Preparation method and application of momordica glycosides compound
High-purity glutinin A, B, C, and D were extracted from the seeds of *Momordica cochinchinensis* using ethanol extraction, ethyl acetate defatting, n-butanol extraction, macroporous resin silica gel column chromatography, and HPLC separation. This method solves the problem of low extraction efficiency in existing technologies and achieves efficient preparation of antibacterial drugs.
Patent Information
- Application Number
- CN202410606615.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-16
- Publication Date
- 2025-11-18
AI Technical Summary
Existing technologies have failed to effectively extract highly active and pure antibacterial components from Gnaphalium affine seeds, and no relevant reports have been found.
Impurities were removed and high-purity gnoctosides A, B, C, and D were separated by a method combining ethanol extraction followed by ethyl acetate defatting, n-butanol extraction, macroporous resin silica gel column chromatography, and HPLC.
The purification effect of gnocchioside was significantly improved, and four antibacterial active compounds with a purity of over 95% were obtained. These compounds were used to prepare antibacterial drugs and effectively inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa.
Smart Images
Figure CN120965788A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of antibacterial drugs, and particularly relates to a preparation method for simultaneously preparing four kinds of momordica cochinchinensis glycosides with a purity of more than 95% and application thereof. BACKGROUND
[0002] Momordica cochinchinensis (Lour.) Spreng is a plant in the family Cucurbitaceae, which is widely distributed in Yunnan, Jiangxi, Zhejiang, Hunan, Guangdong, Guangxi and Sichuan provinces of China, and is also widely cultivated in Vietnam and Thailand. The fruit of Momordica cochinchinensis is a famous fruit, which has the effects of protecting vision, protecting cardiovascular system and protecting skin. However, the seed of Momordica cochinchinensis is toxic and is often discarded. Modern researches have found that the seed of Momordica cochinchinensis has the effects of antibiosis, anti-inflammation and anti-tumor, and particularly has significant antibacterial activity, and has been applied to the treatment of various bacterial infectious diseases in clinic. Therefore, it is of great significance to extract antibacterial components with high activity and high purity from the seed of Momordica cochinchinensis. However, there is no report on the extraction of antibacterial components from the seed of Momordica cochinchinensis. SUMMARY
[0003] The present application provides a preparation method for momordica cochinchinensis glycosides and application thereof, and the seed of Momordica cochinchinensis is subjected to ethanol extraction, ethyl acetate degreasing and n-butanol extraction, so that water-soluble impurities and fat-soluble impurities can be removed at the same time, and the impurity removal efficiency is high. After impurity removal, the seed of Momordica cochinchinensis is subjected to macroporous resin silica gel column chromatography and silica gel column chromatography purification, and then is subjected to preparation HPLC separation, so that four new disaccharide glycosides with antibacterial activity can be obtained at the same time, and the purification effect is significantly improved.
[0004] The present application is realized by the following technical scheme:
[0005] The present application relates to a kind of momordica cochinchinensis glycosides, specifically: momordica cochinchinensis glycosides A, B, C, D (mubeside A, B, C, D), its chemical structure is in turn:
[0006] Momordica cochinchinensis glycoside A
[0007] Momordica cochinchinensis glycoside B
[0008] Momordica cochinchinensis glycoside C
[0009] Momordica cochinchinensis glycoside D
[0010] The present application relates to the above-mentioned extraction method of the compound of lognannin, through the ethanol reflux extraction of the Momordica cochinchinensis seed, the ethanol extract is obtained; the ethanol extract is extracted by ethyl acetate to remove fat, and then extracted by water-saturated n-butanol to obtain n-butanol extract; the n-butanol extract is chromatographed by macroporous resin column to obtain lognannin extract; the lognannin extract is chromatographed by silica gel column to obtain lognannin crude product; finally, the lognannin A-D refined product is obtained through HPLC preparation and separation.
[0011] The Momordica cochinchinensis seed is the dry mature seed of Momordica cochinchinensis (Lour.) Spreng.
[0012] The ethanol reflux extraction is carried out with 95% (v / v) ethanol, and the ratio of the amount of ethanol to the mass of medicinal material is 10:1-20:1, the reflux time is 1-2 h, and the extraction times is 1-2 times.
[0013] The ethyl acetate extraction to remove fat is carried out with the ratio of the volume of ethyl acetate to the volume of water solution being 0.5:1-1:1, and the extraction times is 2-3 times.
[0014] The water-saturated n-butanol extraction is carried out with the ratio of the volume of water-saturated n-butanol to the volume of water solution being 0.5:1-1:1, and the extraction times is 2-3 times.
[0015] The macroporous resin column chromatography is carried out with D101 type macroporous resin and ethanol solution as the eluent, and the amount of macroporous resin is 30-60 times the mass of n-butanol extract; the impurity-removing eluent is 40% ethanol, and the amount of eluent is 3-5 times the column volume (BV); then, 80% ethanol is used for elution, and the amount of eluent is 3-5 times the column volume (BV).
[0016] The silica gel column chromatography is carried out with silica gel particle size being 200-300 mesh, and the sample amount ratio being 1:30-1:60 (sample to silica gel, w / w); first, 4-6 BV of dichloromethane-methanol (17:3, V / V) is used for elution to remove impurities; then, 4-6 BV of dichloromethane-methanol (65:35, V / V) is used for elution at a flow rate of 1 BV / h and 0.25 BV / flow; after the eluate is collected, TLC detection is carried out, the flow with main spot Rf 0.3-0.6 is combined, and the solvent is recovered under reduced pressure to obtain the lognannin crude product.
[0017] The silica gel column chromatography is carried out through TLC detection and the combination of the flow with main spot R f f 0.3-0.6, and dichloromethane-methanol-water (60:37:3, v / v / v) is used as the developing agent.
[0018] The HPLC preparation and separation is carried out with C 18Column (250*20mm, 5μm), mobile phase was acetonitrile-water, gradient elution for 30min (acetonitrile-water 1:3~3:2, v / v); flow rate was 8.0ml / min; DAD detection, four main peaks with maximum absorption wavelength of 248nm were collected respectively, and the refined product of Loganin was obtained by recovering the solvent.
[0019] The present application relates to the application of the above-mentioned Loganin compound, which is used for preparing antibacterial drugs.
[0020] The antibacterial drugs inhibit Staphylococcus aureus, Pseudomonas aeruginosa, etc. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 Loganin A 13 C-NMR spectrum (176MHz, DMSO-d6);
[0022] Figure 2 Loganin B 13 C-NMR spectrum (176MHz, DMSO-d6);
[0023] Figure 3 Loganin C 13 C-NMR spectrum (176MHz, DMSO-d6);
[0024] Figure 4 Loganin D 13 C-NMR spectrum (176MHz, DMSO-d6). DETAILED DESCRIPTION Example 1
[0025] This example is implemented under the following implementation conditions and technical requirement conditions:
[0026] Step 1, 1kg of Logan seed is added with 15L of ethanol for refluxing for 1h, and the ethanol is recovered to obtain an alcohol extract; the alcohol extract is suspended in 400ml of water, and 200ml of ethyl acetate is extracted for 3 times, and the ethyl acetate is recovered, and the extract is discarded; the water layer is continuously extracted with water-saturated n-butanol for 3 times, 200ml each time, and the n-butanol is recovered to obtain an n-butanol extract (15.0g).
[0027] Step 2, the n-butanol extract is dissolved in water, and subjected to macroporous resin (450g) column chromatography, 40% ethanol is used for elution for 3BV to remove impurities; 80% ethanol is used for elution for 3BV, and the ethanol is recovered to obtain a Loganin extract (8.5g), wherein: the amount of macroporous resin is 30-50 times the mass of the extract.
[0028] Step 3, The extract of loganin was subjected to silica gel column chromatography (255 g of silica gel) and eluted with dichloromethane-methanol (17:3, V / V) for 6 BV. The extract was recovered and discarded. The extract was eluted with dichloromethane-methanol-water (65:35, V / V) for 6 BV. The eluate was collected and the main spots were detected by TLC. The main spots were combined and the solvent was recovered to obtain the crude loganin (573 mg).
[0029] Step 4, The crude loganin was subjected to HPLC separation. Four main chromatographic peaks were collected to obtain refined loganin A (15.2 mg, purity 97.4%, HPLC), refined loganin B (11.0 mg, purity 97.6%, HPLC), refined loganin C (8.3 mg, purity 97.2%, HPLC), and refined loganin D (7.5 mg, purity 95.6%, HPLC).
[0030] As shown in Table 1, the physical properties and spectral data of the obtained components are as follows: Figures 1-4
[0031] Loganin A: white powder; 1 H, 13 C-NMR: see Table 1; HR-ESI-MS m / z: 461.1297 ([M-H] - , C 19 H 25 O 13 , calculated value 461.1295).
[0032] Loganin B: white powder; 1 H, 13 C-NMR: see Table 1; HR-ESI-MS m / z: 447.1497 ([M-H] - , C 19 H 27 O 12 , calculated value 447.1503).
[0033] Loganin C: white powder; 1 H, 13 C-NMR: see Table 2; HR-ESI-MS m / z: 597.1847 ([M+CH3COO] - , C 27 H 33 O 15 , calculated value 597.1819).
[0034] Loganin D: white powder; 1 H,13 C-NMR: see Table 2; HR-ESI-MS m / z: 569.1513 ([M+COOH]~, C 70 H 109 O 36 , calculated 569.1506).
[0035] Table 1. HPLC data of loganic acid A, B 1 H-NMR (700MHz), 13 C-NMR (176MHz) data (DMSO-d6)
[0036] Table 2. HPLC data of loganic acid C, D 1 H-NMR (700MHz), 13 C-NMR (176MHz) data (DMSO-d6) Example 2
[0037] This example was carried out under the following implementation conditions and technical requirements:
[0038] Step 1, 1 kg of Semen Loganii was refluxed with 10 L of ethanol for 2 times, 1 h each time, and the extract was recovered by ethanol to obtain an alcohol extract. The alcohol extract was suspended in 500 ml of water, and extracted with 500 ml of ethyl acetate for 2 times, and the extract was discarded after recovering the ethyl acetate; the water layer was further extracted with 500 ml of water-saturated n-butanol for 2 times, and the n-butanol was recovered to obtain an n-butanol extract (18.1 g).
[0039] Step 2, the n-butanol extract was dissolved in water, and subjected to macroporous resin (700 g) column chromatography, eluted with 40% ethanol for 5 BV to remove impurities; eluted with 80% ethanol for 5 BV, and the loganic acid extract (10.4 g) was recovered by ethanol.
[0040] Step 3, the loganic acid extract was subjected to silica gel column chromatography (450 g of silica gel), eluted with dichloromethane-methanol (17:3, V / V) for 5 BV, and the extract was discarded after recovering the solvent. The extract was eluted with dichloromethane-methanol-water (65:35, V / V) for 5 BV, and the eluate was collected. TLC detection was performed, and the main spots were combined in the flow fractions of 0.3-0.6, and the loganic acid crude product (735 mg) was recovered by recovering the solvent.
[0041] Step 4: Separate the crude bromelain by HPLC, and collect the four main chromatographic peaks to obtain 20.8 mg of purified bromelain A (purity 96.4%, HPLC method), 12.6 mg of purified bromelain B (purity 96.2%, HPLC method), 9.1 mg of purified bromelain C (purity 97.0%, HPLC method), and 8.2 mg of purified bromelain D (purity 95.6%, HPLC method). Example 3
[0042] This embodiment is implemented under the following conditions and technical requirements:
[0043] Step 1: Add 1 kg of *Momordica cochinchinensis* seeds to 20 L of ethanol and reflux for 2 h. Recover the ethanol from the extract to obtain an alcohol extract. Suspend the alcohol extract in 400 ml of water and extract three times with 300 ml of ethyl acetate each time. Recover the ethyl acetate and discard the extract. Continue to extract the aqueous layer three times with 300 ml of water-saturated n-butanol each time. Recover the n-butanol to obtain an n-butanol extract (16.0 g).
[0044] Step 2: Dissolve the n-butanol extract in water and perform macroporous resin (800g) column chromatography. Elute with 40% ethanol to remove 4BV and impurities; elute with 80% ethanol to remove 4BV and recover the ethanol to obtain ethanol extract (9.0g).
[0045] Step 3: Perform silica gel column chromatography (540g silica gel) on the above ethanol extract. Elute with dichloromethane-methanol (17:3, V / V) for 4 BV, recover the solvent, and discard the extract. Elute with dichloromethane-methanol-water (65:35, V / V) for 4 BV, and collect the eluent. Detect by TLC, combine fractions with the main spot at 0.3–0.6, recover the solvent, and obtain crude gnocchioside (603mg).
[0046] 4. The crude bromelain was separated by HPLC. Four main chromatographic peaks were collected at 248 nm to obtain 16.0 mg of purified bromelain A (purity 95.7%, HPLC method), 12.1 mg of purified bromelain B (purity 96.3%, HPLC method), 8.4 mg of purified bromelain C (purity 97.1%, HPLC method), and 7.3 mg of purified bromelain D (purity 97.6%, HPLC method).
[0047] This embodiment relates to the antibacterial application of the aforementioned type of bromelain, used to inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa, specifically including:
[0048] 1) Inoculate the preserved Staphylococcus aureus and Pseudomonas aeruginosa strains onto broth medium and incubate overnight at 37°C. Repeat the inoculation process to obtain activated strains (3 generations).
[0049] 2) Wash the fresh cultured Staphylococcus aureus and Pseudomonas aeruginosa with 0.9% sterile sodium chloride solution and adjust the turbidity to 0.1 absorbance at 590nm (108 CFU / mL, 1% bacterial solution).
[0050] 3) Add 2 mL of bacterial suspension to 200 mL of liquid broth medium and mix well. Add 200 μL of the above culture medium to different concentrations of the test sample, positive control, and negative control, respectively. Incubate at 37℃ for 24 h and observe the growth of the strain. Place the reaction plate on a dark, non-reflective surface to determine the experimental results. The minimum concentration of the test sample in the culture medium that inhibits the growth of the strain is taken as the MIC. Each sample is tested in triplicate. The specific results are shown in Table 3.
[0051] Table 3. Minimum inhibitory concentrations (mg / mL, n=3) of gnocchioside Ingredient Staphylococcus aureus Pseudomonas aeruginosa Kurarin A 0.031 0.063 Kurarin B 0.063 0.125 Kurarin C 0.031 0.063 Kurarin D 0.063 0.031 Ampicillin 0.063 0.031
[0052] As shown in Table 3, all four glutenin compounds exhibit significant cytotoxic activity, significantly inhibiting the growth of Staphylococcus aureus and Pseudomonas aeruginosa, with effects comparable to positive control drugs.
[0053] The above-described specific implementations can be partially adjusted by those skilled in the art in different ways without departing from the principles and purpose of the present invention. The scope of protection of the present invention is defined by the claims and is not limited to the above-described specific implementations. All implementation schemes within the scope of the claims are bound by the present invention.
Claims
1. A class of sesquiterpene compounds, characterized in that, Specifically, it is: gnoctoside AD, whose chemical structure is as follows:
2. A method for extracting thymol compounds according to claim 1, characterized in that, The seeds of *Momordica cochinchinensis* were extracted by reflux with ethanol to obtain an ethanol extract; the ethanol extract was defatted by extraction with ethyl acetate and then extracted with water-saturated n-butanol to obtain a n-butanol extract; the n-butanol extract was subjected to macroporous resin column chromatography to obtain a calciposide extract; the calciposide extract was subjected to silica gel column chromatography to obtain crude calciposide; finally, the purified calciposide AD product was obtained by HPLC preparation and separation. The aforementioned Momordica cochinchinensis seeds are the dried, mature seeds of Momordica cochinchinensis (Lour.) Spreng., a plant belonging to the Cucurbitaceae family.
3. The extraction method according to claim 2, characterized in that, The ethanol reflux extraction method uses an ethanol concentration of 95% (v / v); the ratio of ethanol to the mass of medicinal materials is 10:1 to 20:1; the reflux time is 1 to 2 hours; and the extraction is performed 1 to 2 times.
4. The extraction method according to claim 2, characterized in that, The ethyl acetate extraction and defatting process involves a volume ratio of ethyl acetate to aqueous solution of 0.5:1 to 1:1, and the extraction is performed 2 to 3 times.
5. The extraction method according to claim 2, characterized in that, The water-saturated n-butanol extraction method uses a water-saturated n-butanol volume to aqueous solution volume ratio of 0.5:1 to 1:1, and the extraction is performed 2 to 3 times.
6. The extraction method according to claim 2, characterized in that, The macroporous resin column chromatography uses D101 type macroporous resin, with ethanol solution as the eluent. The amount of macroporous resin used is 30 to 60 times the amount of n-butanol extractant. The impurity removal eluent is 40% ethanol, and the eluent volume is 3 to 5 times the column volume (BV). Then, 80% ethanol is used for elution, with the eluent volume being 3 to 5 times the column volume (BV).
7. The extraction method according to claim 2, characterized in that, The silica gel column chromatography method uses silica gel with a particle size of 200-300 mesh and a sample-to-silica gel ratio of 1:30-1:60 (sample to silica gel, w / w). Specifically, it includes: first, eluting with dichloromethane-methanol (17:3, V / V) for 4-6 BV to remove impurities, then eluting with dichloromethane-methanol (65:35, V / V) for another 4-6 BV at a flow rate of 1 BV / h and 0.25 BV / fraction. After collecting the eluent, TLC detection is performed, and fractions with the main spot Rf of 0.3-0.6 are combined. The solvent is recovered under reduced pressure to obtain crude gnoctoside.
8. The extraction method according to claim 2, characterized in that, All silica gel column chromatography analyses were performed by TLC, and the main spot R was combined. f The fraction was 0.3–0.6, with a dichloromethane-methanol-water mixture (60:37:3, v / v / v) as the developing solvent.
9. The extraction method according to claim 2, characterized in that, The HPLC preparative separation described above uses a C10 column. 18 The column (250*20mm, 5μm) was used with acetonitrile-water as the mobile phase and gradient elution for 30 min (acetonitrile-water 1:3 to 3:2, v / v); the flow rate was 8.0 ml / min; DAD detection was performed, and the four main peaks with the maximum absorption wavelength of 248 nm were collected. The solvent was recovered to obtain the zebuxin purified product.
10. An application of a glycoside compound based on the glycoside compound of claim 1 or the glycoside compound prepared by the method of claims 2-9, characterized in that, It is used to prepare antibacterial drugs; The aforementioned antibacterial drug inhibits bacteria including Staphylococcus aureus and Pseudomonas aeruginosa.