Application of white mulberry root-bark extract in preparation of anti-lung cancer drugs

Abstract

The invention discloses an application of a white mulberry root-bark extract in preparation of an anti-lung cancer drug. The white mulberry root-bark extract is separated by using a simple and rapid separation method, and the anti-lung cancer activity of the white mulberry root-bark extract is verified, so that the white mulberry root-bark extract is proved to be a potential anti-lung cancer compound. The white mulberry root-bark extract has remarkable anti-lung cancer activity, can be used as an active component, is applied to treatment of lung cancer, and provides a new choice for preparation of anti-lung cancer drugs. Therefore, the white mulberry root-bark extract prepared by separation can be used for preparing anti-lung cancer drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to an application of a mulberry bark extract in preparing an anti-lung cancer drug. Background Art

[0002] Mulberry bark is the dried root bark of Morus alba L., a plant of the Moraceae family. It was first recorded in "Shennong's Herbal Classic". It is cold in nature, sweet in taste, and belongs to the lung meridian. It has the effects of clearing the lungs and relieving asthma, promoting diuresis and reducing swelling. It is mainly used to treat lung heat, asthma, cough, edema, oliguria, and swelling of the face and skin. It is mainly used clinically to treat respiratory system infections, urinary system infections and diabetes.

[0003] Existing studies have shown that in addition to the traditional pharmacological effects of antitussive, expectorant, antiasthmatic, and diuretic, mulberry bark also has multiple pharmacological effects such as analgesia, anti-inflammatory, hypoglycemic, cardiovascular relaxation, antiviral, anticancer, immunomodulatory, antioxidant, and anti-allergic. Therefore, mulberry bark has a broad research space, and it is expected that through further research and exploration, new active substances can be found to expand the clinical application of mulberry bark.

[0004] Although there are many reports on the pharmacological effects of Morus alba bark, there are few studies on the anti-lung cancer active substances in Morus alba bark. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an application of a Morus alba extract in the preparation of an anti-lung cancer drug. The present invention uses a simple and rapid separation method to separate a Morus alba extract, and verifies its anti-lung cancer activity, confirming that it is a potential anti-lung cancer compound.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The invention provides an application of a Morus alba bark extract in preparing an anti-lung cancer drug.

[0008] The Morus alba extract is obtained by the following separation method:

[0009] (1) Crush the bark of Morus alba, add 100% pure methanol in an amount 10 times the weight of the bark, and perform ultrasonic extraction for 3 times, each time for 4 hours; filter after the end of the ultrasonic extraction to remove the residue; evaporate the filtrate to recover the solvent, and concentrate the extract to obtain the original extract of Morus alba;

[0010] (2) dispersing the mulberry bark extract obtained by step (1) with water as a dispersant, extracting with petroleum ether and ethyl acetate in sequence, using an extractant 3 times the amount of the dispersant, extracting at least 3 times, and recording the mass of the extract after each extraction, until the mass of the extract no longer increases, that is, the extraction is complete;

[0011] (3) Further separation of the ethyl acetate extraction part: wet-mix the ethyl acetate extraction part extract onto an equal amount of dry macroporous resin, separate by ODS column chromatography, sequentially use an acetonitrile-water mixed system gradient elution, and recover the solvent at the same time; the acetonitrile-water mixed system gradient is set to: 20% acetonitrile, 40% acetonitrile, 60% acetonitrile, 80% acetonitrile, 100% acetonitrile;

[0012] (4) Several samples with the same gradient were combined into one section, and the sample with 60% acetonitrile gradient was further separated;

[0013] (5) Purifying the sample by preparative liquid chromatography, wherein the preparative liquid chromatography is equipped with C 18 The column was isocratically eluted with 60% acetonitrile at a flow rate of 10 mL / min and a column temperature of 25° C. The eluate was detected by a dual-channel UV detector at 230 and 280 nm to obtain the Morus alba bark extract.

[0014] The C18 column has specifications of 20×200 mm and 10 μm.

[0015] The mulberry bark extract is used as the sole active ingredient to prepare an anti-lung cancer drug.

[0016] The mulberry bark extract is used in combination with other drugs to prepare anti-lung cancer drugs.

[0017] The medicine comprises a mulberry bark extract and a pharmaceutically acceptable carrier or excipient.

[0018] The auxiliary materials include one or more of wetting agents, diluents, antioxidants, emulsifiers, preservatives, disintegrants or adhesives.

[0019] The wetting agent is selected from at least one of water and ethanol.

[0020] The diluent is selected from at least one of starch, sugar, cellulose or inorganic salts.

[0021] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and lipids thereof.

[0022] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.

[0023] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts or parabens.

[0024] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

[0025] The binder is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0026] The dosage form of the medicine is capsule, tablet, granule, oral solution, injection or infusion.

[0027] The dosage form of the medicine is granules, tablets, capsules, suspensions, oral solutions and injections.

[0028] The effective administration concentration range of the Morus alba extract is 3.625-100 μM.

[0029] The drug of the present invention can be administered in various known ways, such as oral administration, injection, and administration by inhalation spray. The drug of the present invention can be administered alone or in combination with other drugs. The oral composition can be any oral acceptable dosage form, including but not limited to capsules, tablets, granules, pills, suspensions and solutions.

[0030] The medicine of the present invention can be made into common preparations, and can also be made into sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0031] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds and / or materials used in combination with the Morus alba extract, the age, sex, weight, general health and past medical history of the patient being treated, and similar factors well known in the medical field.

[0032] The anti-lung cancer activity of the isolated and prepared Morus alba bark extract was verified on lung cancer cells A549, HCC827, NCI-H1299, LA795, LLC and normal human lung epithelial cells BEAS-2B, and cisplatin was used as a positive drug control, confirming that the isolated and prepared Morus alba bark extract of the present invention has anti-lung cancer activity.

[0033] The inhibitory effect of the mulberry bark extract on the activity of lung cancer cells is concentration-dependent, and the cell survival rate decreases with increasing drug concentration.

[0034] Beneficial effects:

[0035] The mulberry bark extract of the present invention has significant anti-lung cancer activity, can be used as an active ingredient, and is applied to treat lung cancer, providing a new option for preparing anti-lung cancer drugs. Therefore, the mulberry bark extract separated and prepared by the present invention can be used to prepare anti-lung cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a flow chart for the separation and preparation of SBP-C from Morus alba extract;

[0037] Figure 2 This is a high performance liquid chromatography test chart of SBP-C of Morus alba extract;

[0038] Figure 3 Morus alba extract SBP-C 1 H NMR results (Acetone-d6, 500MHz);

[0039] Figure 4 Morus alba extract SBP-C 13 C NMR results (Acetone-d6, 125MHz);

[0040] Figure 5 This is a study on the cytotoxicity of SBP-C extracted from Morus alba and cisplatin, a positive anti-lung cancer drug, on CCK-8 cells; Figure 5 A is the cytotoxicity of SBP-C from Morus alba extract; Figure 5 B is cisplatin cytotoxicity; all data are expressed as means±SDs of experiments repeated at least three times. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0044] Morus alba bark was purchased from Anhui Bozhou Ziting Pharmaceutical Co., Ltd.

[0045] The present invention uses high performance liquid chromatography to test the purity of the Morus alba extract:

[0046] The chromatographic column is Shim-pack VP-ODS250mm×4.6μm;

[0047] Mobile phase A is water, B is methanol; 40%-95% methanol gradient elution for 30 min; flow rate 1 mL / min;

[0048] Detection wavelength: 310 nm; column temperature: 25°C; injection volume: 10 μL.

[0049] Example 1 Extraction and separation of SBP-C from Morus alba extract

[0050] Figure 1 This is a flow chart for the separation and preparation of SBP-C from Morus alba extract in this example.

[0051] (1) 1 kg of dried Morus alba bark was crushed, and 10 L of 100% pure methanol was added for ultrasonic extraction. The extraction was performed 3 times, and each ultrasonic extraction was performed for 4 hours. After the ultrasonic extraction, the extraction was filtered to remove the residue. The filtrates obtained from the 3 extractions were combined, and the solvent was evaporated and recovered using a rotary evaporator, and the extract was concentrated to finally obtain 105.7 g of Morus alba bark extract, with an extract yield of 10.57%.

[0052] (2) The obtained mulberry bark extract was dispersed with 130 mL of water, extracted with petroleum ether and ethyl acetate in turn, with 400 mL of extractant used each time, extracted three times, and obtained 29 g of petroleum ether extract, 11 g of ethyl acetate extract, and 25 g of the remaining extract.

[0053] (3) According to the liquid phase analysis, the SBP-C content of the Morus alba extract in the ethyl acetate extract is relatively high, so the Morus alba extract SBP-C is isolated from this part.

[0054] (4) The ethyl acetate extract was wet-mixed onto an equal amount of dry macroporous resin and separated by ODS column chromatography, using an acetonitrile-water mixture system (20% acetonitrile, 40% acetonitrile, 60% acetonitrile, 80% acetonitrile, 100% acetonitrile) for gradient elution, while recovering the solvent.

[0055] (5) The gradient eluted samples were spotted on reversed phase thin layer chromatography plates (TLC Silica gel 60RP-18F 254 S), developed with 50% acetonitrile, observed the development results under 254 / 365nm ultraviolet light and developed with vanillin-concentrated sulfuric acid colorimetric agent, confirmed that the 6 samples with the same gradient were combined into one section, and divided into 5 sections AE. After high performance liquid chromatography analysis, the samples under 60% acetonitrile gradient were further separated.

[0056] (6) Purify the sample by preparative liquid chromatography, the preparative chromatography is equipped with C 18 Column (20×200mm, 10μm, Shimadzu, Japan), 60% acetonitrile isocratic elution, flow rate of 10mL / min, column temperature of 25°C, eluent was detected by dual-channel UV detector at 230 and 280nm. Finally, 150.7mg of Morus alba extract SBP-C was obtained, and its purity reached 99.17% by high performance liquid chromatography. The chromatographic detection results are shown in Table 1 and Figure 2.

[0057] Table 1 SBP-C HPLC data

[0058]

[0059] The purity of the compound was verified by NMR, and the structure of the compound was analyzed based on the results. The NMR test results are shown in Figure 3 and Figure 4 The specific optical rotation of the compound was measured (the conditions and data for the determination of SBP-C optical rotation are shown in Table 2) to determine the chirality of the carbon atom of the compound, and the structure of the compound was further confirmed as follows:

[0060]

[0061] Table 2 SBP-C optical rotation measurement conditions and data

[0062]

[0063] Example 2 Study on CCK-8 cytotoxicity of SBP-C extracted from Morus alba and cisplatin, a positive anti-lung cancer drug

[0064] Lung cancer cells A549, LA795, LLC in the logarithmic growth phase and normal human lung epithelial cells BEAS-2B were inoculated in 96-well plates (5.0×10 3 cells / well), cultured in DMEM medium, and incubated overnight in a 37°C incubator. Lung cancer cells HCC827 and NCI-H1299 (all from ATCC) in the logarithmic growth phase were inoculated in 96-well plates (5.0×10 3 Cells / well) were cultured in RPMI-1640 medium and incubated in a 37°C incubator overnight.

[0065] The next day, the culture medium was discarded and the cells were incubated with different concentrations of SBP-C (3.625, 7.25, 12.5, 25, 50, 100 μM) and cisplatin (1.25, 2.5, 5, 10, 20, 40 μM) for 48 h. After the incubation, 10 μL CCK-8 was added to each well and cultured for 2 h. Finally, the OD value of each well was measured at a wavelength of 450 nm using a SpectraMax plus384 microplate reader.

[0066] like Figure 5 As shown in the results, within the range of drug concentrations, the inhibitory effects of SBP-C and cisplatin on the activity of various cells were concentration-dependent, and the cell survival rate decreased with the increase of drug concentration. 50 Slightly higher, but still considerable, its IC 50 as follows:

[0067] Cisplatin:

[0068] BEAS-2B:IC 50 =11.099±0.444μM A549:IC 50 =10.952±0.719μM

[0069] HCC827:IC 50 =11.356±0.503μM NCI-H1299:IC 50 =15.596±0.525μM

[0070] LA795:IC 50 =7.005±0.453μM LLC:IC 50 =8.034±0.576μM

[0071] Morus alba extract SBP-C:

[0072] BEAS-2B:IC 50 =28.457±0.649μM A549:IC 50 =14.795±0.099μM

[0073] HCC827:IC 50 =17.260±1.006μM NCI-H1299:IC 50 =17.375±0.685μM

[0074] LA795:IC 50 =12.462±2.969μM LLC:IC 50 =11.815±1.133μM

[0075] In addition, human normal lung epithelial cells BEAS-2B were used as a control cell model to evaluate drug toxicity. The toxicity of SBP-C from Morus alba extract to BEAS-2B cells was significantly lower than that of other lung cancer cells, while cisplatin was not, indicating that SBP-C from Morus alba extract was less toxic to normal lung cells than cisplatin, and could reduce the side effects of treatment to a certain extent. The above results show that SBP-C from Morus alba extract has significant anti-lung cancer activity.

[0076] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Use of a Morus alba bark extract in the preparation of anti-lung cancer drugs.

2. The use according to claim 1, characterized in that: The Morus alba extract is obtained by the following separation method: (1) Crush the bark of Morus alba, add 100% pure methanol in an amount 10 times the weight of the bark, and perform ultrasonic extraction for 3 times, each time for 4 hours; filter after the end of the ultrasonic extraction to remove the residue; evaporate the filtrate to recover the solvent, and concentrate the extract to obtain the original extract of Morus alba; (2) dispersing the mulberry bark extract obtained by step (1) with water as a dispersant, extracting with petroleum ether and ethyl acetate in sequence, using an extractant 3 times the amount of the dispersant, extracting at least 3 times, and recording the mass of the extract after each extraction, until the mass of the extract no longer increases, that is, the extraction is complete; (3) Further separation of the ethyl acetate extraction part: wet-mix the ethyl acetate extraction part extract onto an equal amount of dry macroporous resin, separate by ODS column chromatography, sequentially use an acetonitrile-water mixed system gradient elution, and recover the solvent at the same time; the acetonitrile-water mixed system gradient is set to: 20% acetonitrile, 40% acetonitrile, 60% acetonitrile, 80% acetonitrile, 100% acetonitrile; (4) Several samples with the same gradient were combined into one section, and the sample with 60% acetonitrile gradient was further separated; (5) Purifying the sample by preparative liquid chromatography, wherein the preparative liquid chromatography is equipped with C 18 The column was isocratically eluted with 60% acetonitrile at a flow rate of 10 mL / min and a column temperature of 25° C. The eluate was detected by a dual-channel UV detector at 230 and 280 nm to obtain the Morus alba bark extract.

3. The use according to claim 2, characterized in that: The C18 column has specifications of 20×200 mm and 10 μm.

4. The use according to claim 2, characterized in that: The mulberry bark extract is used as the sole active ingredient to prepare an anti-lung cancer drug.

5. The use according to claim 2, characterized in that: The mulberry bark extract is used in combination with other drugs to prepare anti-lung cancer drugs.

6. The use according to any one of claims 2 to 5, characterized in that: The medicine comprises a mulberry bark extract and a pharmaceutically acceptable carrier or excipient.

7. The use according to any one of claims 2 to 5, characterized in that: The dosage form of the medicine is granules, tablets, capsules, suspensions, oral solutions and injections.

8. The use according to any one of claims 2 to 5, characterized in that: The effective administration concentration range of the Morus alba extract is 3.625-100 μM.

9. The use according to claim 8, characterized in that: The inhibitory effect of the mulberry bark extract on the activity of lung cancer cells is concentration-dependent, and the cell survival rate decreases with increasing drug concentration.

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