Separation, identification and application of novel steroid saponin in allium macrostemon

Abstract

The invention mainly relates to separation, identification and application of active ingredients in traditional Chinese medicines, and particularly discloses separation and purification of a novel steroid saponin compound in allium macrostemon. Structural identification is performed on a compound according to physicochemical properties and spectrum technologies such as high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy (1D- / 2D-NMR) and the like, the structure of the compound is analyzed, the molecular formula of the compound is determined, the compound is finally named as allium macrostemon saponin U, the pharmacological activity of the allium macrostemon saponin U is researched through in-vitro cell experiments, and the application prospect is broad. It is found that allium macrostemon saponin U remarkably improves the activity of mouse hippocampal neuronal cells treated by glutamic acid, has good neuroprotective activity and can be used for preparing and applying nerve injury resisting drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the separation, purification and preparation of a new steroidal saponin substance in Xiebai, and the determination of the neuroprotective activity thereof. Background Art

[0002] Hippocampal neuron damage and death are associated with a variety of neurological diseases, including neurodegenerative diseases such as depression, Alzheimer's disease and anxiety. Currently, the drugs used in clinical practice to improve nerve damage still have problems such as long onset time, persistent side effects, and limited therapeutic effects. Therefore, it is particularly urgent to find effective drugs with few side effects and neuroprotective effects. Allium macrosAemon Bunge) or scallion ( Allium chinense The dried bulb of G. Don has the effects of regulating qi, widening the chest, promoting yang and dispersing nodules. Through in-depth research on the active ingredients in Xiebai in recent years, it was found that steroidal saponins are the important pharmacological material basis for Xiebai to exert its effects, which has gradually intensified the research on steroidal saponins in Xiebai. So far, according to the literature reports on the chemical composition of Xiebai, there are no reports on the compound Xiebai saponin U and its research on neuroprotection. Summary of the invention

[0003] The present invention innovatively discovered and successfully isolated Xiebai 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-galactopyranoside, {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 Xiebai samples.

[0004] The compound Allium saponin U in the present invention has been preliminarily confirmed to have a neuroprotective effect.

[0005] The invention provides a compound in Xiebai and also provides a method for separating the compound from Xiebai.

[0006] The present invention first provides a new compound, which is named as Xylella saponin U and has the molecular formula: 44 H 70 O 18 , the structural formula is as follows: .

[0007] The separation method of the compound of the present invention specifically comprises the following steps: Step 1: Crush the dried Xiebai sample and pass it through a 40-mesh sieve, dry the dried Xiebai powder, soak the dried Xiebai powder in petroleum ether (60-90°C) at a solid-liquid ratio of 1:5 for 1 h, perform 100 W ultrasonic-assisted extraction at room temperature for 30 min, filter, and repeat 6 times; add 80% ethanol solution to the residue after petroleum ether extraction at a solid-liquid ratio of 1:5 and soak it for 1 h, perform 100 W ultrasonic-assisted extraction for 30 min, filter, and repeat 6 times, combine the filtrate, concentrate under reduced pressure at 35°C, and freeze-dry to obtain Xiebai ethanol extract; Step 2: dissolving the ethanol extract of Xiebai in step 1 in 2 times the amount of distilled water, and then extracting with petroleum ether, dichloromethane and n-butanol in a gradient manner, extracting four times with each solvent, combining the extracts, concentrating the n-butanol part under reduced pressure at 35° C., and then freeze-drying to obtain the n-butanol extract of Xiebai; Step 3: The n-butanol extract in step 2 was subjected to column chromatography using 200-300 mesh silica gel (Qingdao Ocean Chemical Co., Ltd.) and gradient elution using dichloromethane-methanol-water at a volume ratio of 5:2:1 to 13:8:2 as the mobile phase to obtain Fr. 3.1 to 3.12, respectively; Step 4: Fr.3.5 in step 3 was subjected to ODS (YMC, Japan) column chromatography, and gradient elution was performed using methanol-water in a ratio of 40:60 to 90:10 as the eluent to obtain 5 components, namely Fr.3.5.1 to 3.5.5; Step 5: Fr.3.5.5 in step 4 was subjected to ODS (YMC Co., Ltd., Japan) column chromatography, and gradient elution was performed using methanol-water in a ratio of 60:40 to 90:10 as the eluent to obtain compound Allium macrostemon saponin U.

[0008] The present invention further provides the use of the compound in preparing a drug for resisting neuron damage, specifically, the use of the compound in preparing a drug for preventing, treating or improving neuron damage.

[0009] As a preferred embodiment of the present invention, the neuron damage-related diseases include depression, Alzheimer's disease, anxiety disorder, etc.

[0010] The beneficial effect of the present invention over the prior art is that: the present invention separates a steroidal saponin compound Allium macrostemon saponin U from Allium macrostemon, and the compound has good protective activity against Glu-induced HT22 cell damage. The present invention lays a foundation for the development and utilization of anti-neuronal damage drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Attached Figure 1 HR-ESI-MS spectrum of the Allium saponin U of the present invention;

[0012] Attached Figure 2 The present invention is the oxalis saponin U 1 H-NMR (600 MHz, Pyridine-d5) spectrum;

[0013] Attached Figure 3 The present invention is the oxalis saponin U 13 C-NMR (150 MHz, Pyridine-d5) spectrum;

[0014] Attached Figure 4 is the HMQC spectrum of the Allium macrostemon saponin U of the present invention;

[0015] Attached Figure 5 is the HMBC spectrum of the Allium macrostemon saponin U of the present invention;

[0016] Attached Figure 6 is the COSY spectrum of the Allium macrostemon saponin U of the present invention;

[0017] Attached Figure 7 It is the NOESY spectrum of Allium macrostemon saponin U of the present invention. DETAILED DESCRIPTION

[0018] Below I will further illustrate the separation and purification method of the present invention in conjunction with specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] Example 1: The separation and preparation of the compound of the present invention, namely, Xanthoxylum saponin U, specifically comprises:

[0020] The structural formula of the compound xiebai saponin U of this embodiment is as follows: .

[0021] The separation and preparation method of the compound specifically comprises the following steps: Step 1: grind the dried Xiebai sample and pass it through a 40-mesh sieve, dry the dried Xiebai powder, soak the dried Xiebai powder in petroleum ether (60-90°C) at a solid-liquid ratio of 1:5 for 1 h, perform 100 W ultrasonic-assisted extraction at room temperature for 30 min, filter, and repeat 6 times, add 80% ethanol solution at a solid-liquid ratio of 1:5 to the residue after petroleum ether extraction for 1 h, perform 100 W ultrasonic-assisted extraction for 30 min, filter, and repeat 6 times, combine the filtrate, concentrate under reduced pressure at 35°C until there is no alcohol taste, and then freeze-dry to obtain the Xiebai ethanol extract; Step 2: dissolving the ethanol extract of Xiebai in step 1 in 2 times the amount of distilled water, and then extracting with petroleum ether, dichloromethane and n-butanol in a gradient manner, extracting four times with each solvent, combining the extracts, concentrating the n-butanol part under reduced pressure at 35° C., and then freeze-drying to obtain the n-butanol extract of Xiebai; Step 3: The n-butanol extract in step 2 was subjected to column chromatography using 200-300 mesh silica gel (Qingdao Ocean Chemical Co., Ltd.) and gradient elution using dichloromethane-methanol-water at a volume ratio of 5:2:1 to 13:8:2 as the mobile phase to obtain Fr. 3.1 to 3.12, respectively; Step 4: Fr.3.5 in step 3 is subjected to ODS column chromatography, and gradient elution is performed using methanol-water in a ratio of 20:80 to 70:30 as the eluent to obtain 5 components, namely Fr.3.5.1 to 3.5.5; Step 5: Fr.3.5.5 in step 4 is subjected to ODS column chromatography, and gradient elution is performed using methanol-water in a ratio of 60:40 to 90:10 as the eluent to obtain compound Allium macrostemon saponin U.

[0022] 2. Structural identification of the obtained compounds, including: Step 1: The obtained compound is in the form of white amorphous powder. It is positive in the Molish and Liebermann-Burchard reactions, but negative in the Ehrlich reagent reaction. After heating with 10% concentrated H2SO4-EtOH on a thin layer silica gel plate, it turns purple-red. After being left for a period of time, it finally turns yellow-green, indicating that the compound may be a spirostanol saponin. Step 2: Positive ion HR-ESI-MS obtained its m / z: 1125.5114 [M+Na]+ (theoretical value is 1125.5094, Figure 1 ), combined with its 1 H and 13 C spectrum, confirming that its molecular formula is C 53 H 82 O 24； The 1H-NMR (δ ppm, 600 MHz, Pyridine-d5) data of this compound (Table 1, Figure 2 and Figure 3) in the high-field region, there are two characteristic signals of tertiary methyl protons at δH 0.83 (3H, s, Me-18), 0.70 (3H, d, J=5.64 Hz, Me-19) and one characteristic signal of secondary methyl proton at δH 1.15 (3H, d, J=6.90 Hz, Me-21). The 13C-NMR (δppm, 150 MHz, Pyridine-d5) data of this compound (Table 1) suggest the presence of 53 carbon signals, of which 29 carbon signals are assigned to sapogenin and 24 carbon signals are assigned to four sugar molecules. In the low-field region, there are double bond characteristic signals at δC 141.39, 122.12, 144.88 and 109.24 and a carbonyl characteristic signal at δC 171.09, which indicates that there may be two double bonds and one carbonyl group in the structure of the compound. There are three methyl signals at 16.83, 17.81 and 15.53, which are respectively assigned to C-18, C-19 and C-21 according to previous studies. At the same time, the parent nucleus of this compound and the compound recorded in the literature have two more carbon signals at δC 171.09 and 21.58. According to HMQC ( Figure 4 ), δC 21.58 correlates with δH 2.06 (3H, s), suggesting the presence of an acetyl group; at the same time, the C-27 methyl signal is missing on the spirostanyl sapogenin nucleus, and the two terminal olefinic proton signals are at 4.82 (2H, m) and are remotely correlated with δC 145.03 (C-25) (HMBC, Figure 5), so it was determined that there was a double bond at C25-C27; the proton signal δH 1.15 (3H, d, J=6.90 Hz) of Me-21 had long-range correlations with δC63.31, 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 had 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) had long-range correlations with δC 171.09, respectively, and the acetyl group was connected to the oxygen atom on C12; the proton signal δH 0.70 (3H, d, J = 5.64 Hz) is remotely correlated 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. In addition, the proton signals of C-4 (δH 1.68), C-7 (δH 1.44) and the olefinic hydrogen proton signal of C-6 (δH5.30) are remotely correlated with C-5 (δC 141.39), so it is determined that there is a double bond between C5 and C6. In the 1H-1H COSY spectrum ( Figure 6 ), H2-1 / H2-2 / H-3 are correlated, and combined with the HMQC spectrum, δC 29.72 is attributed to C-2, and δC 81.56 is attributed to the C-3 signal. At the same time, considering that C-3 is affected by the glycosylation shift, the chemical shift moves to 81.56 ppm downfield, so it is inferred that C-3 is connected to the sugar group; in the NOESY spectrum ( Figure 7 ) has no correlation with the signal of Me-18 (β-type), thus confirming that the oxygen-containing group at C-12 is β-type; Step 3: The results of the acid hydrolysis test 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, 7.56 Hz, H-1a), 5.33 (1H, d, J=7.62 Hz, H-1b), 5.63 (1H, t,J=8.16 Hz, H-1c) and 5.10 (1H, dd, J=3.06, 7.92 Hz, H-1d), it was inferred that there were four sugar molecules, and the correlation between HMQC and the four isomeric carbons was 103.17, 104.85, 105.02 and 105.14 ppm, respectively, which verified this conjecture. In the HMBC spectrum, the terminal proton signal δH 4.90 (H-1a) of galactose a has a long-range correlation with the C-3 of the parent nucleus (δC 81.56), so it is judged that the sugar is connected to the C-3 position of the sapogenin parent nucleus. The terminal proton δH 5.31 (H-1b) of glucose b has a long-range correlation with the C-4a (δC 81.96) of galactose a, and it is speculated that its connection mode is 1→4; the terminal proton δH 5.63 (H-1c) of glucose c has a long-range correlation with the C-2b (δC 80.64) of glucose b, and it is speculated that its connection mode is 1→2; the terminal proton δH 5.10 (H-1d) of rhamnose d has a long-range correlation with the C-3b (δC89.27) of glucose b, and it is speculated that its connection mode is 1→3; 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-galactopyranoside{spirostane-5,25(27)-dien-12β-OAc-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.

[0023] Table 1 The content of scutellaria baicalensis saponin U 1 H-NMR and 13 C-NMR data .

[0024] Example 2: The protective effect of the compound of the present invention, Allium saponin U, on glutamate (Glu)-induced damage in mouse hippocampal neuron cells (HT22), specifically including:

[0025] Step 1: Drug preparation: Allium saponin U was dissolved in DMEM high-glucose medium to make its stock solution concentration of 10 mM / mL, and then diluted to the following concentrations: 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM, 10.0 μM, 20.0 μM, 40.0 μM; Glu was mixed evenly with DMEM high-glucose medium to make its solution concentration of 20 mM.

[0026] Step 2: Cell culture: HT22 cells were grown in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2 humidified air. The complete medium was replaced every 48 h, and all experiments were performed when the cells reached the logarithmic growth phase.

[0027] Step 3: MTT method to determine the effect of Allium sativum saponin U on cell viability: HT22 cells were cultured at 1×10 5 The number of cells was seeded in a 96-well plate, and the supernatant was discarded after incubation in an incubator for 24 h. The cells were pre-treated with different concentrations of Allium saponin U. The control group and the Glu group were given an equal amount of DMEM high-glucose medium. The supernatant was discarded after incubation in an incubator for 24 h. The cells were treated with 20 mM glutamate. The control group was given an equal amount of DMEM high-glucose medium. After incubation in an incubator for 24 h, 20 μL of MTT solution (5 mg / mL) was added to each well under light-proof conditions. After incubation in an incubator for 3.5 h, the supernatant was discarded, 100 μL of DMSO was added to each well to dissolve formazan, and the absorbance at 490 nm was measured using a microplate reader after sufficient shaking.

[0028] Step 4: Data processing: The experimental data were compared between groups using the mean ± SD method and the one-way analysis of variance (ANOVA) method was used for statistical analysis. p <0.05 or p <0.01 is statistically significant.

[0029] Step 5. Result: The activity of Xylella saponin U on Glu-induced HT22 cell injury is shown in Table 2. After Glu treatment, cell viability was significantly decreased ( ### p <0.001), and administration of Allium macrostemon U showed better protective activity (*** p <0.001, ** p <0.01, * p <0.05); It can be seen that Allium saponin U has a good activity in protecting HT22 cell damage induced by Glu; Table 2 Comparison of the activity of Allium macrostemon saponin U on Glu-induced HT22 cell injury .

[0030] The present invention is described in detail above in combination with preferred embodiments and exemplary examples. However, it should be stated that these specific embodiments are only illustrative explanations of the present invention and do not constitute any limitation to the protection scope of the present invention. Various improvements, equivalent substitutions or modifications may be made to the technical content and implementation methods of the present invention without exceeding the spirit and protection scope of the present invention, all of which fall within the protection scope of the present invention. The protection scope of the present invention shall be subject to the attached claims.

Claims

1. A novel 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 scutellariae saponin U), and its molecular formula is: C 53 H 82 O 24 , the structural formula is as follows: .

2. The method for preparing a compound according to claim 1, characterized in that: The method comprises: Step 1: The dried sample of Xiebai was crushed and passed through a 40-mesh sieve, and degreased using petroleum ether (60-90°C). The residue of the medicinal material after extraction with petroleum ether was extracted with 80% ethanol solution by ultrasonic assisted extraction, and freeze-dried to obtain Xiebai ethanol extract; Step 2: dissolving the ethanol extract of Xiebai in water, then extracting with petroleum ether, dichloromethane and n-butanol in a gradient manner, and concentrating and freeze-drying the n-butanol part to obtain the n-butanol extract of Xiebai; Step 3: After the n-butanol extract is dissolved, it is separated by silica gel column chromatography and gradient eluted with dichloromethane-methanol-water. During the elution process, TLC is used to track and merge, and a total of 12 fractions Fr. 3.1~12 are obtained; Step 4: Fr. 3.5 was subjected to ODS column chromatography and gradient eluted with methanol and water as the mobile phase to obtain 5 elution fractions Fr.3.5.1~3.5.5; Step 5: Fr. 3.5.5 was subjected to ODS column chromatography with methanol and water as the mobile phase for gradient elution to obtain compound Allium saponin U.

3. The compound isolated from Xiebai according to claim 2, characterized in that: The number of extractions with each solvent in step 2 is four times, the extraction time is 2 hours, and the concentrate is concentrated under reduced pressure.

4. The n-butanol extract in step 2 is chromatographed on a 200-300 mesh silica gel column, characterized in that: Gradient elution was performed using dichloromethane-methanol-water as the mobile phase in a volume ratio of 5:2:1 to 13:8:2 to obtain Fr. 3.1 to 3.12, respectively.

5. In step 4, Fr.3.5 is subjected to ODS column chromatography, characterized in that: Gradient elution was performed using methanol-water in a ratio of 40:60 to 90:10 as the eluent to obtain five components, namely Fr.3.5.1 to 3.5.

5.

6. In step 5, Fr.3.5.5 is subjected to ODS column chromatography, characterized in that: The compound Allium saponin U was obtained by gradient elution using methanol-water in a ratio of 60:40 to 90:10 as the eluent.

7. The compound according to claim 1, characterized in that It can significantly improve glutamate-induced neuronal cell damage.

8. The activity of the compound according to claim 7, characterized in that: Application in the preparation of drugs for preventing, treating or improving diseases related to neuronal damage.

9. The use according to claim 8, characterized in that: The neuron damage-related diseases include depression, Alzheimer's disease, anxiety disorder, etc.

10. A pharmaceutical composition, comprising the compound of claim 1, and optionally comprising one or more auxiliary materials, wherein the auxiliary materials include excipients, and the excipients include any one or more of a binder, a filler, and a lubricant.

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