A new compound isolated from Morus alba bark and its preparation method and application

Abstract

The invention discloses a new compound separated from white mulberry bark, a preparation method and application thereof, and relates to the technical field of plant chemistry. The chemical structural formula of the new compound is as follows: The preparation method comprises: S1. crushing white mulberry bark, extracting with methanol reflux for multiple times, filtering and combining the extracts, and concentrating under reduced pressure to obtain extract a; dispersing extract a with ethyl acetate and water, and then extracting with ethyl acetate for multiple times, and recovering the solvent to obtain an extract; S2. completely dissolving the extract with methanol, and then mixing the sample with silica gel as a carrier, performing normal phase silica gel column chromatography after forced air drying, using silica gel as a filler, wet loading, performing normal pressure gradient elution, combining the collected liquids containing the target compound, and concentrating under reduced pressure to obtain extract b; S3. dissolving extract b with methanol, filtering with a filter membrane, and then preparing and separating the filtrate with a C18 reverse phase chromatography system, and concentrating under reduced pressure to obtain the new compound. The method has simple operation steps, is easy to control, and the entire production process is short in time.

Description

Technical Field

[0001] The invention relates to the technical field of plant chemistry, and in particular to a new compound separated from mulberry bark and a preparation method and application thereof. Background Art

[0002] Morus alba bark is the dried root bark of Morus alba L., a plant of the Moraceae family. It is mainly produced in Henan, Anhui, Sichuan, Hunan, Hebei and Guangdong provinces. The Chinese Pharmacopoeia states that Morus alba bark is cold in nature and sweet in taste. It belongs to the lung meridian and is used to relieve lung and asthma, promote diuresis and reduce swelling. Morus alba bark contains rich chemical components, mainly flavonoids, coumarins, stilbenes and other components, among which flavonoids have the highest content.

[0003] In recent years, researchers have isolated Morusinol, Kuwanon H, Albanol B, Sanggenone G, Kuwanon O, Kuwanon K, Moracin P, Steppogenin, Moracin M, Moracin O, 3,5-Dihydroxybenzaldehyde, Esculetin, 5,7-Dihydroxycoumarin, Resveratrol, Aromadendrin, Mulberrofuran G, Albanin A, Mulberrin, Kuwanon W, Kuwanon G, Mulberrofuran Q from Morus alba bark. Q) and other characteristic compounds, and modern pharmacological research has been carried out. Morus alba bark has multiple pharmacological effects. It can effectively relieve the symptoms of respiratory diseases such as cough and asthma, promote the discharge of sputum, and has a certain therapeutic effect on diseases such as bronchitis and asthma; it can act as a diuretic, thereby alleviating the symptoms of diseases such as edema and hypertension; it can dilate blood vessels and lower blood pressure; it can inhibit inflammatory reactions and pain transmission in the body, thereby alleviating the pain and discomfort of diseases such as arthritis and neuralgia; it can also inhibit the growth and proliferation of tumor cells.

[0004] This application conducts further research and development on mulberry bark, explores the anti-tumor potential of mulberry bark by extracting the active ingredients in mulberry bark, and seeks the possibility of applying it to tumor treatment, providing more treatment options for clinical practice. Summary of the invention

[0005] To address the deficiencies in the prior art, the present invention provides a new compound separated from Morus alba bark and a preparation method and application thereof. The preparation method can obtain a new medicinal compound with pharmacological activity; the method has simple operating steps, is easy to control, and the entire production process is time-saving.

[0006] In order to achieve the purpose of the present invention, the following scheme is proposed:

[0007] A new compound isolated from Morus alba bark, the chemical structure is as follows:

[0008]

[0009] The compound is a white solid. Electrospray ionization mass spectrometry (ESI-MS) shows: positive ion 477.17 [M+Na] + , negative ion 453.21[MH] - , that is, the molecular weight of the compound is 454 and the molecular formula is C 25 H 26 O8, after consulting the literature, it was found that the compound was a new compound and was named Sanggenol S.

[0010] A method for preparing a new compound isolated from Morus alba bark comprises the following steps:

[0011] S1. The mulberry bark was crushed, extracted with 50%-90% methanol under reflux several times, the extracts were filtered and combined, and concentrated under reduced pressure to obtain extract a; the extract a was dispersed with ethyl acetate and water, and then extracted with ethyl acetate several times, and the solvent was recovered to obtain an ethyl acetate extract;

[0012] S2. The ethyl acetate extract was completely dissolved in methanol, and then the sample was mixed with 1-2 times the mass of silica gel as a carrier. The obtained silica gel mixture was air-dried and then subjected to normal phase silica gel column chromatography. 5-7 times silica gel was used as a filler, and the sample was wet loaded. The dichloromethane-methanol (100:0-0:1) solvent system was used as an eluent for normal pressure gradient elution. The fractions were collected. According to the results of thin layer chromatography, the collected solutions containing the target compound were combined, and concentrated to dryness under reduced pressure at 45°C-50°C to obtain extract b;

[0013] S3. Dissolve the extract b with methanol, filter it with a filter membrane, separate the filtrate by C18 reverse phase chromatography system, and concentrate under reduced pressure at 45°C-50°C to obtain Morus alchohol S.

[0014] Furthermore, in step S1, 50% methanol was used for reflux extraction 3 times, and ethyl acetate was used for extraction 4 times.

[0015] Furthermore, in step S2, the sample is mixed with 1.5 times the amount of silica gel as the extract, and 6 times the amount of silica gel is used as filler during normal phase silica gel column chromatography.

[0016] Furthermore, in step S3, filtration is performed using a 0.45 μm filter membrane and concentration is performed under reduced pressure at 45° C.

[0017] Furthermore, in step S3, the C18 reverse phase chromatography system used, A: methanol B: water, A: B (55:45) V / V as the mobile phase, the detection wavelength was 288 nm, and the corresponding chromatographic peaks were collected.

[0018] Furthermore, after obtaining Morus alcohol S, the purity of the product was re-tested using analytical liquid chromatography (RP-HPLC), and the result was above 98.90%.

[0019] The invention discloses an application of a new compound separated from mulberry bark in the preparation of anti-tumor drugs, wherein the tumors include liver cancer, breast squamous cell carcinoma, cervical cancer, gastric cancer, intestinal cancer, glioma and leukemia.

[0020] The beneficial effects of the present invention are as follows: using mulberry bark as a raw material, a new medicinal compound with pharmacological activity can be obtained through methanol reflux extraction, ethyl acetate extraction, silica gel column chromatography separation, C18 reverse phase chromatography system preparation and other process steps; the preparation method has simple operation steps, is easy to control, and the entire production process is short in time. DETAILED DESCRIPTION

[0021] Example 1

[0022] S1. Crush 50 kg of Morus alba bark and extract it with 50% methanol reflux for 3 times, filter and combine the extracts, and concentrate under reduced pressure to obtain extract a; disperse the extract a with ethyl acetate and water, and then extract it with ethyl acetate for 4 times, recover the solvent to obtain ethyl acetate extract, 1.2 kg.

[0023] S2. The ethyl acetate extract was completely dissolved in methanol, and the sample was mixed with silica gel at 1.5 times the mass of the extract. The obtained silica gel sample was air-dried and then subjected to normal phase silica gel column chromatography. 6 times silica gel was used as filler, and the sample was loaded by wet method. Dichloromethane-methanol (100:0-0:1) solvent system was used as eluent for normal pressure gradient elution. The fractions were collected and the collected solutions containing the target compound were combined according to the thin layer chromatography results, and concentrated under reduced pressure at 50°C to extract b, 21 g.

[0024] S3. Dissolve the extract b with methanol, filter it with a 0.45μm filter membrane, and separate the filtrate by C18 reverse phase chromatography system, with A: methanol B: water, A:B (55:45) V / V as the mobile phase, and the detection wavelength is 288nm. Collect the corresponding chromatographic peaks, and concentrate under reduced pressure at 45℃ to obtain Sanggenol S (50mg).

[0025] The entire production process for this compound takes about five days.

[0026] The purity of the product was re-tested by analytical liquid chromatography (RP-HPLC), and the result was 99.14%.

[0027] Example 2

[0028] S1. Crush 50 kg of Morus alba bark and extract it with 70% methanol reflux for 3 times, filter and combine the extracts, and concentrate under reduced pressure to obtain extract a; disperse the extract a with ethyl acetate and water, and then extract it with ethyl acetate for 4 times, recover the solvent to obtain ethyl acetate extract, 1.7 kg.

[0029] S2. The ethyl acetate extract was completely dissolved in methanol, and the sample was mixed with silica gel at 1.5 times the mass of the extract. The obtained silica gel sample was air-dried and then subjected to normal phase silica gel column chromatography. 6 times silica gel was used as filler, and the sample was loaded by wet method. Dichloromethane-methanol (100:0-0:1) solvent system was used as eluent for normal pressure elution. The fractions were collected and the collected solutions containing the target compound were combined according to the thin layer chromatography results, and concentrated under reduced pressure at 50°C to extract b, 33 g.

[0030] S3. Dissolve extract b in methanol, filter with a 0.45 μm filter membrane, and separate the filtrate by C18 reverse phase chromatography system, with A: methanol B: water, A:B (55:45) V / V as the mobile phase; detect at 288 nm, collect the corresponding chromatographic peaks, and concentrate under reduced pressure at 45°C to obtain Sanggenol S (85 mg).

[0031] The entire production process for this compound takes about five days.

[0032] The purity of the product was re-tested by analytical liquid chromatography (RP-HPLC), and the result was 98.90%.

[0033] Example 3

[0034] S1. Crush 50 kg of Morus alba bark and extract it with 90% methanol reflux for 3 times, filter and combine the extracts, and concentrate under reduced pressure to obtain extract a; disperse the extract a with ethyl acetate and water, and then extract it with ethyl acetate for 4 times, recover the solvent to obtain ethyl acetate extract, 2.5 kg.

[0035] S2. The ethyl acetate extract was completely dissolved in methanol, and the sample was mixed with silica gel at 1.5 times the mass of the extract. The obtained silica gel sample was air-dried and then subjected to normal phase silica gel column chromatography. 6 times silica gel was used as filler, and the sample was loaded by wet method. Dichloromethane-methanol (100:0-0:1) solvent system was used as eluent for normal pressure elution. The fractions were collected and the collected solutions containing the target compound were combined according to the thin layer chromatography results, and concentrated under reduced pressure at 50°C to extract b, 42 g.

[0036] S3. Dissolve the extract b in methanol, filter it with a 0.45 μm filter membrane, and separate the filtrate by C18 reverse phase chromatography system, with A: methanol, B: water, A:B (55:45) V / V as the mobile phase; the detection wavelength is 288 nm, and the corresponding chromatographic peaks are collected. Concentrate under reduced pressure at 45°C to obtain Sanggenol S (130 mg).

[0037] The entire production process for this compound takes about five days.

[0038] The purity of the product was re-tested by analytical liquid chromatography (RP-HPLC), and the result was 99.04%.

[0039] The structure was identified as follows:

[0040] A new compound isolated from Morus alba bark, the chemical structure is as follows:

[0041]

[0042] The compound is a white solid. Electrospray ionization mass spectrometry (ESI-MS) shows: positive ion 477.17 [M+Na] + , negative ion 453.21[MH] - , that is, the molecular weight of the compound is 454 and the molecular formula is C 25 H 26 O8, after consulting the literature, it was found that the compound was a new compound and was named Sanggenol S.

[0043] Table 1 Compound data attribution (DMSO, TMS, δppm, J=Hz)

[0044]

[0045]

[0046] The H and C spectra in Table 1 indicate that the compound is a dihydroflavonoid compound.

[0047] From the hydrogen spectrum, there are three methyl substituents, δH 1.20, 1.24, 1.35 (eacH 3H, s); in the low field part, there are proton signals of four benzene rings, δH5.89 (2H, s), 6.27 (1H, s), 7.51 (1H, s). Through HSQC, the carbon and hydrogen are fully assigned, and HMBC finds that δH 6.27 (H-2′) is related to δC 73.7 (C-2), 116.5 (C-1′), 123.5 (C-9), 153.2 (C-5′); at the same time, δH7.51 (H-6′) is related to δC 73.7 (C-2), 123.5 (C-4′), 154.3 (C-2′). In addition, δH 3.77 (H-18) correlates with δC132.0 (C-10), 123.5 (C-9), 69.4 (C-16), 29.2 (C-15); combined with NOESY, δH4.14 (H-16-OH) correlates with 4.97 (H-18-OH), indicating that the configuration of hydroxyl groups at positions 16 and 18 is α. After consulting the literature, the compound is a new compound and is named Sanggenol S.

[0048] NMR HMBC and NOESY:

[0049]

[0050] The invention discloses an application of a new compound separated from mulberry bark in the preparation of anti-tumor drugs, wherein the tumors include liver cancer, breast squamous cell carcinoma, cervical cancer, gastric cancer, intestinal cancer, glioma and leukemia.

[0051] In vitro antitumor activity test of Sanggenol S:

[0052] The MTT method was used, with cisplatin as the positive control drug. Human liver cancer HepG2 cells, human breast squamous cancer MCF-7 cells and HCC1086 cells, human cervical cancer H460 cells and A549 cells, human gastric cancer MGC-803 cells, human intestinal cancer HCT116 cells, human glioma SHG44 cells, and human leukemia CEM cells in logarithmic growth phase were taken and serum-free medium was added to adjust the cell concentration to 5×10 4 / mL, inoculated in 96-well culture plates, 200μL per well, cultured at 37℃, 5% CO2 for 12 hours, aspirated the culture medium, took 20mg / mL WM-AC DMSO solution, diluted it with serum-free medium, and added new compounds and cisplatin drugs (6.25, 12.5, 25, 50, 100 μg / mL), and set 3 replicates for each concentration. Continue to culture for 24 hours, aspirate the drug-containing culture medium, add 100 μL of serum-free medium, 5 mg / mL to each well. Take the new compound and configure it into (100, 10, 1, 0.1, 0.01, 0.001 μg / mL).

[0053] 20 μL of MTT solution was added and incubated in an incubator for 4 hours. The culture medium was discarded, and 100 μL of DMSO was added to each well and shaken thoroughly. The absorbance (A) value was read at 570 nm. All compounds were tested in parallel for 3 times at each concentration, and the inhibition rate of the tested drugs on tumor cells was calculated.

[0054] The S-shaped dose-effect curve was drawn using a nonlinear regression model, and the half-maximal inhibitory concentration (IC) was calculated using OriginPro software. 50 )value.

[0055] Inhibition rate = (A 对照 -A 给药 ) / A 对照

[0056] lgIC 50 =X m -I[P-(3-P m -P n ) / 4]

[0057] X m is lg maximum dose; I is lg (maximum dose / adjacent dose); P is the sum of inhibition rates; P m is the maximum inhibition rate; P n is the minimum inhibition rate.

[0058] The in vitro antitumor activity results of the new compound Sanggenol S are shown in Table 2. According to the test results, it can be seen that the new compound Sanggenol S shows a strong inhibitory effect on human liver cancer HepG2 cells, human breast squamous carcinoma MCF-7 cells and HCC1086 cells, human cervical cancer H460 cells and A549 cells, human gastric cancer MGC-803 cells, human intestinal cancer HCT116 cells, human glioma SHG44 cells, and human leukemia CEM cells. Among them, the inhibitory effect on human gastric cancer MGC-803 cells and human glioma SHG44 cells is stronger than that of the positive drug cisplatin.

[0059] Table 2 Inhibitory effect of Morus alcohol S on tumor cells

[0060]

[0061] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

Claims

1. A compound isolated from Morus alba bark, characterized in that: The molecular weight of the compound is 454 and the molecular formula is C 25 H 26 O8, self-named Sanggenol S, has the following chemical structure: 。 2. A method for preparing a compound isolated from Morus alba bark as claimed in claim 1, characterized in that: The following steps are involved: S1. The mulberry bark was crushed, extracted with 50%-90% methanol under reflux several times, the extracts were filtered and combined, and concentrated under reduced pressure to obtain extract a; the extract a was dispersed with ethyl acetate and water, and then extracted with ethyl acetate several times, and the solvent was recovered to obtain an ethyl acetate extract; S2. The ethyl acetate extract was completely dissolved in methanol, and then the sample was mixed with 1-2 times the mass of silica gel as a carrier. The obtained silica gel mixture was air-dried and then subjected to normal phase silica gel column chromatography, with 5-7 times silica gel as filler, wet loading, and dichloromethane-methanol solvent system as eluent, and dichloromethane: methanol = 100:0-0:1 for normal pressure gradient elution, and segmented collection. According to the results of thin layer chromatography, the collected solutions containing the target compound were combined, and concentrated to dryness under reduced pressure at 45°C-50°C to obtain extract b; S3. Dissolve the extract b with methanol, filter it with a filter membrane, separate the filtrate by C18 reverse phase chromatography system, and concentrate under reduced pressure at 45°C-50°C to obtain Morus alchohol S.

3. The preparation method according to claim 2, characterized in that: In step S1, 50% methanol was used for reflux extraction 3 times, and ethyl acetate was used for extraction 4 times.

4. The preparation method according to claim 2, characterized in that: In step S2, the sample is mixed with silica gel of 1.5 times the mass of the extract, and 6 times the mass of silica gel is used as filler during normal phase silica gel column chromatography.

5. The preparation method according to claim 2, characterized in that: In step S3, the product was filtered using a 0.45 μm filter membrane and concentrated under reduced pressure at 45°C.

6. The preparation method according to claim 2, characterized in that: The C18 reverse phase chromatography system used in step S3, A: methanol B: water, with A and B as mobile phases, the volume ratio of the two is 55:45, the detection wavelength is 288nm, and the corresponding chromatographic peaks are collected.

7. The preparation method according to claim 2, characterized in that: After obtaining Morus alcohol S, the purity of the product was re-tested by analytical liquid chromatography (RP-HPLC), and the result was above 98.90%.

8. A use of the compound isolated from Morus alba bark as claimed in claim 1, characterized in that: In the application of preparing anti-tumor drugs, the tumor is any one or more of liver cancer, breast squamous cell carcinoma, cervical cancer, gastric cancer, intestinal cancer, glioma, and leukemia.

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