molecules. In the low-field region, characteristic signals of double bonds at δC 141.39, 122.12, 144.88, and 109.24 and a carbonyl group at δC 171.09 are present, suggesting the possible presence of two double bonds and one carbonyl group in the compound's structure. In the high-field region, δC... Three methyl signals are present at 16.83, 17.81, and 15.53, which, based on previous studies, are assigned to C-18, C-19, and C-21, respectively. Meanwhile, the parent nucleus of this compound and the compounds described in the literature have two additional carbon signals at δC 171.09 and 21.58, according to HMQC (… Figure 4 The correlation between δC 21.58 and δH 2.06 (3H, s) suggests the presence of an acetyl group; simultaneously, the methyl signal at C-27 is missing from the spirostane saponin core, and the two terminal olefin proton signals are at 4.82 (2H, m), showing a long-range correlation with δC 145.03 (C-25) (HMBC). Figure 5Therefore, it was determined that a double bond exists between C25 and C27; the proton signal δH 1.15 (3H,d,J=6.90Hz) of Me-21 showed long-range correlations with δC 63.31, 42.42, and 109.74, which were assigned to C-17, C-20, and C-22, respectively; the proton signal δH 0.83 (3H,s) of Me-18 showed long-range correlations with δC 78.05, 40.89, 57.05, and 63.31, which were assigned to C-12, C-13, C-14, and C-17, respectively; at the same time, δH 3.88 (H-12) and 2.06 (Ac-Me) showed long-range correlations with δC 171.09, and this acetyl group was connected to the oxygen atom on C12; the proton signal δH of Me-19 showed long-range correlations with δC 171.09, which were associated with ... The wavelength 0.70 (3H, d, J = 5.64 Hz) shows long-range correlations with δC 37.48, 141.39, 50.67, and 36.25, which are assigned to C-1, C-5, C-9, and C-10, respectively. Furthermore, the proton signals at C-4 (δH 1.68) and C-7 (δH 1.44), and the olefinic proton signal at position C-6 (δH 5.30) all show long-range correlations with C-5 (δC 141.39), thus confirming the presence of a double bond between C5 and C6. In the 1H-1H COSY spectrum (… Figure 6 The correlation between H2-1 / H2-2 / H-3, combined with the HMQC spectrum, leads to the attribution of δC 29.72 to C-2 and δC 81.56 to the C-3 signal. Furthermore, considering the influence of glycosylation shift on C-3, causing a chemical shift to the lower field to 81.56 ppm, it is inferred that the C-3 position is connected to a glycosyl group. In the NOESY spectrum (…),… Figure 7 The hydrogen signal at C-12 in the sample is not correlated with the Me-18 (β-type) signal, thus determining that the oxygen-containing group at C-12 is β-type.

[0036] Step 3: The acid hydrolysis test results showed that the ratio of galactose, glucose, and rhamnose was 1:2:1. Based on the four isomeric proton signals δH 4.90 (1H,dd,J=1.86,7.56Hz,H-1a), 5.33 (1H,d,J=7.62Hz,H-1b), 5.63 (1H,t,J=8.16Hz,H-1c), and 5.10 (1H,dd,J=3.06,7.92Hz,H-1d), it was inferred that there were four sugar molecules. The correlation degrees between HMQC and the four isomeric carbons were 103.17, 104.85, 105.02, and 105.14 ppm, respectively, verifying this conjecture. In the HMBC spectrum, the terminal proton signal δH 4.90 (H-1a) of galactose a showed a long-range correlation with the C-3 position of the parent nucleus (δC 81.56), indicating that this sugar is linked to the C-3 position of the saponin nucleus. The terminal proton δH 5.31 (H-1b) of glucose b showed a long-range correlation with the C-4a position of galactose a (δC 81.96), suggesting a 1→4 linkage. The terminal proton δH 5.63 (H-1c) of glucose c showed a long-range correlation with the C-2b position of glucose b (δC 80.64), suggesting a 1→2 linkage. The terminal proton δH 5.10 (H-1d) of rhamnose d showed a long-range correlation with the C-3b position of glucose b (δC 89.27), suggesting a 1→3 linkage.

[0037] In summary, the structure of this compound was determined to be spirostane-5,25(27)-dien-12β-O-Ac-3-O-β-D-glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→3)]-O-β-D-glucopyranosyl(1→4)-O-β-D-galactopyranosid}, and named Macrostemonoside U.

[0038] Table 1. 1H-NMR and 13C-NMR data of Allium macrostemon saponin U

[0039]

[0040] Example 2: The protective effect of the compound Allium macrostemon saponin U of the present invention against glutamate (Glu)-induced damage to mouse hippocampal neurons (HT22) specifically includes:

[0041] Step 1: Drug Preparation

[0042] Allium macrostemon saponin U was dissolved in DMEM high-glucose medium to make the stock solution concentration 10 mM / mL, and then diluted to concentrations of 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM, 10.0 μM, 20.0 μM, and 40.0 μM, respectively; Glu was mixed evenly with DMEM high-glucose medium to make the solution concentration 20 mM.

[0043] Step 2, Cell Culture:

[0044] HT22 cells were grown in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, at 37°C and humidified with 5% CO2 air. The complete medium was replaced every 48 hours. All experiments were performed when the cells reached the logarithmic growth phase.

[0045] Step 3: Determination of the effect of Allium macrostemon saponin U on cell viability using the MTT assay:

[0046] HT22 cells were seeded at a density of 1×10⁵ cells in 96-well plates. After incubation for 24 h, the supernatant was discarded. Cells were pretreated with different concentrations of allium saponin U. The control group and the Glu group were given the same amount of DMEM high-glucose medium. After incubation for 24 h, the supernatant was discarded. Cells were then treated with 20 mM glutamate. The control group was given the same amount of DMEM high-glucose medium. After incubation for 24 h, 20 μL of MTT solution (5 mg / mL) was added to each well under dark conditions. After incubation for 3.5 h, the supernatant was discarded. 100 μL of DMSO was added to each well to dissolve formazan. After thorough shaking, the absorbance at 490 nm was measured using a microplate reader.

[0047] Step 4: Data Processing

[0048] The mean and standard deviation (Mean ± SD) were used for intergroup analysis of experimental data. One-way ANOVA was used for statistical analysis, and p < 0.05 or p < 0.01 was considered statistically significant.

[0049] Step 5, Results: The activity of allium saponin U against Glu-induced HT22 cell damage is shown in Table 2. Cell viability decreased significantly after Glu treatment (###p<0.001), while allium saponin U showed good protective activity (***p<0.001,**p<0.01,*p<0.05).

[0050] Therefore, it can be seen that Allium macrostemon saponin U has good activity in protecting against Glu-induced HT22 cell damage;

[0051] Table 2 Comparison of the activities of Allium macrostemon saponin U on Glu-induced HT22 cell damage

[0052]

[0053] The present invention has been described in detail above with reference to preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are merely illustrative explanations of the present invention and do not constitute any limitation on the scope of protection of the present invention. Various improvements, equivalent substitutions or modifications can be made to the technical content and implementation of the present invention without departing from the spirit and scope of protection of the present invention, and all of these fall within the scope of protection of the present invention. The scope of protection of the present invention is determined by the appended claims.

Claims

1. A compound, characterized in that, The compound is named as spirostane-5, 25(27)-dien-12β-O-Ac-3-O-β-D- glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→3)]-O-β-D-glucopyranosyl(1→4)-O-β-D- galactopyranosid {spirostane-5, 25(27)-dien-12β-O-Ac-3-O-β-D-glucopyranosyl(1→2)-O-[α-L- rhamnopyranosyl(1→3)]-O-β-D-glucopyranosyl(1→4)-O-β-D-galactopyranosid}, macrostemonoside U or Allium macrostemon Bunge saponin U, with a molecular formula of C 53 H 82 O 24 , and a structural formula as follows:

2. The method for preparing the compound according to claim 1, characterized in that, The method comprises: Step one: after crushing the dried sample of Allium bakeri, pass through a 40 mesh sieve, use petroleum ether for degreasing treatment at 60-90 DEG C, use 80% ethanol solution for ultrasonic-assisted extraction of the residue after petroleum ether extraction, freeze-drying, and obtain the ethanol extract of Allium bakeri; Step two: dissolve the ethanol extract of Allium bakeri in water, then use petroleum ether, dichloromethane and n-butanol for gradient extraction in sequence, and freeze-dry the n-butanol part to obtain the n-butanol extract of Allium bakeri; Step three: after dissolving the n-butanol extract, separate by silica gel column chromatography, use dichloromethane-methanol-water for gradient elution, and use TLC tracking to combine in the elution process, and obtain 12 fractions Fr.3.1-12 in total; Step four: use ODS column chromatography for Fr.3.5, use methanol-water as the mobile phase for gradient elution, and obtain 5 elution parts Fr.3.5.1-3.5.5; Step five: use ODS column chromatography for Fr.3.5.5, use methanol-water as the mobile phase for gradient elution, and obtain compound Allium bakeri saponin U.

3. The method of claim 2, wherein the compound is prepared by the process comprising: The number of times of extraction of each solvent in step two is four, and the extraction time is 2h, and the concentrated liquid is concentrated under reduced pressure.

4. The method of claim 2, wherein the compound is prepared by the process comprising: The n-butanol extract in step two is separated by 200-300 mesh silica gel column chromatography, use dichloromethane-methanol-water as the mobile phase for gradient elution at a volume ratio of 5:2:1-13:8:2, and obtain Fr.3.1-3.12 in sequence.

5. The method for preparing the compound according to claim 2, characterized in that, In step four, Fr.3.5 is separated by ODS column chromatography, use methanol-water as the eluent for gradient elution at a ratio of 40:60-90:10, and obtain 5 components, which are Fr.3.5.1-3.5.

5.

6. The method of claim 2, wherein, In step five, Fr.3.5.5 is separated by ODS column chromatography, use methanol-water as the eluent for gradient elution at a ratio of 60:40-90:10, and obtain compound Allium bakeri saponin U.

7. Use of the compound of claim 1 in the preparation of a drug for preventing, treating or improving a disease related to neuron injury.

8. Use according to claim 7, characterized in that, The disease related to neuron injury is selected from depression, Alzheimer's disease and anxiety.

9. A pharmaceutical composition containing the compound of claim 1, and optionally containing one or more than one auxiliary material, wherein the auxiliary material includes an excipient, and the excipient includes any one or more of a binder, a filler and a lubricant.

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