CN111407784A

CN111407784A - Method for preparing PD-1/PD-L1 inhibitor from peganum harmala

Info

Publication number: CN111407784A
Application number: CN202010217556.1A
Authority: CN
Inventors: 才让南加; 吴疆; 林鹏程; 姚政; 叶萍; 扎西吉
Original assignee: Qinghai Nationalities University
Current assignee: Qinghai Nationalities University
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2020-07-14

Abstract

The invention discloses an active ingredient extracted from a natural plant peganum harmala and an extraction method thereof, and more particularly relates to an active ingredient extracted from peganum harmala, which can effectively block the combination of PD1-PD L1, thereby eliminating, inhibiting, reducing or blocking the interaction and subsequent actions of PD1-PD L1, and a pharmaceutical composition of the active ingredient and a pharmaceutically acceptable carrier can effectively become a novel PD1-PD L1 inhibitor, so that the inhibitor can be applied to the anti-tumor related diseases, and can provide reference for a clinical treatment scheme.

Description

Method for preparing PD-1/PD-L1 inhibitor from peganum harmala

The technical field is as follows:

the invention relates to the technical field of application of natural products, in particular to a pharmaceutical composition which is used for extracting active ingredients from a natural plant peganum harmala and has the functions of eliminating, inhibiting, reducing or blocking PD1-PD L1 combination and a pharmaceutically acceptable carrier of the active ingredients, and application of the pharmaceutical composition to related diseases.

Background art:

in normal organisms, immune checkpoints on the one hand maintain immune tolerance to self antigens, avoid autoimmune diseases due to too strong immune responses, and on the other hand negatively feed back immune responses, avoid tissue damage due to overstimulation, however, tumor cells may abnormally upregulate co-inhibitory molecules and their related ligands, such as PD-1, PD-L, inhibit immune activity of T cells, cause tumor immune escape, known immune checkpoints including PD-1/PD-56, CT 29A-4/CD 80/CD86 and TCR-L, inhibit immune activity of T cells, cause tumor immune escape, and T-1/PD-L, CT 394/CD 80/CD86 and tra &t-T.

PD-1 and its ligand PD-L1 are relatively well studied immune checkpoint molecules in recent years, which play an important negative regulatory role in tumor microenvironment, after PD-1 and PD-L are combined, a significant inhibitory effect on T cells through mTOR and PI3K/AKT pathway is generated, PD-1 is a cell which attenuates the response of T cells at the later stage of the activation process after T cells enter the tumor microenvironment, the blocking of PD-1/PD-L interaction can restore the immune response of the body to tumors, since adaptive immune resistance inhibits the killing effect of T cells on tumor cells in the tumor microenvironment, PD-1 and its ligand PD-L can effectively reduce the immune response involved by immune T cells, the tumor cells escape immune surveillance of the body by highly expressing PD-L, blocking the interaction of PD1 and PD-L can significantly increase the activity of CD8+ cytotoxic T cells to kill tumor cells, PD-L is mainly expressed in T cells, B cells, dendritic cells, endothelial cells, epithelial cells, and endothelial cells, epithelial cells, and epithelial cells.

PD-L1 is expressed on the surface of a variety of tumor cells including lung, liver, ovary, cervix, skin, bladder, colon, breast, glioma, kidney, stomach, esophagus, oral squamous cell carcinoma, head and neck cancer.

Since 2014, the first PD-1 monoclonal antibody Pembrolizumab was marketed, a total of 15 PD-1/PD-L antibody drugs have been approved so far, wherein 9 monoclonal antibody drugs, 3 small molecule peptide inhibitors and non-small molecule peptide drugs each, and 3 PD-1/PD-L drugs have become important components in the field of tumor immunity, and according to 2018 statistics, from 2017 9 to 2018 9, a PD-1/PD-L drug clinical trial has been newly developed, and clinical trials in an active state have 2250 items in total.

However, the inherent disadvantages of antibody therapy, such as high cost, poor oral administration, immune-related side effects, and a first-line overall population response rate of a single drug of only 20% -30%, the development of non-peptide small molecule inhibitors of the PD-1/PD-L pathway is a promising option for treating patients with tumors.

Peganum harmala, which is a general name of plants and Chinese medicinal herbs. The peganum harmala is a perennial herb of Zygophyllaceae, and has special odor in the whole plant. The root is fat and long. Multiple branches, spreading out, lying on the lower part, growing obliquely on the upper part, round stem and branch with edge, smooth and hairless. Distributed in Ningxia, inner Mongolia Bayan Yan (Union), Alahineman, Gansu Hexi, Xinjiang and Tibet (Gongsong, Zedang). Mongolia, Central Asia, Western Asia, Iran, India (northwest), Mediterranean and northern Africa.

The herba Pegani Harmalae is the whole herb of herba Pegani Harmalae of Zygophyllaceae and herba Pegani Harmalae of the same genus. Has the effects of relieving cough and asthma, expelling wind-damp and eliminating swelling and toxin. The alkaloid contained in peganum harmala is protoplasmic toxin, is a monoamine oxidase inhibitor, has effects on both central nervous system and cardiovascular system, and has anti-tumor effect. In addition, the seeds of the plant peganum harmala can also be used for medicine, the traditional Chinese medicine peganum harmala has the efficacies of relieving cough and asthma, dispelling wind and dampness and relieving depression, also has the anti-tumor effect, and can be used for treating stomach cancer, esophageal cancer and other diseases.

Disclosure of Invention

The invention discloses an active ingredient extracted from a natural plant peganum harmala and an extraction method thereof, and more particularly relates to an active ingredient extracted from peganum harmala, which can effectively block the combination of PD1-PD L1, thereby eliminating, inhibiting, reducing or blocking the interaction and subsequent actions of PD1-PD L1, and a pharmaceutical composition of the active ingredient and a pharmaceutically acceptable carrier can effectively become a novel PD1-PD L1 inhibitor and can be applied to the anti-tumor related diseases.

In order to achieve the purpose, the technical scheme is that the peganum harmala effectively blocks active ingredients bound by PD1-PD L1 and is characterized in that the active ingredients are derived from a peganum harmala water-phase extract part, the peganum harmala water-phase extract part is further separated by D101 macroporous adsorption resin and eluted by ethanol-water systems with different proportions, and the active ingredients mainly come from pure water and 20 percent of eluted parts.

In brief, the active ingredient of peganum harmala, which effectively blocks PD1-PD L1 binding, is derived from the pure water, 20% elution site of the aqueous extract.

The preparation method of the active component of peganum harmala which can effectively block the combination of PD1-PD L1 comprises the following steps:

(1) extraction: selecting high-quality peganum harmala, cleaning with pure water, drying in the air, pulverizing, soaking with 60-80% ethanol for 4-24h, extracting twice, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 60-80% ethanol into the residue, ultrasonic extracting for 2 times, each time for 1-5h, and concentrating under reduced pressure to obtain 60-80% ethanol extract; extracting with 85-98% ethanol by the same method, concentrating under reduced pressure to obtain 85-98% ethanol extract, and mixing to obtain total ethanol extract. Dissolving the total ethanol extract with water at ratio of 1:1-5, extracting with ethyl acetate at ratio of 0.5-1:1 to obtain ethyl acetate part and water phase extract part;

(2) separation: separating ethyl acetate part with silica gel column chromatography, eluting with chloroform-methanol system (pure chloroform, 50:1, 25:1, 10:1, 5:1, methanol) at different ratio to obtain 6 eluate parts; eluting the water phase extract with D101 macroporous adsorbent resin in ethanol-water system (pure water, 20%, 40%, 60%, 80%, 95%) at different ratio to obtain 6 eluates.

Wherein the reduced pressure recovery condition is 0.1-0.5MPA and the temperature is 25-40 ℃; the ultrasonic extraction condition is 200-800HZ, the ultrasonic treatment is carried out for 30-45min, and the temperature is 30-50 ℃.

Wherein, the extraction conditions are as follows: the extraction pressure is 20MPa to 40MPa, the extraction temperature is 30 ℃ to 50 ℃, and the extraction time is 0.8h to 2.5 h.

Wherein, the silica gel column chromatography separation conditions are as follows: using chloroform-methanol system as eluent, gradient eluting with pure chloroform, 50:1, 25:1, 10:1, 5:1 and methanol at flow rate of 0.2-1.0 ml/min.

Wherein, the separation conditions of the D101 macroporous adsorption resin are as follows: eluting with ethanol-water system as eluent, gradient eluting with pure water, 20%, 40%, 60%, 80%, 95%, and flow rate of 0.3-1.5 ml/min.

The invention also relates to a pharmaceutical composition containing the peganum harmala active ingredient and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for preventing or treating a disease or condition by abolishing, inhibiting, reducing or blocking PD1-PD L1 binding activity, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of a peganum harmala active extract fraction of the present invention together with a pharmaceutically acceptable carrier.

Wherein the disease or disorder is selected from cancer; preferably, the cancer is selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal pelvis, neoplasms of the central nervous system, neoplasms of the head or neck, malignant melanoma, cancer of, Primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, including combinations of said cancers.

The term "preventing or treating a disease or disorder by eliminating, inhibiting, reducing or blocking PD1-PD L1 binding activity" as used herein is intended to mean a disease or disorder caused by PD-1 expression or symptomatic/characteristic of the PD1-PD L1 binding.

As used herein, "therapeutically effective amount" refers to a dose sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the condition being treated. Prescription of treatment (e.g., decisions on dosage, etc.) is ultimately the responsibility of and depends on general practitioners and other physicians, often taking into account the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herou, geranium, crane, etc.), domestic animals (e.g., ducks, geese, etc.) or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, etc.).

The term "pharmaceutical composition" as used herein denotes a combination of at least one drug, optionally together with a pharmaceutically acceptable carrier or adjuvant, combined together to achieve a specific purpose. In embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. For example, the ingredients contained in the pharmaceutical composition (e.g. the pharmaceutical composition of the peganum harmala aqueous phase extract fraction according to the invention and the pharmaceutically acceptable carrier) may be administered to the subject in whole or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.

The invention also provides application of the medicinal composition of the peganum harmala active extract part and the medicinal carrier in preparing medicaments for treating diseases or symptoms, in particular application of antitumor medicaments for treating tumor diseases, and application of the medicinal composition of the peganum harmala active extract part and the medicinal carrier in effectively eliminating, inhibiting, reducing or blocking antitumor medicaments combined by PD1-PD L1.

The anti-tumor drug of the pharmaceutical composition of the peganum harmala active extract part and the pharmaceutically acceptable carrier can be an oral preparation or an injection preparation or other preparation types.

The anti-tumor medicinal preparation of the medicinal composition of the peganum harmala active extract part and the medicinal carrier is prepared by combining the peganum harmala active extract part and pharmaceutically acceptable medicinal auxiliary materials.

The pharmaceutic adjuvant is one or more of excipient, diluent, carrier, flavoring agent, adhesive, filler, drug carrier, solubilizer, cosolvent, emulsifier, suspending agent, clarifier, deflocculant, correctant, colorant, preservative, chemical sterilizing agent, adsorbent, filter aid, antioxidant, pH regulator, isotonic regulator, diluent, adhesive, wetting agent, disintegrant, lubricant, antioxidant, controlled release agent, coating material, film-forming material and capsule material.

The oral preparation can be tablets, capsules, granules, oral liquid solvents, liposomes and the like.

The injection preparation can be liposome injection, nano injection, microsphere injection, etc.

The preferred dosage form is an oral dosage form or an injectable dosage form. Oral dosage forms and injectable dosage forms are the most preferred routes of administration. The oral dosage form refers to a dosage form for intestinal administration, and preferably, the oral dosage form is a sustained-release or controlled-release oral preparation. Wherein the capsule dosage form comprises soft capsule or hard capsule.

In addition, the preparation of the active ingredient of the peganum harmala, which can effectively eliminate, inhibit, reduce or block the combination of PD1-PD L1, can also be prepared by other administration routes, such as respiratory tract administration preparations (sprays, aerosols and powder sprays), skin administration preparations (external solution, lotion, liniment, ointment, plaster, paste and patches), film administration preparations (eye drops, nose drops, eye ointment, gargle and sublingual tablets) and cavity administration preparations (suppositories).

The injection can be a conventional injection preparation; preferably, the injection is a liposome injection, a nano injection or a microsphere injection, and the injections respectively adopt liposome, nano particles and microspheres as drug carriers, so that the circulation time of the drug-loaded particles in the body is prolonged, the retention time of the drug-loaded particles at an absorption part is prolonged, the burst effect of the drug at the initial release stage is controlled, and the drug effect is enhanced. Preferably, the nanoparticles used as the carrier in the nanoparticle injection are nanocrystallized colloidal particles.

By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that:

1. the peganum harmala active extract part can effectively eliminate, inhibit, reduce or block the combination of PD1-PD L, can be used for preparing antitumor drugs for generating cancers due to the combination of PD1-PD L, is suitable for developing antitumor drugs, and the applicant finds that the raw materials for preparing the antitumor drugs are deficient at present, but in the prior art, the traditional Chinese medicine peganum harmala has the effects of relieving cough and asthma, dispelling rheumatism and eliminating swelling toxin, wherein the peganum harmala contains alkaloid protoplasm toxin and is a monoamine oxidase inhibitor, has the effects on a central nervous system and a cardiovascular system and also has the antitumor effect in the aspect of medicine application.

2. The method has the advantages of rapid separation of various components of peganum harmala, simple and stable separation and extraction process, suitability for industrial continuous production, high product yield, high-efficiency and simple separation of various active ingredients, separation of the active ingredients in a water phase, and reduction of the influence of an organic solvent on the activity of the medicine.

3. The application of the peganum harmala active extract part in preparing the antitumor drug for eliminating, inhibiting, reducing or blocking the combination of PD1-PD L1 expands the combination inhibitor or raw material drug channel of PD1-PD L1, increases the providers of effective drugs, expands the medicinal value of peganum harmala, develops the peganum harmala into a new raw material for inhibiting the combination of PD1-PD L1 with the antitumor drug, and can obviously improve the economic value and the use value of the peganum harmala.

Description of the drawings:

FIG. 1 shows the performance test of various extraction parts of peganum harmala;

FIG. 2 is IFN gamma detection, which is an experiment on the function of T cells stimulated outside the body extracted from peganum harmala;

FIG. 3 shows the in vitro T cell stimulation test-I L2 test of various extracts from Peganum harmala;

FIG. 4 shows that in vitro stimulated T cells of various extracts of Peganum harmala kill tumor cells.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific embodiments. It should be understood that the described embodiments are part of the present invention, and are intended to be illustrative only and not limiting in scope. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1 preparation of crude extract of active fraction of Peganum harmala

(1) extraction: selecting high-quality peganum harmala, cleaning with pure water, drying in the air, then crushing, soaking with 60% ethanol for 8h, extracting twice, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 70% ethanol into filter residue, performing ultrasonic extraction for 2 times, extracting for 3h each time, and concentrating under reduced pressure to obtain 70% ethanol extract; extracting with 83% ethanol once, concentrating under reduced pressure to obtain 83% ethanol extract, and mixing to obtain total ethanol extract. Dissolving the total ethanol extract with water at ratio of 1:3, extracting with ethyl acetate at ratio of 0.4:1 to obtain ethyl acetate part and water phase extract part;

(2) separating, namely separating a certain amount of ethyl acetate part by silica gel column chromatography, eluting by chloroform-methanol systems (pure chloroform, 50:1, 25:1, 10:1, 5:1 and methanol) with different proportions to obtain 6 elution parts, eluting the water phase extract part by D101 macroporous adsorption resin by ethanol-water systems (pure water, 20%, 40%, 60%, 80% and 95%) with different proportions to obtain 6 elution parts, wherein the pure water elution part and the elution part of the ethyl acetate extract 50:1 in the water phase extract are active parts of peganum harmala which can effectively block the combination of PD1-PD L1.

Wherein the reduced pressure recovery condition is 0.28MPA and the temperature is 28 ℃; the ultrasonic extraction conditions are 550HZ, 30min of ultrasonic treatment and 30 ℃.

Wherein, the extraction conditions are as follows: the extraction pressure is 28MPa, the extraction temperature is 36 ℃, and the extraction time is 2 h.

Wherein, the silica gel column chromatography separation conditions are as follows: using chloroform-methanol system as eluent, gradient eluting with pure chloroform, 50:1, 25:1, 10:1, 5:1 and methanol at flow rate of 0.5 ml/min.

Wherein, the separation conditions of the D101 macroporous adsorption resin are as follows: eluting with ethanol-water system as eluent, gradient eluting with pure water, 20%, 40%, 60%, 80%, 95%, and flow rate of 0.8 ml/min.

Example 2 crude extract preparation of active fraction of Peganum harmala

(1) extraction: selecting high-quality peganum harmala, cleaning with pure water, drying in the air, then crushing, soaking with 74% ethanol for 4h, extracting twice, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 80% ethanol into filter residue, performing ultrasonic extraction for 2 times, extracting for 4h each time, and concentrating under reduced pressure to obtain 80% ethanol extract; extracting with 85% ethanol once, concentrating under reduced pressure to obtain 85% ethanol extract, and mixing to obtain total ethanol extract. Dissolving the total ethanol extract with water at ratio of 1:4, extracting with ethyl acetate at ratio of 0.5:1 to obtain ethyl acetate part and water phase extract part;

Wherein the reduced pressure recovery condition is 0.35MPA and the temperature is 30 ℃; the ultrasonic extraction conditions are 300HZ, 35min of ultrasonic treatment and 35 ℃.

Wherein, the extraction conditions are as follows: the extraction pressure is 30MPa, the extraction temperature is 40 ℃, and the extraction time is 1 h.

Wherein, the silica gel column chromatography separation conditions are as follows: using chloroform-methanol system as eluent, gradient eluting with pure chloroform, 50:1, 25:1, 10:1, 5:1 and methanol at flow rate of 0.8 ml/min.

Wherein, the separation conditions of the D101 macroporous adsorption resin are as follows: eluting with ethanol-water system as eluent, gradient eluting with pure water, 20%, 40%, 60%, 80%, 95%, and flow rate of 1.0 ml/min.

Example 3 detection of Peganum harmala blocking PD1-PD L1 binding activity site Performance

The peganum harmala active site extracted and purified in the examples 1 and 2 is used as a drug performance detector, and the following experiments are carried out:

the detection principle of HTRF PD1/PD L1 detection kit (purchased from CISbIO) and HTRF PD1/PD L1 detection kit in experiments is that HTRF PD1/PD L1 detection kit is used to detect the interaction of PD1 and PD L1 proteins, the properties of each compound and antibody blocker can be described quickly, simply and at high throughput using HTRF (homogeneous time-resolved fluorescence) technology, the interaction between Tag 1-PD-L1 and Tag2-PD1 is detected by using anti-Tag 1-europium (HTRF donor) and anti-Tag2-X L665 (HTRF acceptor), when the donor antibody and the acceptor antibody are brought close together due to the binding of PD-L1 and PD1, the excitation of the donor antibody triggers the transfer of Fluorescence Resonance Energy (FRET) to the acceptor antibody, the acceptor antibody releases a specific signal at nm, this specific signal of 1/PD-L1 interacts to the extent of PD-3536, and thus the compound L1/1 reduces the signal.

The experiment is divided into 3 groups, namely an experiment group (2 mu L extract), a positive control group (2 mu L kit solvent, positive) and a negative control group (2 mu L DMSO, negative), wherein each group is provided with three compound holes.

The experimental group comprises transferring 2 mu L ethanol-water extract and ethyl acetate into a 96-well plate, sequentially adding 4 mu L Tag 1-PD-L1 protein (kit) and 4 mu L Tag2-PD-1protein (kit), standing at room temperature for 15min, adding 10 mu L solution prepared in advance (kit pre-mixed anti-Tag1-Eu3+ and anti-Tag2-X L665), and standing at room temperature for 2 h.

The positive control group is prepared by adding 2 mu L kit solvent dilent, 4 mu L Tag 1-PD-L1 protein and 4 mu L Tag2-PD-1protein into a 96-well plate, standing at room temperature for 15min, adding 10 mu L solution prepared in advance (kit pre-mixed anti-Tag1-Eu3+ and anti-Tag2-X L665), and standing at room temperature for 2 h.

The negative control group comprises 2 mu L DMSO, 4 mu L Tag 1-PD-L1 protein, 96 pore plate, room temperature for 15min, 10 mu L solution prepared in advance (kit pre-mixed anti-Tag1-Eu3+ and anti-Tag2-X L665), room temperature for 2h, measuring values of Signal 665nm and Signal 620nm by a microplate reader, calculating according to the following formula

Results analysis shows that the ratio of each active site is less than 0, and the representative site has the active component, which can block the binding interaction of PD1-PD L1, block the signal path of PD1-PD L1, and prevent the disease from developing.

As shown in figure 1, the parts 1-6 are sequentially active parts obtained by eluting and collecting water phase extract parts by pure water, 20%, 40%, 60%, 80% and 95% ethanol in an elution system water-ethanol, the parts 7-12 are parts obtained by eluting ethyl acetate parts by pure chloroform, 50:1, 25:1, 10:1 and 5:1 and methanol in a chloroform-methanol system and collecting the parts, and the influence of each part on the binding interaction of PD1-PD L1 is detected by using an HTRF PD1/PD L1 detection kit.

As can be seen from fig. 1, in each of the collection sites of the ethyl acetate extraction site and the aqueous phase extract site of peganum harmala, pure water and 20% elution site of the aqueous phase extract are shown to have the function of eliminating, inhibiting, reducing or blocking the PD1-PD L1 binding interaction, so that the pure water and 20% elution site of the aqueous phase extract of peganum harmala are active sites that effectively eliminate, inhibit, reduce or block the PD1-PD L1 binding interaction, and the active ingredients can develop an antitumor inhibitor by blocking the PD1-PD L1 binding interaction.

Example 4 Peganum harmala active extract in vitro stimulation of T cell function assay-tetanus toxin stimulation assay

Freshly prepared PBMC were plated in 96-well flat-bottom plates and after overnight incubation, concentrations (100ug/ml) of peganum harmala aqueous extract, ethyl acetate extract and 100ng/ml of Tetanus Toxin (TT) (L ist Biological L organisms) were added and after three days supernatants were harvested and assayed for IFN γ content by Elisa using the IFN γ kit from R & D systems, where the positive control group used atezolizumab, as can be seen from the results in fig. 2, and pure water from the aqueous extract and 20% of the eluted fraction extract from peganum harmala greatly increased cytokine secretion by immune cells stimulated by tetanus toxin TT while the other fractions did not significantly after elimination, inhibition, reduction or blocking of PD1-PD L1 signals.

Example 5 Peganum harmala Activity extraction in vitro stimulation of T cell function

The freshly prepared PBMC (obtained according to the conventional preparation method) are plated into a 96-well flat-bottom plate, after being incubated overnight, 100ug/ml concentrations of peganum harmala aqueous phase extract, ethyl acetate extract and 100ng/ml Tetanus Toxin (TT) are added, supernatant is collected after 3 days of culture, the secretion levels of peganum harmala aqueous phase extract, ethyl acetate extract and positive control (Atezolizumab) I L2 are detected by a L uminex instrument of L ife Technology company and a CD8+ cytokine detection kit of EMD company, the results of FIG. 3 show that pure water and 20% of elution part extract of the peganum harmala aqueous phase extract greatly increase cytokine I L2 which stimulates immune cells to secrete, can stimulate T cell functions, and the effects of other parts are not significant after eliminating, inhibiting, reducing or blocking PD1-PD L1 signals.

Example 6 Peganum harmala active extract can stimulate T cells to kill tumor cells in vitro

The method comprises the steps of infecting SW1417 cells with lentivirus (Qiagen) expressing PD-L1 protein to obtain a SW1417 cell line stably expressing PD-L1, introducing GFP gene to the cell line so that the GFP protein is stably expressed, extracting Dendritic Cells (DC) from fresh human peripheral blood cells, co-culturing the extracted T cells (1000/well) and 100ug/ml of peganum harmala aqueous phase extract, ethyl acetate extract and a positive control (Atezolizumab) in a 96-well plate for 3 days at 300 cells/well and the modified SW1417 cells (300/well), and co-culturing for 3 days to read GFP fluorescence values, wherein the result (shown in figure 4) shows that pure water in the peganum harmala and 20% of elution positions extract the T cells are externally stimulated to kill tumor cells obviously, and other positions of the T cells do not kill tumor cells obviously.

Example 7 antitumor inhibitor tablet blocking the binding of PD1-PD L1

The preparation method of the peganum harmala active site tablet comprises the steps of uniformly mixing 480mg of peganum harmala active extraction site and 160mg of starch, adding 76.8mg of starch paste with the mass percentage of 12% to prepare a soft material, sieving to obtain wet granules, drying to obtain dry granules, adding 10mg of magnesium stearate, uniformly mixing, and tabletting to obtain the anti-tumor inhibitor for blocking the combination of PD1-PD L1.

Example 8 antitumor inhibitor pellet Capsule blocking PD1-PD L1 binding

The pellet capsule consists of the following raw materials in percentage by weight:

the active extraction part of the peganum harmala is 400 mg;

80mg of lecithin;

50mg of taurocholate sodium;

45mg of microcrystalline cellulose;

the preparation method comprises the steps of uniformly mixing the active extraction part of peganum harmala, lecithin, sodium taurocholate and microcrystalline cellulose according to the proportion, adding an ethanol water solution, uniformly stirring to obtain a pasty material, pouring the pasty material into an extruder, extruding, obtaining granules by a roller, obtaining pellets by an extrusion rotating speed of 380rpm/min, a roller rotating speed of 900r/min and a roller time of 30min, drying, obtaining pellets by a 24-35-mesh sieve, and filling the pellets into a capsule shell to obtain the antineoplastic inhibitor pellet capsule for blocking the combination of PD1-PD L1.

Example 9 antitumor inhibitor liposomes that block PD1-PD L1 binding

An antitumor inhibitor liposome for blocking the combination of PD1-PD L1 comprises an antioxidant, a peganum harmala active site, phospholipid, a cholesterol compound, a surfactant and an aqueous medium, wherein the mass ratio of the peganum harmala active site to the antioxidant to the phospholipid to the cholesterol to the surfactant is 1:3-50:5-100:1-50:0-200, preferably the mass ratio of the peganum harmala active site to the phospholipid to the cholesterol to the surfactant is 1:3-30:5-50:1-30:0-100, and further preferably 1:4-20:5-25:1-10: 50-100.

Wherein the phospholipid may be a natural phospholipid such as: one or more of soybean lecithin, yolk lecithin and hydrogenated soybean lecithin; synthetic phospholipids such as: dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), phosphatidylserine (DOPS), Distearoylphosphatidylethanolamine (DSPE), Dioleoylphosphatidylethanolamine (DOPE), Dipalmitoylphosphatidylglycerol (DPPG), (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), and the like; optionally, phospholipid with functional modification such as PEG-DSPE can be added into phospholipid.

The surfactant can be Tween 80, Span 20, sodium cholate or the like.

The aqueous phase medium is a subacid PBS buffer solution, preferably, the pH value of the PBS buffer solution is 3-6, and further preferably, the pH value of the PBS buffer solution is 4.5-6.0.

The co-carried liposome can be prepared by an injection method or a thin film hydration method, and the preparation method comprises the following specific steps:

1. injection method

a. Dissolving phospholipid, cholesterol and surfactant in organic reagent at a certain ratio to obtain organic phase, wherein the organic reagent can be one or more of ethanol, chloroform, etc.;

b. dissolving the active part of the peganum harmala in a water phase medium to prepare a solution of the active part of the peganum harmala;

c. adding the peganum harmala active site solution into the organic phase in the step a, performing ultrasonic treatment to form a uniform emulsion phase, and performing rotary evaporation to remove the organic solvent to obtain a liposome solution;

d. filtering the liposome solution with microporous filter membranes of 0.8 μm, 0.45 μm, 0.22 μm and 0.1 μm to obtain stable co-carried liposome.

2. Thin film hydration process

a. Dissolving phospholipid, cholesterol and surfactant in an organic reagent according to a certain ratio to prepare an organic phase, and removing the organic reagent by spinning to obtain a uniform lipid membrane, wherein the organic reagent can be one or a mixture of ethanol, chloroform and the like;

c. b, adding the peganum harmala active site solution into the lipid membrane in the step a, and hydrating at room temperature to obtain a liposome solution;

d. reducing the particle size of the liposome by ultrasonic treatment, and sequentially filtering with microporous filter membranes of 0.8 μm, 0.45 μm, 0.22 μm and 0.1 μm to obtain stable liposome solution.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. An active ingredient of peganum harmala which effectively blocks the binding of PD1-PD L1 is characterized in that the active ingredient is derived from an aqueous extract fraction of peganum harmala.

2. The active component of peganum harmala L, which effectively blocks the binding of PD1-PD L1, according to claim 1, is characterized in that the water phase extract part of the peganum harmala L is separated by D101 macroporous adsorption resin and eluted by ethanol-water systems with different proportions, and the active component mainly comes from pure water and 20 percent of eluted parts.

3. The active ingredient of peganum harmala which effectively blocks the combination of PD1-PD L1 according to claim 1 is characterized in that the active ingredient of peganum harmala which effectively blocks the combination of PD1-PD L1 is prepared by the following method:

(1) extraction: selecting high-quality peganum harmala, cleaning with pure water, drying in the air, pulverizing, soaking with 60-80% ethanol for 4-24h, extracting twice, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 60-80% ethanol into the residue, ultrasonic extracting for 2 times, each time for 1-5h, and concentrating under reduced pressure to obtain 60-80% ethanol extract; extracting with 85-98% ethanol by the same method, concentrating under reduced pressure to obtain 85-98% ethanol extract, and mixing to obtain total ethanol extract; dissolving the total ethanol extract with water at ratio of 1:1-5, extracting with ethyl acetate at ratio of 0.5-1:1 to obtain ethyl acetate part and water phase extract part;

4. The peganum harmala L active ingredient drug which effectively blocks the binding of PD1-PD L1 according to any one of claims 1 to 3 is characterized by comprising a pharmaceutical composition of a peganum harmala L active ingredient and a pharmaceutically acceptable carrier.

5. The application of the peganum harmala active ingredient capable of effectively blocking the binding of PD1-PD L1 according to any one of claims 1 to 4, wherein the pharmaceutical composition of the peganum harmala active ingredient and a pharmaceutically acceptable carrier is applied to an anti-tumor medicament capable of effectively eliminating, inhibiting, reducing or blocking the binding of PD1-PD L1.

6. The use of peganum harmala as an active ingredient for effectively blocking the binding of PD1-PD L1 according to claim 5, wherein the tumor is a cancer selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, carcinoma of the esophagus, carcinoma of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, and combinations thereof.

7. The peganum harmala L active ingredient drug which effectively blocks the combination of PD1-PD L1 according to claim 4, is characterized in that the pharmaceutical composition of the peganum harmala L active ingredient and the pharmaceutically acceptable carrier is an oral preparation or an injection preparation.

8. The application of the active component of peganum harmala which effectively blocks the combination of PD1-PD L1 according to claim 5 is characterized in that the antitumor pharmaceutical preparation which effectively blocks the combination of PD1-PD L1 is prepared by combining the active component of peganum harmala with pharmaceutically acceptable pharmaceutical excipients.

9. The peganum harmala L active ingredient drug which effectively blocks the combination of PD1-PD L1 according to claim 6, is characterized in that the oral preparation can be tablets, capsules, granules, oral liquid solvents, liposomes and the like.

10. The peganum harmala L.var.harmala L.var.L.var.L.var.L.var.L.var.L.var.In effective as an active ingredient medicament for blocking the binding of PD1-PD L1, wherein the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic salt solution such as PBS (phosphate buffered saline), glucose, mannitol, dextroglucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like.