CN111214581A

CN111214581A - Preparation method and application of solanum nigrum fruit total alkaloids

Info

Publication number: CN111214581A
Application number: CN201811413396.7A
Authority: CN
Inventors: 刘锐; 李茂�; 谢博君; 孙传腾; 范宸华
Original assignee: Research Institute Branch Of Tianjin Zhongxin Pharmaceutical Group Co Ltd
Current assignee: Research Institute Branch Of Tianjin Zhongxin Pharmaceutical Group Co Ltd
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2020-06-02

Abstract

The invention relates to a preparation method of a black nightshade fruit total alkaloid extract and application thereof in preparing a medicament for treating leukemia.

Description

Preparation method and application of solanum nigrum fruit total alkaloids

Technical Field

The invention relates to an alkaloid extract, a preparation method thereof and application thereof in preparing a medicament for treating leukemia, belonging to the technical field of natural medicaments.

Background

The Solanum nigrum fruit is Solanum nigrum (Solanum nigrum L.) of SolanaceaeSolanum nigrumL.) fruit. According to records, the black nightshade fruit in the Uighur medicine has the efficacies of regulating blood and detoxifying, clearing heat and quenching thirst, and astringing and reducing swelling, and is often combined with other traditional Chinese medicines to play the roles of resisting cancer and resisting inflammation clinically^[1-2]. The black nightshade fruits are distributed nationwide, the resources are rich, the content of steroid alkaloid is high, and the potential development value is realized.

The Solanum nigrum fruit mainly contains steroidal alkaloid, steroidal saponin, anthocyanin, polysaccharide and other components. The content of steroid alkaloid solasonine and solamargine is very high^[3]. In recent years, the successive development of steroid alkaloids containing solasonine and solamargine as active ingredients has become a clinical practice in the preparation of preparationsBed studies. Such as Australian scholars Cham B. E. from devil apple (Solanum linnaeanum) The obtained mixture of steroid alkaloids BEC001 is developed into cream Curaderm BEC5 for treating non-melanoma skin cancer^[4-5]. The product coramsine/SBP002 developed by Solbec pharmaceuticals corporation is composed of 1:1 solasonine and solamargine, and is administered by injection for treating melanoma and metastatic renal cell carcinoma^[6]. Taiwan delta-Chinesemedicine Co., Ltd is from Solanum torvum (Hill. Ex Fr.) (Solanum incanum) The water soluble extract is developed into gel preparation SR-T100 for treating solar keratosis and squamous cell carcinoma in situ^[7]. A large number of documents report that solasonine and solamargine show strong antitumor activity in vivo and in vitro, and especially have potential therapeutic effects on liver cancer, breast cancer, lung cancer and the like^[8-12]. However, no reports on the effect on leukemia have been reported.

Some documents also report extraction methods and pharmacological effects of solanum nigrum alkaloids therein.

CN200610041072 discloses that the whole plant or fruit of Solanum nigrum, or Solanum xanthocarpum, or Solanum australosum is extracted by 50-90% ethanol, the extract is concentrated to a certain density, acid solution is added, stirring and filtering are carried out, the filtrate is absorbed by weak polarity or medium polarity resin (or the ethanol extract is concentrated and then alkali is added for precipitation, the precipitation is dissolved by acid solution and then resin is used for absorption), alkali washing or low concentration alkali alcohol washing is firstly carried out, then 50-90% ethanol eluent is collected and concentrated to have no alcohol smell, alkali solution is added for adjusting the pH value to 8-10, standing, refrigeration and collection of precipitation. Drying the precipitate, dissolving with organic solvent, and performing silica gel column chromatography to obtain solamargine extract with content of more than 90.0%. The extract is administered by gastric perfusion and intraperitoneal injection to treat S₁₈₀The mouse with sarcoma and Hep liver cancer transplantation tumor has strong anti-tumor effect, and the solamargine extract also has certain anti-inflammatory effect.

CN200610041070 discloses extracting whole plant or fruit of Solanum nigrum, or Solanum xanthocarpum, or Solanum australianum with 50-90% ethanol, concentrating the ethanol extractive solution to certain density, adding acidic solution, stirring, filtering, adsorbing the filtrate with weak or medium polar resin (or concentrating the ethanol extractive solution)Adding alkali for precipitation, adding acidic solution for dissolving the precipitate, adsorbing with resin), washing with alkali or low concentration alkali alcohol, collecting 50-90% ethanol eluate, concentrating until no alcohol smell is observed, adding alkaline solution to adjust pH to 8-10, standing, refrigerating, and collecting precipitate. Drying the precipitate, dissolving with organic solvent, and performing silica gel column chromatography to obtain solasonine extract with content of more than 90.0%. The extract is administered by gastric perfusion and intraperitoneal injection to treat S₁₈₀The mouse with sarcoma and Hep liver cancer transplantation tumor has strong anti-tumor effect, and the solasonine extract also has certain anti-inflammatory effect.

CN200810033619 discloses that the whole plant or fruit of Solanum nigrum is extracted by ethanol, the extract is concentrated and then is chromatographed by cation exchange resin, the elution part containing alkali and ethanol is collected, the ethanol is recovered to obtain concentrated solution, the precipitate is obtained by centrifugal separation, and the Solanum nigrum extract is obtained by washing water and acetone. The extract contains solasonine, solamargine and solasonine as active ingredients. The extract can be administered subcutaneously and intraperitoneally at 40 mg/kg level to significantly reduce tissue levels of mouse liver cancer H22 and Lewis lung cancer. Can obviously reduce the postprandial blood sugar level of normal mice and experimental diabetic mice at the level of 10 mg/kg and 20 mg/kg, reduce the serum NO, T-NOS and iNOS levels of asthma guinea pigs, and has protective effect on myocardial ischemia-reperfusion injury.

CN200610041072 and CN200610041070 report that the solasonine/solamargine with high content is obtained by extracting, purifying and chromatographing whole plant or fruit of Solanum nigrum, or Solanum xanthocarpum, or Solanum australianum. But the silica gel column chromatography has high cost, low yield and great environmental pollution. CN200810033619 reports that the ethanol extract of the whole plant or fruit of solanum nigrum is processed by cation exchange resin to obtain solanum nigrum extract, although the method is simple, the yield is low, and is only 0.03% to 0.15%. The inventor uses a thin-layer scanning method to measure the alkaloid content, because the steroid alkaloid and the steroid saponin contained in the black nightshade are R on the thin layer_fThe values are very close, the measurement result of the thin layer scanning method is inaccurate, and the repeatability is poor.

Although the extraction method of steroid alkaloid from the whole plant or fruit of Solanum nigrum is reported, the obtained extract has low yield and poor repeatability, and is not suitable for industrial production. In addition, the pharmacological activity of alkaloid extracts reported is directed against S₁₈₀Sarcoma, Hep liver cancer and Lewis lung cancer transplantable tumor, and no activity report on leukemia is found.

Disclosure of Invention

The invention aims to overcome the defects of the existing preparation method and provides the preparation method of the solanum nigrum fruit total alkaloids, which has the advantages of simple method, high content of the obtained total alkaloids, high yield, low cost and good reproducibility. And has strong inhibiting effect on various leukemia cell strains, and is expected to be applied to the field of leukemia.

the solanum nigrum fruit total alkaloid comprises solasonine, solamargine and β 2-solamargine, wherein the sum of the content of the solasonine, the content of the solamargine and the content of the β 2-solamargine is 45-70% (w/w), the content of the solasonine is 10-20% (w/w), the content of the solamargine is 20-30% (w/w) and the content of the β 2-solamargine is 15-20% (w/w) based on the total weight of the composition.

The method comprises the following specific steps:

extracting the fruit of Solanum nigrum with methanol and ethanol by refluxing, ultrasonic or percolation for 1-4 times, wherein the weight of the extraction solvent is 6-30 times of that of the Solanum nigrum fruit. Concentrating the extracting solution to obtain concentrated solution, adjusting the pH value of the concentrated solution to 1-3 by using 1-20% (w/w) solution of formic acid, acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid or nitric acid, extracting by using petroleum ether, dichloromethane, chloroform, ethyl acetate or diethyl ether, adsorbing the extracted water solution by using medium-polarity or weak-polarity macroporous resin, eluting by using 17-25% (v/v) methanol or ethanol solution, eluting by using 65-95% (v/v) methanol or ethanol solution, collecting eluent, concentrating, adjusting the pH value of the concentrated solution to 8-12 by using ammonia water, sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide, extracting by using ethyl acetate, diethyl ether or n-butyl alcohol, washing the extract to be neutral by using water, concentrating to remove an organic solvent, and drying to obtain the black nightshade fruit total alkaloid extract.

the total alkaloid yield obtained by the preparation method is 0.6-1.2% (w/w), and the sum of the contents of the solasonine, the solamargine and the β 2-solamargine is 45-70% (w/w), wherein the content of the solasonine is 10-20% (w/w), the content of the solamargine is 20-30% (w/w), and the content of the β 2-solamargine is 15-20% (w/w), and is shown in the attached figures 1, 2 and 3.

The solanum nigrum fruit total alkaloids can be used for preparing medicines for preventing and treating leukemia.

Compared with the prior art, the invention has the following beneficial effects: the medicinal materials are extracted by alcohol, concentrated and then adjusted in pH value by an acid solution, and the step of extracting by an organic solvent is added, so that about 12% (w/w) of non-alkaloid impurities can be removed without losing target components. The extracted water liquid is clear, the macroporous resin is not blocked, and the macroporous resin can be conveniently reused.

The invention only uses acid and alkali once, has simple and convenient operation, high content of total alkaloid obtained, high yield, controllable quality and good repeatability, and is suitable for industrialized mass production.

Drawings

FIG. 1 shows solasonine;

FIG. 2 shows solamargine

FIG. 3 is β 2-solamargine;

FIG. 4 is a total alkaloid detection map.

Detailed Description

The invention is further illustrated with reference to the following specific examples, which are not intended to limit the invention in any way.

Example 1

3000g of solanum nigrum fruits are added with 30 times of 50 percent ethanol for percolation extraction;

② concentrating the extract to about 1.05 relative density, adding 20% formic acid aqueous solution to adjust the pH value to 3, extracting 4 times by using dichloromethane;

③ adsorbing the acid water part after extraction by using AB-8 macroporous resin, eluting 3 column volumes by using 17 percent methanol solution, eluting 3 column volumes by using 65 percent methanol, and collecting 65 percent eluent;

fourthly, concentrating the eluent, adding ammonia water to adjust the pH value to 8, and extracting with ethyl acetate;

fifthly, washing the ethyl acetate extraction part with water to be neutral, concentrating and drying to obtain 19.5g of total alkaloid with the yield of 0.65%, wherein the content of solasonine is 10.2% (w/w), the content of solamargine is 20.0% (w/w), and the content of β 2-solamargine is 15.3% (w/w).

Example 2

3000g of solanum nigrum fruit is added with 90 percent methanol which is 15 times the weight of the solanum nigrum fruit for reflux extraction;

② concentrating the extract to the relative density of about 1.05, adding 5 percent sulfuric acid water solution to adjust the pH value to 1, and extracting with ether for 4 times;

③, adsorbing the acid water part after extraction by using D101 macroporous resin, eluting 3 column volumes by using 25% ethanol solution, eluting 3 column volumes by using 95% ethanol, and collecting 95% eluent;

fourthly, concentrating the eluent, adding potassium hydroxide to adjust the pH value to 12, and extracting with ether;

fifthly, washing the ether extraction part with water to be neutral, concentrating and drying to obtain 36.3g of total alkaloid with the yield of 1.2%, wherein the content of solasonine is 19.8% (w/w), the content of solamargine is 29.9% (w/w), and the content of β 2-solamargine is 20.0% (w/w).

Example 3

30000g of solanum nigrum fruits are added with 8 times of 80 percent ethanol for reflux extraction;

② concentrating the extract to about 1.1 relative density, adding 10% hydrochloric acid to adjust the pH value to 2, extracting 4 times with ethyl acetate;

③ adsorbing the acid water part after extraction by AB-8 macroporous resin, eluting 3 column volumes by 20 percent ethanol solution, eluting 3 column volumes by 75 percent ethanol, and collecting 75 percent eluent;

fourthly, concentrating the eluent, adding sodium hydroxide to adjust the pH value to 10, and extracting for 3 times by using normal butanol;

fifthly, washing the n-butanol extraction part to be neutral, concentrating and drying to obtain 300g of total alkaloid with the yield of 1.0%, wherein the solasonine content is 15.2% (w/w), the solamargine content is 24.9% (w/w), and the β 2-solamargine content is 14.9% (w/w).

Example 4

The total alkaloids in example 3 were tested, and the results are as follows:

the instrument comprises the following steps: alltech liquid phase, 426HPLC Pump, UVIS-201 Detector

chromatographic column Venusil ASB C18, 5 μm, 150 Å, 4.6X 150mm

Flow rate: 0.8 mL/min wavelength: 205 nm

Mobile phase: 34.5% acetonitrile-water (containing 2mmol/L Na)₂HPO₄）

The chromatogram is shown in figure 4, wherein 3 main chromatographic peaks are solasonine (t)_R18.7 min), solamargine (t)_R22.4 min), β 2-solamargine (t)_R38.7min）。

Example 5

And (3) testing a sample: the extract obtained in example 3 was sampled;

leukemia cell lines: purchased from Shanghai Life sciences research institute of Chinese academy of sciences;

the test method comprises the following steps: taking 6T-CEM, HL-60, HuT78, MOLT-4 and KU812 as human leukemia cells in logarithmic growth phase, centrifuging, diluting with culture solution containing 10% calf serum to 2 × 10⁵·mL^-1Single cell suspension, which is inoculated into 96-well culture plate with each well at 100 μ L;

to the above 96-well plate, 100. mu.L of sample solution of different concentrations, each concentration being in parallel to 4 wells, was added, and to the cell control group, an equal volume of culture solution containing 10% calf serum was added to make the reaction volume 200. mu.L. 37 ℃ and 5% CO₂After culturing for 72 hours in an incubator, 5 mg/mL of the solution was added to each well^-1mu.L of MTT solution is incubated for 4h at 37 ℃, centrifuged, the supernatant is carefully aspirated, 180. mu.L of DMSO is added to each well, the mixture is shaken, and the absorbance (OD) value is measured at 570nm of a microplate reader. Calculating the inhibition rate, and calculating the IC of the sample to the cell to be detected by using the logarithm value of the sample concentration and the Inhibition Rate (IR) as a regression equation₅₀A value;

IR (%) = (1-experimental OD value/cell control OD value) × 100%;

the experimental results are as follows:

IC of test samples on various leukemia cells₅₀Value (microgram. mL)^-1）

The result shows that the solanum nigrum fruit total alkaloids provided by the invention have obvious inhibition effect on 5 leukemia cells, and especially have stronger inhibition effect on human acute lymphoblastic leukemia cell MOLT-4.

Example 6

Taking 100g of the extract in the embodiment 3, crushing, sieving by a 80-mesh sieve, adding 350g of microcrystalline cellulose, 100g of hydroxypropyl methylcellulose, 10g of sodium carboxymethyl starch, 2g of aerosil and 3g of magnesium stearate, uniformly mixing, and directly pressing into tablets by a tablet machine.

Reference documents:

1. the pharmacological basis and clinical application research progress of fresh solanum nigrum fruit for treating tumors [ J ] the precious national medicine, 2016, 27 (7): 1713-1716.

2. Clinical observations of the anti-inflammatory effects of the drug solanum nigrum fruit [ J ] chinese national journal of medicine, 1999, 5 (4): 11-12.

3. Content determination of solasonine and solamargine in solanum nigrum medicinal materials [ J ]. Chinese natural medicines, 2007, 5 (5): 360-362.

4.Cham B E, Wilson L. HPLC of glycoalkaloids from solanum sodomaeum[J].Planta Medica, 1987, 53(1): 59-62.

5.Cham B E. Drug therapy: solamargine and other solasodine rhamnosylglycosides as anticancer agents[J]. Modern Chemotherapy, 2013, 2(2): 33-49.

6.Millward M, Powell A, Tyson S, et al. Phase I trial of coramsine(SBP002) in patients with advanced solid tumors[C]. Journal of ClinicalOncology, 2005 ASCO Annual Meeting Proceedings, 2005, 23, 16S (June 1Supplement) : 3105.

7.Wu C H, Liang C H, Shiu L Y, et al. Solanum incanum extract (SR-T100)induces human cutaneous squamous cell carcinoma apoptosis through modulatingtumor necrosis factor receptor signaling pathway[J]. Journal ofDermatological Science, 2011, 63(2): 83-92.

8.Hu K, Kobayashi H, Dong A J, et al. Antineoplastic agents. III:Steroidal glycosides from Solanum nigrum[J]. Planta Medica, 1999, 65(1): 35-38.

9.Munari C C, Oliveira P F, Campos J C L, et al. Antiproliferativeactivity of Solanum lycocarpum alkaloidic extract and their constituents,solamargine and solasonine, in tumor cell lines[J]. Journal of NaturalMedicines, 2014, 68(1): 236-241.

10. Investigation of the effects of solamargine on human hepatoma cell proliferation and apoptosis [ D ]. zhenjiang: jiangsu university, 2015

11.Ding X, Zhu F S, Li M, et al. Induction of apoptosis in human hepatomaSMMC-7721 cells by solamargine from Solanum nigrum L. [J]. Journal ofEthnopharmacology, 2012, 139(2): 599-604.

12. Li Ming Hui, Sun Shi hectare, Cao Lian, etc. Solanum Nigri steroid alkaloid to S₁₈₀And effects of Lewis Lung cancer transplantable tumor mice [ J]Chinese natural drug, 2008, 16 (3): 223-226.

Claims

1. A preparation method of a solanum nigrum fruit total alkaloid extract is characterized by comprising the following steps:

step 1: extracting dried near-ripe black nightshade fruit with a practical solvent A, concentrating the extract, adjusting the pH value with an acid solution B, extracting with a solvent C, and separating a water layer;

step 2: adsorbing the water layer obtained in the step 1 by using macroporous resin, eluting by using a solvent D, then eluting by using a solvent E, collecting the eluted part of the solvent E, concentrating, adjusting the pH value of a concentrated solution by using alkali F, then extracting by using a solvent G, washing an extract liquid by using water to be neutral, concentrating and drying to obtain the extract.

2. The method of claim 1, wherein in step 1:

the solvent A is methanol or ethanol, preferably ethanol;

the concentration of the solvent A is 50-90% (v/v), preferably 80% (v/v);

the acid solution B is aqueous solution of formic acid, acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid or nitric acid, preferably hydrochloric acid;

the concentration of the acid liquid B is 1-20% (w/v), preferably 10% (w/v);

the pH value after being adjusted by acid liquor B is 1-3, preferably 2;

the solvent C is petroleum ether, dichloromethane, chloroform, ethyl acetate or diethyl ether, preferably ethyl acetate.

3. The method of claim 1, wherein in step 2:

the solvent D is methanol or ethanol, preferably ethanol;

the concentration of the solvent D is 17-25% (v/v), preferably 20% (v/v);

the solvent E is methanol or ethanol, preferably ethanol;

the concentration of the solvent E is 65-95% (v/v), preferably 75% (v/v);

the alkali F is ammonia water, sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide, and preferably sodium hydroxide;

the pH value after being adjusted by the alkali F is 8-12, and is preferably 10;

the solvent G is ethyl acetate, diethyl ether or n-butanol, preferably n-butanol.

4. The method of claim 1, wherein the extraction is reflux extraction, ultrasonic extraction or percolation extraction, the extraction times are 1 to 4 times, and the weight of the extraction solvent is 6 to 30 times of the weight of the solanum nigrum fruit.

5. The process of claim 1, wherein the macroporous resin comprises a weakly polar macroporous resin and a moderately polar macroporous resin, preferably AB-8, D101, HPD-100 macroporous resin.

6. The method as set forth in claim 1, wherein the extract is prepared in a yield of total alkaloids of 0.6% to 1.2% (w/w) based on the weight of the Solanum nigrum fruit used.

7. the method of claim 1, wherein the extract comprises solasonine, solamargine, and β 2-solamargine, wherein the sum of the amounts of solasonine, solamargine, and β 2-solamargine, based on the total weight of the extract, is 45-70% (w/w).

8. the method of claim 1, wherein the extract is prepared to have a solasonine content of 10-20% (w/w), a solamargine content of 20-30% (w/w), and a β 2-solamargine content of 15-20% (w/w), based on the total weight of the extract.

9. A pharmaceutical composition, which is prepared by using the extract as claimed in claim 7 and 8 as an active ingredient and adding pharmaceutically reasonable auxiliary materials.

10. Use of the extracts and pharmaceutical compositions according to claims 8, 9, 10 for the treatment of leukemia.