Sodium momordica saponin compound as well as preparation and application thereof

Abstract

The invention relates to a sodium momordica saponin compound and preparation and application thereof, the sodium momordica saponin compound comprises sodium momordica saponin I and sodium momordica saponin II, the sodium momordica saponin is obtained through one-step alkaline hydrolysis by fully utilizing the characteristics of good water solubility, high content and easy conversion in an alkaline environment of momordica glycosides I and II, and the preparation process is simple and convenient. The obtained product is high in purity, good in water solubility and remarkable in anti-tumor and antibacterial activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibacterial and antitumor drugs, and particularly to a preparation method and application of a sodium momordica saponin compound. BACKGROUND

[0002] Momordica saponin I and II are main and characteristic components of Momordicae Semen, a commonly used traditional Chinese medicine. In the 1980s, foreign scholars first isolated the components from Momordicae Semen. The chemical structure was determined by using partial acid hydrolysis, complete acid hydrolysis, enzymatic hydrolysis, methylation, acetylation, and a series of methods such as 1D NMR and MS. In recent years, it has been found that momordica saponin I and II have good water solubility and significant antibacterial and antitumor activities. However, momordica saponin I and II are five-ring triterpenoid saponins with eight glycosyl groups, and the chemical structure is very complex, which makes the separation and purification very difficult, thereby limiting the further application. SUMMARY

[0003] The present application proposes a sodium momordica saponin compound, a preparation method and application thereof, to make full use of the characteristics of momordica saponin I and II, such as good water solubility, high content and easy conversion in an alkaline environment, so that the sodium momordica saponin is obtained through one-step alkaline hydrolysis, and the preparation process is simple. The obtained product has high purity, good water solubility, and significant antitumor and antibacterial activities.

[0004] The present application is achieved by the following technical solutions:

[0005] The present application relates to a sodium momordica saponin compound, specifically sodium mubesidate I and sodium mubesidate II, and the chemical structures are as follows:

[0006]

[0007]

[0008] The present application relates to an extraction method of the above-mentioned sodium momordica saponin compound. The sodium momordica saponin I and II are obtained by adding sodium hydroxide to the water decoction of the momordica seed, heating, adjusting to neutral with hydrochloric acid, and then performing macroporous resin column chromatography and HPLC preparation and separation to collect two main chromatographic peaks.

[0009] The momordica seed is the dried mature seed of Momordica cochinchinensis (Lour.) Spreng. of the Cucurbitaceae family.

[0010] The water decocting is carried out for 0.5-1h, and the water is used in the proportion of 10:1-16:1 to the mass of the medicinal materials.

[0011] The sodium hydroxide is added and heated, and the amount of the sodium hydroxide is 1%-2% of the mass of the medicinal materials, the temperature is 70-90℃, and the heating time is 1-3h.

[0012] The macroporous resin column chromatography is carried out by using D101 type macroporous resin and ethanol solution as the eluent, and the amount of the macroporous resin is 1-2 times of the mass of the medicinal materials.

[0013] The HPLC preparation separation is carried out by using C 18 column (250*20mm, 5um), the mobile phase is acetonitrile-water, gradient elution for 30min (acetonitrile-water 1:3-3:2, v / v), the flow rate is 12.0ml / min, DAD detection, and two main peaks with the maximum absorption wavelength of 210nm are collected respectively, and the sodium aescine refined product is obtained by recovering the solvent.

[0014] The present application relates to the application of the above-mentioned sodium aescine compound, which is used for preparing an antitumor and / or antibacterial drug.

[0015] The antitumor and / or antibacterial effect includes inhibiting the cell growth of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, lung cancer A549 cells, osteosarcoma 143B cells and breast cancer 4T1 cells. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The sodium aescine I is 13 C-NMR chart (DMSO-d6, 176MHz);

[0017] Figure 2 The sodium aescine II is 13 C-NMR chart (DMSO-d6, 176MHz). DETAILED DESCRIPTION Example 1

[0018] This example is implemented under the following implementation conditions and technical requirement conditions:

[0019] Step 1: Crush 500g of Momordica cochinchinensis seeds, add 6L of water and decoct for 1 hour. Concentrate the extract to 500ml, centrifuge, add 5g of sodium hydroxide and heat at 80℃ for 2 hours. Let it cool to room temperature, adjust to neutral with hydrochloric acid, and centrifuge again. Perform macroporous resin (1.0kg) column chromatography, eluting successively with water and 40% ethanol (4 BV each) to remove impurities; elute with 80% ethanol for 4 BV, and recover the ethanol to obtain crude sodium Momordica cochinchinensis saponin (0.40g).

[0020] Step 2: Separate the crude sodium gaccharin by HPLC and collect the two main chromatographic peaks to obtain 34 mg of refined sodium gaccharin I (purity 95.6%, HPLC method) and 22 mg of refined sodium gaccharin II (purity 96.6%, HPLC method).

[0021] like Figure 1 and Figure 2 As shown, the physical properties and spectral data of the obtained components are as follows:

[0022] Sodium gaccharin I: white powder; 1 H, 13 C-NMR: See Table 1; HR-ESI-MS m / z: 975.4542 ([MH]) - C 48 H 72 NaO 19 (Calculated value: 975.4565).

[0023] Sodium gaccharin II: White powder; 1 H, 13 C-NMR: See Table 2; HR-ESI-MS m / z: 991.4505 ([MH]) - C 48 H 72 NaO 20 (Calculated value: 991.4515).

[0024] Table 1. Sodium Gynostemin I 1 H-NMR (700MHz), 13 C-NMR (176MHz) data (DMSO-d6)

[0025] Table 2. Sodium Gnaphalium II 1 H-NMR (700MHz), 13 C-NMR (176MHz) data (DMSO-d6) Example 2

[0026] This example was carried out under the following implementation conditions and technical requirements:

[0027] Step 1, 500g of Semen Momordicae was added to 5L of water and decocted twice for 0.5h each time, and the two extraction solutions were combined. The extraction solution was concentrated to 500ml, centrifuged, 10g of sodium hydroxide was added and heated at 70°C for 3h, then left to room temperature, neutralized with hydrochloric acid, and centrifuged. The supernatant was subjected to column chromatography on a macroporous resin (1.0kg), and eluted with water, 40% ethanol (5BV each) in sequence to remove impurities; eluted with 80% ethanol for 5BV, and the sodium aescinate crude product (0.56g) was recovered by ethanol recovery.

[0028] Step 2, the sodium aescinate crude product was separated by HPLC, and two main chromatographic peaks were collected, namely 45mg of refined sodium aescinate I (purity 95.3%, HPLC method) and 30mg of refined sodium aescinate II (purity 96.2%, HPLC method). Example 3

[0029] This example was carried out under the following implementation conditions and technical requirements:

[0030] Step 1, 500kg of Semen Momordicae was added to 8L of water and decocted for 0.75h, the extraction solution was centrifuged, the extraction solution was concentrated to 500ml, centrifuged, 7.5g of sodium hydroxide was added and heated at 90°C for 1h, then left to room temperature, neutralized with hydrochloric acid, and centrifuged. The supernatant was subjected to column chromatography on a macroporous resin (0.75kg), and eluted with water, 40% ethanol (6BV each) in sequence to remove impurities; eluted with 80% ethanol for 6BV, and the sodium aescinate extract (0.50g) was recovered by ethanol recovery.

[0031] Step 2, the sodium aescinate crude product was separated by HPLC, and two main chromatographic peaks were collected, namely 37mg of refined sodium aescinate I (purity 96.7%, HPLC method) and 26mg of refined sodium aescinate II (purity 95.8%, HPLC method).

[0032] This example relates to the antibacterial application of the above-mentioned sodium aescinate, which is used to inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, specifically including:

[0033] Step a) The preserved Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans strains were inoculated on culture medium and incubated at 37°C overnight, and the activated strains (3 generations) were obtained by repeated inoculation of the culture medium.

[0034] Step b) Fresh culture of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans was washed with 0.9% sterile sodium chloride solution, and the turbidity was adjusted to 0.1 at 590 nm.

[0035] Step c) 2 mL of bacterial solution was added to 200 mL of liquid medium and mixed evenly. Different concentrations of samples to be tested, positive control and negative control were added to 200 μL of the above culture solution, and incubated at 37°C for 24 h to observe the growth of the strains. The reaction plate was placed on the surface of a dark, non-reflective object to determine the experimental results, and the minimum inhibitory concentration (MIC) was determined. Each sample was operated in parallel for 3 times. The specific results are shown in Table 3.

[0036] This example relates to the anti-tumor application of the above-mentioned sodium hemsleyansaponin, for inhibiting the growth of osteosarcoma 143B cells, lung cancer cells A549, breast cancer 4T1 cells, specifically comprising:

[0037] Step 1) Lung cancer cells A549, osteosarcoma 143B cells, breast cancer 4T1 cells were inoculated in RPMI 1640 culture medium containing 10% fetal bovine serum, and the medium was changed every 24 h. The cells in the logarithmic growth phase were digested into single cells with 0.25% trypsin, counted, and diluted with culture medium to a final concentration of 1×10 5 / mL of single cell suspension, 100 μL per well was inoculated in a 96-well culture plate, and incubated in a 37°C, 5% CO2 incubator overnight. After the cells adhered, the test was performed.

[0038] Step 2) After the lung cancer cells A549, osteosarcoma 143B cells, breast cancer 4T1 cells were cultured for 24 h, the culture medium was carefully aspirated, and 100 μL of drug solution (5 replicates for each concentration) was added, and 100 μL of culture medium was added to the blank control group. Continue to incubate in a 37°C, 5% CO2 incubator. After 48 h, add 20 μL of 5 mg / mL MTT solution to each well, continue to incubate in the incubator, remove after 4 h, aspirate the supernatant in each well, add 100 μL of DMSO to each well, shake on a horizontal shaker at low speed for 10 min (90 rpm, 37°C), and then measure the OD value of each well at 490 nm wavelength with a microplate reader.

[0039] The drug solution concentration is 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 μM. The tumor cell growth inhibition rate was calculated according to the following formula: inhibition rate (%) = (1- average OD value of the drug administration group / average OD value of the control group) × 100%. On this basis, the half inhibitory concentration (IC 50 ) was calculated by SPSS. The specific results are shown in Table 3.

[0040] Table 3 Anti-bacterial and cytotoxic effects of sodium akeboside (μg / mL, mean ± SD, n = 3)

[0041] As shown in Table 3, both sodium akebosides have significant cytotoxic activity and can significantly inhibit the growth of S. aureus, P. aeruginosa and C. albicans, and the effect is comparable to that of the positive drug. In addition, they also have significant cytotoxic activity and can significantly inhibit the growth of A549, 143B and 4T1 tumor cells, and the effect is comparable to that of 5-fluorouracil.

[0042] The above specific embodiments can be adjusted in different ways by those skilled in the art without departing from the principles and purposes of the present application. The protection scope of the present application is subject to the claims and is not limited by the above specific embodiments. Each implementation within the scope is subject to the constraints of the present application.

Claims

1. A class of sodium gac fruit saponin compounds, characterized in that, Specifically, sodium mubesidate I and sodium mubesidate II have the following chemical structures:

2. A method for extracting sodium sesquiterpene saponin compound according to claim 1, characterized in that, Sodium gaccharin I and II were obtained by boiling the seeds of Gnaphalium affine in water, adding sodium hydroxide to the decoction and heating, then adjusting the pH to neutral with hydrochloric acid, followed by macroporous resin column chromatography, and finally using HPLC to prepare and separate two main chromatographic peaks. 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 water decoction method is described above, with a water-to-medicinal-material ratio of 10:1 to 16:1, a decoction time of 0.5 to 1 hour, and a decoction frequency of 1 to 2 times.

4. The extraction method according to claim 2, characterized in that, The process involves adding sodium hydroxide and heating, with the amount of sodium hydroxide being 1% to 2% of the weight of the medicinal material, the temperature being 70℃ to 90℃, and the heating time being 1 to 3 hours.

5. The extraction method according to claim 2, characterized in that, The macroporous resin column chromatography uses D101 type macroporous resin and ethanol solution as eluent. The amount of macroporous resin used is 1 to 2 times the amount of the drug material. The eluents for removing impurities are water and 40% ethanol in sequence, and the amount of eluent used is 4 to 6 times the column volume (BV). Then, 80% ethanol is used for elution, and the amount of eluent used is 4 to 6 times the column volume (BV).

6. 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 12.0 ml / min; DAD detection was performed, and the two main peaks with the maximum absorption wavelength of 210 nm were collected. The solvent was recovered to obtain the refined sodium gaccharin.

7. The application of the sodium sesquiterpene saponin compound according to claim 1 or the sodium sesquiterpene saponin compound prepared by any of the methods described in 2-6, characterized in that, It is used to prepare anti-tumor drugs; The anti-tumor effects include inhibiting the growth of lung cancer A549 cells, osteosarcoma 143B cells, and breast cancer 4T1 cells.

8. The application of the sodium sesquiterpene saponin compound according to claim 1 or the sodium sesquiterpene saponin compound prepared by any of the methods described in 2-6, characterized in that, It is used to prepare antibacterial drugs; The antibacterial properties include inhibiting the cell growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.

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