WO2020052526A1 - 荷叶碱和荷叶提取物作为制备治疗萎缩性胃炎和/或阻断胃炎癌转化发生药物的应用 - Google Patents

荷叶碱和荷叶提取物作为制备治疗萎缩性胃炎和/或阻断胃炎癌转化发生药物的应用 Download PDF

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WO2020052526A1
WO2020052526A1 PCT/CN2019/104997 CN2019104997W WO2020052526A1 WO 2020052526 A1 WO2020052526 A1 WO 2020052526A1 CN 2019104997 W CN2019104997 W CN 2019104997W WO 2020052526 A1 WO2020052526 A1 WO 2020052526A1
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lotus leaf
gastritis
cancer
leaf extract
gastric
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李梢
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/62—Nymphaeaceae (Water-lily family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of natural small molecule compounds and Chinese herbal extracts of traditional Chinese medicine, and particularly relates to lotus leaf alkali and lotus leaf extract as preparation medicines and health products for treating chronic atrophic gastritis, treating gastric precancerous lesions and / or blocking the transformation of gastritis cancer. And food applications.
  • Chronic inflammation is closely related to tumorigenesis [1].
  • the current epidemiological survey and a number of scientific research results show that in addition to genetic factors, most tumors are also closely related to environmental factors and lifestyles, such as smoking, diet, and infection. These carcinogenic factors can cause different forms of Chronic inflammation, rather than controlled inflammation, can promote tumorigenesis [2,3].
  • the occurrence of gastric cancer is closely related to gastritis and Helicobacter pylori infection [4,5].
  • gastritis and Helicobacter pylori infection According to statistics from the National Cancer Registry Center, more than half of the tumor-related deaths in China are digestive system tumors. Among them, the incidence and mortality of gastric cancer are at the forefront, and almost half of global gastric cancers occur in China. Gastric cancer is the first tumor with the highest incidence and mortality in rural areas of China [6,7]. Early prevention and control of gastric cancer is crucial. The lack of effective prevention and treatment measures for the transformation of gastritis cancer is an important reason for the high incidence of gastric cancer.
  • immune cells can release a variety of cytokines and chemokines.
  • key transcription factors such as NF- ⁇ B, STAT, and SMAD
  • somatic cells By activating key transcription factors such as NF- ⁇ B, STAT, and SMAD in somatic cells, they cause changes in somatic genome and epigenetics. causes cell proliferation, apoptosis and differentiation.
  • ROS reactive oxygen species
  • reactive nitrogen and other groups in the inflammatory microenvironment, which not only affect the epigenomic changes of somatic cells by affecting the chemical modification of DNA and proteins, but also play a key role in DNA damage and eventually cause the body Cell mutation and tumorigenesis [8-10].
  • How to treat mild inflammation to severe inflammation and block the transformation of inflammatory cancer is of great significance to prevent tumorigenesis.
  • the discovery of drugs that have the effect of treating chronic inflammation and then blocking or inhibiting the transformation of inflammation and cancer has also become the focus of much attention.
  • the lotus leaves are the leaves of the lotus family Nelumbo nuficera Gaertn, also known as lotus leaves and loquat leaves, which are grown in the north and south of China.
  • Lotus leaf has been included in the list of "food and medicine” medicine and food homogeneous traditional Chinese medicines in the No. 45 document of the Ministry of Health of China (1991) since November 1991. Recently, the National Health and Family Planning Commission announced it in 2018.
  • Lotus leaf alkali is an apomorphine alkaloid compound, and it is one of the main active ingredients of traditional Chinese medicine lotus leaf.
  • Lotus leaf alkali can be dissolved in organic solvents such as methanol and chloroform. The extraction process generally uses dried and crushed lotus leaves as raw materials.
  • the organic solvent method is used.
  • ultrasound, critical extraction and supercritical extraction are generally used as auxiliary methods.
  • Rhodocrine can improve hyperlipidemia [12] and reduce lipase activity [13].
  • scopolamine has pharmacological activity in treating many diseases, such as cardiovascular disease and cancer.
  • lotus leaf alkali has biological activities such as anti-atherosclerosis, bacteriostasis, anti-virus and anti-tumor [14-16].
  • the lotus leaf extract refers to a substance extracted by using lotus leaf as a raw material and water or an organic solvent.
  • the main components include lotus leaf alkaloids, lotus leaf flavones, citric acid, and oxalic acid.
  • Lotus leaf extracts have been reported to have biological activities such as blood lipid lowering, weight loss, and free radical scavenging [17,18].
  • Lotus leaf alkali and lotus leaf extract as substances in current weight-loss health products, are inexpensive and safe, providing greater possibilities for its clinical use in the treatment of chronic atrophic gastritis and blocking the transformation of gastritis cancer.
  • the inventor's previous research found that for the first time, lotus leaf alkali has an inhibitory effect on the transformation of enteritis cancer, and has obtained an invention patent license [19].
  • enteritis cancer transformation and gastritis cancer transformation are two distinctly different disease processes.
  • the literature investigation has not found any reports on the therapeutic effects of lotus leaf alkali and lotus leaf extract on gastric cancer, gastric precancerous lesions, and gastric cancer transformation caused by gastritis.
  • Anti-tumor drugs that block the transformation of inflammatory cancers are a specialized area independent of anti-tumor drugs. Because the research of anti-tumor drugs is directly using tumor cell lines or tumor tissues, the corresponding clinical treatment is the treatment of advanced tumors. This is a completely different concept from preventing tumorigenesis, especially blocking the transformation of inflammatory cells or tissues into tumors.
  • the purpose of the present invention is to provide lotus leaf alkali and lotus leaf extract as applications for treating precancerous lesions of chronic atrophic gastritis accompanied by intestinalization or dysplasia, and blocking the occurrence of gastritis cancer transformation, which can be used for the prevention of gastric cancer.
  • the medicinal and food homologous Chinese medicines involved in the present invention are lotus leaf alkali and lotus leaf extract.
  • the extraction technology is mature, the extraction rate is high, and the cost is low.
  • Lotus leaf alkali and lotus leaf extract are mainly prepared from natural plant lotus leaves.
  • lotus leaf alkali and lotus leaf extract have the effects of treating chronic atrophic gastritis and gastric precancerous lesions, and preventing the transformation of gastritis to gastric cancer.
  • the present invention provides the application of a substance for preventing the occurrence of gastric cancer.
  • the product is one selected from the group consisting of medicines, health products, and food.
  • the substance is selected from the following: A mixture of derivatives and lotus leaf extracts.
  • the prevention of gastric cancer includes at least one of treating chronic atrophic gastritis, protecting gastric mucosa, blocking the transformation of gastritis cancer, preventing the transformation of gastritis to gastric cancer, treating gastric precancerous lesions, and preventing the occurrence of gastric cancer.
  • a substance for the preparation and treatment of chronic atrophic gastritis, gastric precancerous lesions, and blocking the transformation of gastritis cancer.
  • the substance is one selected from the following:
  • the mixture includes at least two selected from the group consisting of lotus leaf alkali, lotus leaf alkali derivative, and lotus leaf extract.
  • the lotus leaf base belongs to apomorphine alkaloid compounds, the molecular formula is C 19 H 21 NO 2 , and the molecular weight is 295.38 g / mol; the structural formula is:
  • the product is one selected from the group consisting of medicines, health products, and foods, and the substance is one selected from the following Species:
  • the mixture includes at least two selected from the group consisting of lotus leaf alkali, lotus leaf alkali derivative, and lotus leaf extract.
  • the above-mentioned prevention of tumor formation includes at least one of treating chronic atrophic gastritis, protecting gastric mucosa, blocking gastritis cancer transformation, treating gastric precancerous lesions, and preventing the occurrence of gastric cancer.
  • a product characterized in that one of the following substances is selected as an active ingredient:
  • the mixture includes at least two selected from the group consisting of lotus leaf alkali, lotus leaf alkali derivative, and lotus leaf extract.
  • the product is selected from the group consisting of medicine, health products and food.
  • the above medicine has a dosage form selected from the group consisting of a tablet, a capsule, a pill, an injection, a sustained release agent, a controlled release agent, a powder, a beverage and the like.
  • the above health care product has a dosage form selected from the group consisting of a tablet, a capsule, a pill, an injection, a sustained release agent, a controlled release agent, a powder, a beverage and the like.
  • FIG. 1 shows the statistical results of the pathological state of chronic inflammation of gastric tissue in the experiment of the treatment of chronic gastritis atrophy with intestinal atrophy with intestinalization in one embodiment of the present invention.
  • 2A-2E show HE staining diagrams of gastric tissue pathological sections under normal, chronic gastritis atrophy with intestinalization, and lotus leaf alkali intervention in a representative group of individuals in an embodiment of the present invention.
  • Figure 3 shows the statistical results of the pathological state of chronic inflammation of gastric tissue in the experiment of chronic leaf gastritis atrophy with dysplasia in the experiment of lotus leaf extract in one embodiment of the present invention.
  • FIGS. 4A-4D show HE staining diagrams of gastric tissue pathological sections under normal and chronic gastritis atrophy with dysplasia and lotus leaf extract intervention in a representative group of individuals in an embodiment of the present invention.
  • Figures 5A-5E show the improvement of gastric mucosal damage in a representative group of individuals in the corresponding group in a normal, gastric mucosal injury model, lotus leaf alkali intervention, and lotus leaf extract intervention.
  • FIG. 6 shows the effect of statistical scopolamine on inhibiting abnormal proliferation of gastrointestinal stem cells and mucosal ulceration of Drosophila melanogaster in one embodiment of the present invention.
  • FIG. 7 shows the effect of statistical lotus leaf extract on inhibiting abnormal proliferation of gastrointestinal stem cells and mucosal ulcers of Drosophila melanogaster in one embodiment of the present invention.
  • 8A-8G show fluorescence staining diagrams of a representative group of individuals in the corresponding group in the model group, the intervention with lotus leaf alkali and the intervention with lotus leaf extract to inhibit the abnormal proliferation of gastrointestinal stem cells and the improvement of mucosal ulcers under the intervention of lotus leaf extracts.
  • FIG. 9 shows the statistical results of the inhibitory effect of lotus leaf alkali on the growth of transforming cells of gastritis cancer in one embodiment of the present invention.
  • FIG. 10 shows the results of a half-inhibition concentration (IC50) of the inhibitory effect of scopolamine on gastritis cancer transformed cells in one embodiment of the present invention is 92.72 ⁇ mol / L.
  • FIG. 11 shows the improvement of lotus leaf extracts in patients with chronic atrophic gastritis or gastric precancerous lesions according to an embodiment of the present invention.
  • a new medicine for treating atrophic gastritis, gastric precancerous lesions, and blocking the transformation of gastritis cancer comprises nastrine and lotus leaf extract.
  • the lotus leaf extract is derived from the lotus leaf of the plant and is obtained by water or organic solvent extraction.
  • the inventors confirmed through experiments that lotus leaf alkaloids and lotus leaf extracts are safe and can effectively treat chronic atrophic gastritis, protect gastric mucosa, block the transformation of gastritis cancer, prevent the transformation of gastritis to gastric cancer, treat gastric precancerous lesions and prevent Gastric cancer occurs.
  • a plant or natural product containing nicotine as a medicament for treating chronic atrophic gastritis, inhibiting gastritis cancer transformation, and / or preventing the occurrence of gastric cancer.
  • the nuciferine (English name nuciferine) used in order to achieve the above aspects of the present invention belongs to apomorphine alkaloid compounds, and has a molecular formula of C 19 H 21 NO 2 and a molecular weight of 295.38 g / mol; the structural formula is:
  • the prevention of tumorigenesis includes at least one of treating chronic atrophic gastritis, protecting gastric mucosa, blocking gastritis cancer transformation, treating gastric precancerous lesions, and preventing tumorigenesis.
  • the present inventors conducted a series of experiments. The results of these experiments are given in the following description of the examples. These experimental results show that lotus leaf alkali and lotus leaf extract can significantly treat atrophic gastritis (especially chronic atrophic gastritis with intestinal metaplasia or dysplasia) and / or provide a blocking gastritis cancer at a safe dose. Transformation function.
  • the inventors' findings include:
  • the animal model of chronic atrophic gastritis in rats was induced by the combination of sodium deoxycholate, ethanol, and ammonia [21]. After successful modeling, the lotus leaf extract was administered to the stomach, and pathological results showed that the lotus leaf extract can effectively treat chronic atrophic partners. Rats with dysplasia gastritis returned to a normal state in some rats, and gastritis in some rats was reduced.
  • Dextran sodium sulfate was used to induce the abnormal proliferation of gastrointestinal stem cells in Drosophila to simulate the tumorigenesis process.
  • lotus leaf alkali and lotus leaf extract were given. Microscopic results showed that lotus leaf alkali and lotus leaf extract can effectively inhibit the abnormal proliferation of gastrointestinal stem cells and mucosal ulceration of Drosophila gastrointestinal tract.
  • Scopolamine inhibits gastritis cancer transformation-related cell proliferation
  • N-methyl-N-nitrosoguanidine N-methyl- N'-nitro-N-nitrosoguanidine (MNNG) stimulated human gastric epithelial cells GES-1 inflammatory cancer transformation model [24], after MTT method (3- (4,5-dimethylthiazole-2) -2 , 5-Diphenyltetrazolium bromide colorimetric method), the half inhibitory concentration (IC50) of lotus leaf base on GES-1 gastritis cancer transformation model cells stimulated by MNNG was 92.72 ⁇ mol / L. The results show that lotus leaf alkali can inhibit the proliferation of gastritis cancer-related cells and block the gastritis cancer transformation process.
  • Rhodocrine has the effect of treating chronic atrophic rats with enteritis gastritis
  • the experimental animals are from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Thirty SD rats with specific pathogen free (SPF) grade 200 ⁇ 20g were selected, male and female, divided randomly into 5 groups: normal control group, chronic atrophic gastritis (CAG) model group, The positive drug celecoxib intervention group and nifedipine two-dose intervention group, 6 in each group.
  • Lotus leaf alkali was purchased from Chengdu Ruifensi Biotechnology Co., Ltd., with a specification of 5g and a purity of more than 98%.
  • Celecoxib was purchased from Beijing Bailingwei Technology Co., Ltd. under the brand J & K, with a specification of 1g and a purity of more than 98%.
  • Sodium deoxycholate was purchased from Amresco of the United States with a specification of 100 g.
  • the model group, the celecoxib intervention group and the lotus leaf alkali intervention group were administered sodium deoxycholate solution and ammonia water to induce chronic atrophic gastritis in rats.
  • 20mmol / L sodium deoxycholate was administered orally daily at a dose of 6ml / kg, twice a week on an empty stomach, 0.1% ammonia water was freely drinkable, and the model was continuously formed for 8 weeks.
  • Rat models of gastric mucosa atrophy and contraction occurred in different degrees. Associated with intestinal metaplasia.
  • the celecoxib group and the nasturtine intervention group were given celecoxib 10 mg / kg, 40 mg / kg, 10 mg / kg orally once a day. Changes in body weight and food intake were observed during the experiment.
  • the rats gained weight, their food intake recovered compared to the modeling stage, and their hair color returned to normal.
  • the stomach tissues were quickly removed, and pathological sections were prepared.
  • the obtained gastric tissue was cut along the great curvature of the stomach, rinsed with normal saline, blotted dry with filter paper, and fixed with formalin solution (10% formaldehyde solution).
  • the sections were embedded, dehydrated with graded alcohol, and then cleared with xylene. Immerse in wax and embed in paraffin.
  • the sections were soaked in xylene solution for 10 minutes each time, a total of 3 times; then they were soaked in absolute ethanol and rinsed in tap water. Finally, hematoxylin (stained nucleus) and eosin (stained cytoplasm) were used for staining, and the sections were sealed with neutral gum. Finally, pathological changes of gastric tissue in rats were observed under light microscope.
  • Rats in the chronic atrophic gastritis model group exhibited physical wasting, dull hair, atrophy, and loss of appetite. Two rats in the normal group and the model group were tested and found that the rats in the model group had chronic atrophy with different degrees. Intestinal metaplasia.
  • the body weight of the rats in the model group was significantly lower than that in the normal group.
  • the administration of celecoxib and lotus leaf base could increase the weight, restore appetite and increase the activity of the rats, indicating that celecoxib and lotus leaf base could improve the rats.
  • the appearance of chronic atrophic gastritis has a certain therapeutic effect on chronic atrophic gastritis.
  • the gastric mucosa surface of the normal group was smooth, the structure was intact, and the epithelial cells were arranged tightly without obvious inflammatory cell infiltration and erosion ulcer formation. See the normal group shown in Figure 2A.
  • lymphocytes, plasma cells, eosinophils, and a large number of goblet cells can be seen in the lamina intestinal of the gastric mucosa. Most of the inflammatory cells aggregate, intestinal metaplasia, and localized hyperplasia appear, as shown in Figure 2B Model group.
  • Example 2 The effect of lotus leaf extract in treating chronic atrophy and dystrophic gastritis rats
  • mice were from Beijing Spaifu Biotechnology Co., Ltd. Twenty-four SD rats (Specific Pathogen Free (SPF) 200 ⁇ 20g) were selected, male and female, randomly divided into 4 groups: normal control group, chronic atrophic gastritis (CAG) model group, Dutch Leaf extracts were administered in two dose intervention groups, six in each group, as shown in Table 2.
  • Lotus leaf extract was purchased from Hebei Chenguang Biotechnology Group Handan Co., Ltd., specifications: 1kg, lotus leaf alkali content above 0.4%.
  • Sodium deoxycholate was purchased from Amresco of the United States with a specification of 100 g.
  • rats were given sodium deoxycholate solution, ammonia and ethanol to induce chronic atrophic gastritis in rats.
  • 20mmol / L sodium deoxycholate was orally administered with 60% ethanol, a dose of 6ml / kg, twice a week on an empty stomach, 0.1% ammonia water was freely drinkable, and the model was given for 12 weeks.
  • the gastric mucosa of the model rats atrophied. Accompanied by varying degrees of intestinal metaplasia or dysplasia.
  • the lotus leaf extract intervention group was administered with high doses (containing 20 mg / kg of the nicotine) and low doses (containing 10 mg / kg of the nicotine) by gavage once a day. Changes in body weight and food intake were observed during the experiment.
  • the rats' food intake and body weight increased compared with the modeling stage, the coat color returned to light, and the phenotypes such as recovery of activity ability returned to normal.
  • the stomach tissues were quickly removed and pathological sections were prepared. The obtained gastric tissue was cut along the great curvature of the stomach, rinsed with normal saline, blotted dry with filter paper, and fixed with formalin solution (10% formaldehyde solution). The sections were embedded, dehydrated with graded alcohol, and then cleared with xylene. Immerse in wax and embed in paraffin.
  • the sections were soaked in xylene solution for 10 minutes each time, a total of 3 times; then they were soaked in absolute ethanol and rinsed in tap water. Finally, hematoxylin (stained nucleus) and eosin (stained cytoplasm) were used for staining, and the sections were sealed with neutral gum. Finally, pathological changes of gastric tissue in rats were observed under light microscope.
  • Example 3 Lotus leaf alkali and lotus leaf extract have protective effect on gastric mucosal damage
  • the experimental animals were from Beijing Spaifu Biotechnology Co., Ltd., and 30 SD rats (Specific Pathogen Free (SPF)) of 200 ⁇ 20g were selected. Males were randomly divided into 5 groups: normal control group, stomach The mucosal injury model group, low dose of lotus leaf alkali group, high dose of lotus leaf alkali group and lotus leaf extract group are shown in Table 3. The low-dose alkali group, the high-dose alkali group and the lotus leaf extract group were administrated intragastrically for 14 days, once a day. Lotus leaf alkali was purchased from Chengdu Ruifensi Biotechnology Co., Ltd., with a specification of 5g and a purity of more than 98%.
  • Lotus leaf extract was purchased from Hebei Chenguang Biotechnology Group Handan Co., Ltd., specifications: 1kg, lotus leaf alkali content above 0.4%. Rats in the low-dose alkali group, high-dose alkali group, and lotus leaf extract group were administered for 1 hour after continuous administration. The rats in the control group and the model group were strictly fasted for 24 hours (without water). During this period, the rats were also prohibited. Give medication. Rats in the model group, the low-dose alkali group, the high-dose alkali group and the lotus leaf extract group were administrated with 1.0 mL of absolute ethanol per gavage; 1 hour after the end of the modeling, the rats were sacrificed by abdominal aorta and sacrificed.
  • FIGs 5A-5E The normal group had no congestion and bleeding, as shown in Figure 5A.
  • the gastric mucosa was locally hyperemic and bleeding, and submucosal edema was seen, as shown in Fig. 5B.
  • Local congestion and hemorrhage in gastric mucosa were reduced in the low-dose sine base group, as shown in Figure 5C.
  • the gastric mucosa of the high-dose linaline group had no congestion and bleeding, as shown in Figure 5D.
  • Gastric mucosal hyperemia and submucosal edema were reduced in the lotus leaf extract group, as shown in Figure 5E.
  • Example 4 Lotus leaf alkali and lotus leaf extract can inhibit abnormal proliferation of gastrointestinal stem cells
  • Dextran sodium sulfate was used to induce the abnormal proliferation of gastrointestinal stem cells in Drosophila to simulate tumorigenesis.
  • the experimental fruit fly was esg-GFP, which was purchased from Tsinghua Drosophila Center. GFP (green fluorescent protein driven by esg-Gal4) can specifically label intestinal stem cells and intestinal cells.
  • a solution containing 20% sucrose, 3% DSS (average molecular weight 40kDa), and 0.5% dimethyl sulfoxide (DMSO) was mixed with the solution of the fruit fly food, and then left to dry for feeding the fruit fly for modeling. Different groups were given lotus leaf alkali and lotus leaf extract during the modeling.
  • Lotus leaf alkali was purchased from Chengdu Ruifensi Biotechnology Co., Ltd., with a specification of 5g and a purity of more than 98%.
  • Lotus leaf extract was purchased from Hebei Chenguang Biotechnology Group Handan Co., Ltd., specifications: 100g, lotus leaf alkali content above 0.4%.
  • Dextran sodium sulfate was purchased from Beijing Zhongke Keao Biotechnology Co., Ltd., brand mp, specification 50g.
  • Dimethyl sulfoxide was purchased from Amresco of the United States, with a specification of 500 ml.
  • Lotus leaf extract low-dose group medium + 0.5% DMSO + 3% DSS + lotus leaf extract (containing 100 ⁇ M lotus leaf alkali)
  • Lotus leaf extract high dose group medium + 0.5% DMSO + 3% DSS + lotus leaf extract (containing 300 ⁇ M lotus leaf alkali)
  • FIG 8A A severe abnormal proliferation of gastrointestinal stem cells in the Drosophila experiment DSS model group was shown in Figure 8A. Abnormal proliferation of gastrointestinal stem cells in Drosophila low-dose group was reduced, as shown in Figure 8B. Proliferation of gastrointestinal stem cells of the Drosophila high-dose group returned to normal (see Figure 8C). The DSS model group of lotus leaf extract experiment showed that the gastrointestinal mucosa of Drosophila ulcers and the abnormal proliferation of stem cells were abnormal, as shown in Figure 8D. Abnormal proliferation of gastrointestinal stem cells in Drosophila melanogaster low-dose group was reduced (see Figure 8E).
  • Drosophila gastrointestinal mucosal ulcers disappeared in the dose group of lotus leaf extract, and abnormal proliferation of gastrointestinal stem cells was reduced, as shown in Figure 8F.
  • Example 5 Lotus leaf has a significant inhibitory effect on gastritis cancer transformed cell model
  • MNNG human gastric epithelial cells
  • GES-1 human gastric epithelial cells
  • DMSO Dimethyl sulfoxide
  • FBS Fetal bovine serum
  • MTT 3- (4,5-dimethylthiazole-2) -2,5-diphenyltetrazolium bromide
  • GES-1 cells were seeded into a 96-well plate (8000 cells / well, leaving a row of blank wells, the marginal wells were discarded and an equal volume of PBS was added to reduce evaporation), the old solution was aspirated the next day, and 10% FBS was added.
  • Double antibody medium was added with 2 ⁇ 10 -5 MNNG to induce stimulation, and cultured for 24 hours in the dark, and the medium containing MNNG was discarded.
  • the dosing group was set with 7 lotus leaf alkali concentration gradients (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1000 ⁇ M), each gradient had 3 repeating wells; each well was added with the prepared lotus leaf alkali for 48 h, and Aspirate the old solution, add 0.5 mg / ml MTT complete medium (containing 10% FBS, total volume 100 ⁇ l / well), incubate at 37 ° C for 4 hours, aspirate the old solution, add 100 ⁇ l / DMSO, and mix with a horizontal shaker for 10 min The microplate reader detects the absorbance.
  • Example 6 Lotus leaf extract improves symptoms and objective indicators in patients with chronic atrophic gastritis or gastric precancerous lesions
  • Serum gastrin 17 has a promoting role in the occurrence and development of gastric cancer. When the serum gastrin 17 level increases, it indicates the risk of gastric cancer. Serum gastrin 17 can promote the occurrence and development of gastric cancer. When the level of serum gastrin 17 rises, it indicates the risk of gastric cancer [25]. A total of 10 patients were tested for symptoms in this experiment.
  • the overall condition of the test patients was: 3 males and 7 females; 2 were 45-50 years old, 1 was 50-55 years old, 4 were 60-65 years old, and 3 were 65-70 years old; diagnosed before the test was There were 3 patients with chronic atrophic gastritis, 3 with chronic atrophic gastritis with intestinal, and 4 with chronic atrophic gastritis with intestinal and dysplasia. Among them, 2 patients were tested for blood index. Patient 1 was 60 years old, female of gender, and was diagnosed with chronic atrophic gastritis with moderate intestinalization and mild dysplasia before the test. Patient 2 was 52 years old. , Gender female, diagnosed with chronic atrophic gastritis before the test.
  • the improvement rate of gastric pain is 100%, and the improvement rate of radon gas swallowing acid 87.5%, the bloating improvement rate was 83.3%.
  • the above experimental results show that lotus leaf extract can effectively improve the symptoms and objective indicators of patients with chronic atrophic gastritis accompanied by intestinal or dysplasia.
  • lotus leaf alkali and lotus leaf extract can effectively treat chronic atrophic gastritis and gastric precancerous lesions, prevent and / or block the transformation of gastritis cancer; and, lotus leaf alkali and lotus leaf extract Good physical safety, can protect gastric mucosal damage.
  • lotus leaf alkali and lotus leaf extract can be used as medicines, health products and / or foods for effectively treating chronic atrophic gastritis, treating gastric precancerous lesions and / or blocking gastritis cancer; lotus leaf alkali and lotus leaf extract The substance can be used as an effective component of drugs, health products and / or foods for effectively treating chronic atrophic gastritis, treating precancerous lesions of the stomach and / or blocking the development of gastritis cancer.
  • Li MY, Xu ZT The inhibition of the identity containing the lotus leaf-derived inhibitor-on-periodontitis-related bacteria in vitro. Int Dent, 2007, 57 (10): 303-306.

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Abstract

本发明提供了一种物质作为制备治疗慢性萎缩性胃炎、保护胃黏膜、治疗胃癌前病变、阻断胃炎癌转化、预防胃癌发生的产品的应用，所述产品为选自药品、保健品、食品的一种，该物质是从下列中选出的：荷叶碱，荷叶碱的衍生物，荷叶提取物，以及前述物质中选出的至少两种的混合物。所述应用包括治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、治疗胃癌前病变、阻止胃癌发生中的至少一种。体外实验表明，荷叶碱能够阻断胃炎癌转化细胞增殖。体内实验表明，荷叶碱、荷叶提取物能够治疗大鼠萎缩性胃炎（尤其是慢性萎缩性胃炎伴有肠上皮化生或异型增生）、保护大鼠胃黏膜损伤、抑制果蝇胃肠道干细胞异常增殖。临床测试表明，荷叶提取物能够改善萎缩性胃炎或胃癌前病变患者的症状和客观指标。

Description

荷叶碱和荷叶提取物作为制备治疗萎缩性胃炎和/或阻断胃炎癌转化发生药物的应用 技术领域
本发明属于中药天然小分子化合物和中药提取物领域，具体涉及荷叶碱及荷叶提取物作为制备治疗慢性萎缩性胃炎、治疗胃癌前病变和/或阻断胃炎癌转化发生的药物、保健品、食品的应用。
背景技术
慢性炎症与肿瘤发生具有密切的相关性[1]。目前流行病学调查和多项科学研究结果表明，大部分的肿瘤发生除了遗传因素之外，还与环境因素和生活方式息息相关，如吸烟、饮食和感染等，这些致癌因素均可导致不同形式的慢性炎症，而非可控性炎症可促使肿瘤发生[2,3]。尤其典型的是，胃癌的发生和胃炎、幽门螺杆菌感染具有密切关联[4,5]。全国肿瘤登记中心统计，我国半数以上的肿瘤相关死亡是消化系统肿瘤。其中胃癌发病率和死亡率均位居前列，全球胃癌几乎一半发生于中国。胃癌是我国农村地区发生率和死亡率均居第一位的肿瘤[6,7]。胃癌的早期防控至关重要。而缺乏胃炎癌转化有效的防治措施，则是导致胃癌发病率居高不下的重要原因。
在炎症环境下，免疫细胞能够释放多种细胞因子和趋化因子等，通过激活体细胞内的NF-κB、STAT、SMAD等关键转录因子，引起体细胞基因组、表观遗传学发生变化，进而导致细胞增殖、凋亡和分化等异常。同时，炎症微环境中存在大量的活性氧ROS、活性氮等基团，不仅通过影响DNA和蛋白质的化学修饰来影响体细胞的表观基因组变化，而且在DNA损伤中发挥关键作用，最终引起体细胞变异和肿瘤发生[8-10]。 如何治疗轻度炎症到重度炎症进而阻断炎癌转化对于预防肿瘤发生具有极为重要的意义。近年来，发现具有治疗慢性炎症进而阻断或抑制炎癌转化作用的药物也成为备受人们关注的重点。
预防肿瘤和治疗肿瘤是两个截然不同的概念。目前虽然有很多的抗肿瘤药物，但能够有效抑制炎癌转化、预防肿瘤发生的药物迄今为止还很少见。有研究报道显示，长期服用阿司匹林的人群结肠癌发病率降低24％[11]。此外，临床上用于治疗关节炎的COX-2抑制剂塞来昔布(商品名西乐葆，英文商品名Celebrex，全球第一个上市的选择性环氧化酶-2抑制剂)也被用于预防肠炎引起的肠癌。但是消化系统不同部位(如食管、胃、肠、肝、胰腺等)炎癌转化的机制不同，阿司匹林、塞来昔布针对的是肠炎癌转化，并非胃炎癌转化，而且这两种药物对于胃均具有一定的副作用。总之，目前尚缺乏有效防治胃炎癌转化发生的药物。
荷叶为睡莲科植物莲(Nelumbo nuficera Gaertn)的叶，又名莲叶、藕叶，在我国南北方均有种植。荷叶1991年11月开始被列入中国卫生部卫监发(1991)第45号文件中的“既是食品又是药品”的药食同源中药名单，最近国家卫生与计划生育委员会2018年公布的“药食同源”目录里也有荷叶。荷叶碱，是一种阿朴啡型生物碱类化合物，是中药荷叶的主要活性成分之一。荷叶碱能够溶解于甲醇、氯仿等有机溶剂，其提取一般以晒干粉碎的荷叶为原料，采用有机溶剂法，为了提高得率，一般辅助以超声、临界萃取和超临界萃取等方式。荷叶碱能够改善血脂过多[12]、降低脂肪酶活性[13]。许多研究表明，荷叶碱具有治疗多种疾病的药理活性，诸如心血管疾病和癌症等。目前报道称，荷叶碱具有抗动脉粥样硬化、抑菌、抗病毒和抗肿瘤等生物活性[14-16]。荷叶提取物指的是以荷叶为原料、经水或有机溶剂等方式提取得到的物质，主要成分包括荷叶生物碱、荷叶黄酮、柠檬酸、草酸等。荷叶提取物已报道具有降血脂、减肥、清除自由基等生物活性[17,18]。荷叶碱和荷叶提取物作为目前减肥保健品中的物质，其价格低廉，安全性好，为其临床上用于治疗慢性萎缩性胃炎和阻断胃炎癌转化发生提供了更大可能性。本发明人前期研究首次发现荷叶碱对于肠炎癌转化具有抑制作用，并获得发明专利授权[19]。但肠炎癌转化与胃炎癌转化是两类明显不同的疾病过程，文献调研尚未发现荷叶碱、荷叶提取物对胃炎引发的胃癌、胃癌前病变、胃炎癌转化有关治疗作用的报道。
发明内容
阻断炎癌转化的预防肿瘤药物是独立于抗肿瘤药物的一个专门领域。由于抗肿瘤药物研究是直接用肿瘤细胞系或肿瘤组织来进行研究，临床上对应的是肿瘤中晚期的治疗。这与预防肿瘤发生，尤其是阻断发生炎症的细胞或组织向肿瘤的转化功能是完全不同的概念。
本发明的目的在于提供荷叶碱、荷叶提取物作为治疗慢性萎缩性胃炎伴有肠化或异型增生的癌前病变、和阻断胃炎癌转化发生的应用，可用于胃癌的预防。本发明涉及的药食同源中药是荷叶碱及荷叶提取物，其提取技术成熟，提取率较高，成本低廉。荷叶碱和荷叶提取物制备主要是从天然植物荷叶中提取获得。
本发明人经过一系列实验，证实了荷叶碱、荷叶提取物具有治疗慢性萎缩性胃炎和胃癌前病变，以及阻止胃炎向胃癌转化的功效。
本发明提供了一种物质作为制备预防胃癌发生的产品的应用，所述产品为选自药品、保健品、食品的一种，该物质是从下列中选出的：荷叶碱、荷叶碱的衍生物及荷叶提取物的混合物。所述预防胃癌发生包括治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、预防胃炎转变为胃癌、治疗胃癌前病变和阻止胃癌发生中的至少一种。
根据本发明的一个方面，提供了一种物质作为制备治疗慢性萎缩性胃炎、胃癌前病变和阻断胃炎癌转化的应用，该物质是从下列中选出的一种：
荷叶碱，
荷叶碱的衍生物，
荷叶提取物，
混合物，包含荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种。
根据本发明的一个进一步的方面，上述荷叶碱属于阿朴啡型生物碱类化合物，分子式为C 19H 21NO 2，分子量为295.38g/mol；结构式为：
Figure PCTCN2019104997-appb-000001
根据本发明的另一个方面，提供了一种物质作为制备预防肿瘤发生的产品的应用，所述产品为选自药品、保健品、食品的一种，所述物质是从下列中选出的一种：
荷叶碱，
荷叶碱的衍生物，
荷叶提取物，以及
混合物，包含荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种。
根据本发明的一个进一步的方面，上述预防肿瘤发生包括治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、治疗胃癌前病变和阻止胃癌发生中的至少一种。
根据本发明的又一个方面，提供了一种产品，其特征在于：以下列物质中选出的一种作为活性成分：
荷叶碱，
荷叶碱的衍生物，
荷叶提取物，以及
混合物，包含荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种。
并含有其他辅料，其中所述产品选自药物、保健品、食品中的一种。
根据本发明的一个进一步的方面，上述药物具有选自片剂、胶囊、丸剂、针剂、缓释剂、控释剂、粉剂、饮料等剂型。
根据本发明的一个进一步的方面，上述保健品具有选自片剂、胶囊、丸剂、针剂、缓释剂、控释剂、粉剂、饮料等剂型。
附图说明
图1显示了本发明的一个实施例中荷叶碱治疗慢性胃炎萎缩伴肠化实验中胃组织慢性炎症病理状态的统计结果。
图2A－2E显示了本发明的一个实施例中相应组有代表性的个体在正常、慢性胃炎萎缩伴肠化发生、荷叶碱干预下的胃组织病理切片HE染色图。
图3显示了本发明的一个实施例中荷叶提取物治疗慢性胃炎萎缩伴异型增生实验中胃组织慢性炎症病理状态的统计结果。
图4A－4D显示了本发明的一个实施例中相应组有代表性的个体在正常、慢性胃炎萎缩伴异型增生发生、荷叶提取物干预下的胃组织病理切片HE染色图。
图5A－5E显示了本发明的一个实施例中相应组有代表性的个体在正常、胃黏膜损伤模型、荷叶碱干预和荷叶提取物干预下的胃黏膜损伤改善情况。
图6显示了本发明的一个实施例中统计荷叶碱抑制果蝇胃肠道干细胞异常增殖和粘膜溃烂的作用。
图7显示了本发明的一个实施例中统计荷叶提取物抑制果蝇胃肠道干细胞异常增殖和粘膜溃烂的作用。
图8A－8G显示了本发明的一个实施例中相应组代表性个体在模型组、荷叶碱干预和荷叶提取物干预下抑制胃肠道干细胞异常增殖和粘膜溃烂改善情况的荧光染色图。
图9显示了本发明的一个实施例中荷叶碱对胃炎癌转化细胞生长抑制作用的统计结果。
图10显示了本发明的一个实施例中荷叶碱对胃炎癌转化细胞生长抑制作用的半数抑制浓度(IC50)为92.72μmol/L的结果。
图11显示了本发明的一个实施例中荷叶提取物对慢性萎缩性胃炎或胃癌前病变患者相关病情改善情况。
具体实施方式
根据本发明的一个方面，提供了一种新型治疗萎缩性胃炎、胃癌前病变和阻断胃炎癌转化的药物，该药物所包含的活性物质包含荷叶碱、 荷叶提取物。荷叶提取物来源于植物荷叶，通过水或有机溶剂方式提取得到。本发明人通过试验证实荷叶碱、荷叶提取物安全性好，且能够有效地治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、预防胃炎转变为胃癌、治疗胃癌前病变和阻止胃癌发生。
根据本发明的另一个方面，提供了含有荷叶碱的植物或天然产物作为制备治疗慢性萎缩性胃炎、抑制胃炎癌转化和/或预防胃癌发生的药物的应用。
为了实现本发明上述方面而采用的荷叶碱(英文名nuciferine)属于阿朴啡型生物碱类化合物，分子式为C 19H 21NO 2，分子量为295.38g/mol；结构式为：
Figure PCTCN2019104997-appb-000002
在根据本发明所述用途中，所述预防肿瘤发生包括治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、治疗胃癌前病变和阻止肿瘤的发生中的至少一种。
在进行并完成本发明的过程中，本发明人进行了一系列实验。在下文中对实施例的相关说明中，给出了这些实验的结果。这些实验结果表明了，荷叶碱和荷叶提取物在安全的剂量下能够显著治疗萎缩性胃炎(尤其是慢性萎缩性胃炎伴有肠上皮化生或异型增生)和/或提供阻断胃炎癌转化的功能。在上述实验中，以及在与本发明相关的其他工作中，本发明人的发现包括：
(1)荷叶碱治疗慢性萎缩性胃炎伴有肠上皮化生
选择脱氧胆酸钠和氨水联合诱导的大鼠慢性萎缩性胃炎动物模型[20]，造模成功后给予塞来昔布、荷叶碱灌胃，病理结果显示荷叶碱能够有效治疗慢性萎缩性胃炎大鼠、逆转肠上皮化生，使部分大鼠胃部恢复正常状态，大部分大鼠炎症减轻到轻度慢性非萎缩性胃炎状态。
(2)荷叶提取物治疗慢性萎缩性胃炎伴有异型增生
利用脱氧胆酸钠和乙醇、氨水联合诱导的大鼠慢性萎缩性胃炎动物模型[21]，造模成功后给予荷叶提取物灌胃，病理结果显示荷叶提取物能够有效治疗慢性萎缩性伴异型增生胃炎大鼠，使部分大鼠胃部恢复正常状态，部分大鼠胃炎减轻。
(3)荷叶碱、荷叶提取物保护胃黏膜损伤
在大鼠连续给予荷叶碱、荷叶提取物灌胃后，通过无水乙醇诱导大鼠胃黏膜损伤[22]，解剖后在镜下观察胃黏膜损伤情况。可见模型组大鼠出现不同程度胃黏膜损伤，给予荷叶碱组、给予荷叶提取物组大鼠胃黏膜损伤较模型组有明显改善、大鼠活动能力增多。
(4)荷叶碱、荷叶提取物抑制胃干细胞异常增殖
选择《细胞》出版社的刊物《细胞干细胞》(Cell Stem Cell)曾报道的胃肠道肿瘤发生模型[23]。利用葡聚糖硫酸钠(Dextran sulfate sodium，DSS)诱导果蝇胃肠道干细胞异常增殖模拟肿瘤发生过程。造模期间给予荷叶碱和荷叶提取物。显微镜下结果显示，荷叶碱、荷叶提取物均能够有效抑制果蝇胃肠道干细胞异常增殖和粘膜溃烂情况。
(5)荷叶碱抑制胃炎癌转化相关细胞增殖
选择肿瘤研究知名刊物、英国自然出版社《致癌基因》(Oncogene)曾报道的胃炎癌转化的细胞模型——以化学诱变剂N-甲基N-硝基亚硝基胍(N-methyl-N'-nitro-N-nitrosoguanidine，MNNG)刺激的人胃上皮细胞GES-1炎癌转化模型为对象[24]，经MTT法(3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐比色法)测定，荷叶碱对MNNG刺激下的GES-1胃炎癌转化模型细胞的半数抑制浓度(IC50)为92.72μmol/L。结果表明荷叶碱能够抑制胃炎癌转化相关细胞增殖、阻断胃炎癌转化过程。
(6)荷叶提取物改善萎缩性胃炎伴肠化或异型增生患者的症状和指标
依据新悉尼标准，纳入内镜病理诊断为慢性萎缩性胃炎伴肠上皮化生或异型增生的患者(年龄在18岁-70岁)，在受试患者填写知情同意书后，进行荷叶提取物治疗胃炎癌转化的临床观察试验，记录症状和化验胃炎相关血液指标(血清胃泌素17)。结果表明荷叶提取物能够改善慢性萎缩性胃炎伴肠化或异型增生患者的症状和客观指标。
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
实施例1：荷叶碱具有治疗慢性萎缩性伴有肠化胃炎大鼠的作用
实施方案：实验动物来自北京维通利华实验动物技术有限公司。选择无特定病原体动物(Specific pathogen free，SPF)级200±20g SD大鼠30只，雌雄各半，随机分为5组：正常对照组、慢性萎缩性胃炎(Chronic atrophic gastritis，CAG)模型组、阳性药塞来昔布干预组和荷叶碱两个剂量干预组，每组6只。荷叶碱购自成都瑞芬思生物科技有限公司，规格5g，纯度98％以上。塞来昔布购自北京百灵威科技有限公司，品牌J&K，规格1g，纯度98％以上。脱氧胆酸钠购自美国Amresco公司，规格100g。模型组、塞来昔布干预组和荷叶碱干预组采用大鼠给予脱氧胆酸钠溶液、氨水，诱发大鼠发生慢性萎缩性胃炎。20mmol/L的脱氧胆酸钠每日灌胃，剂量6ml/kg，每周两次空腹灌胃，0.1％氨水自由饮用，连续造模8周，造模大鼠胃粘膜出现不同程度的萎缩并伴有肠上皮化生。在造模结束后，塞来昔布组和荷叶碱干预组分别给予塞来昔布10mg/kg，荷叶碱40mg/kg，10mg/kg灌胃给药，一天一次。实验期间观察大鼠体重和摄食量的改变。
治疗性给药4周后，大鼠体重增加，摄食量较造模阶段有恢复，毛色恢复光亮等外观正常状态。给药实验结束阶段，将大鼠脱椎处死后，迅速取出胃组织，并进行病理切片准备。将获取的胃组织沿胃大弯剪开，用生理盐水冲洗，滤纸吸干，用福尔马林溶液(10％甲醛溶液)固定。包埋切片，逐级酒精脱水，然后二甲苯透明。浸蜡，进行石蜡包埋。染色阶段，将切片放入二甲苯溶液中浸泡，每次10分钟，共3次；随后放入无水乙醇浸泡和自来水中漂洗。最后用苏木精(染细胞核)和伊红(染细胞浆)染色，用中性树胶封片。最后在光镜下观察大鼠胃部组织病理变化。
表1荷叶碱治疗慢性萎缩性胃炎实验分组
Figure PCTCN2019104997-appb-000003
实验结果：
1.慢性萎缩性胃炎组大鼠表型的观察
慢性萎缩性胃炎模型组大鼠出现了体态消瘦，毛发无光泽、精神萎靡和食欲减退等表现，其中正常组和模型组各取2只进行检验发现，模型组大鼠出现不同程度慢性萎缩并伴有肠上皮化生状态。
实验中模型组大鼠体重相比正常组明显下降，给予塞来昔布和荷叶碱可以增加大鼠体重、食欲有所恢复和活动增多，表明塞来昔布和荷叶碱可以改善大鼠慢性萎缩性胃炎的外观表现，对慢性萎缩性胃炎有一定的治疗作用。
2.光镜下观察胃组织病理切片
根据病理切片判读结果统计，相比正常组大鼠，造模阶段大鼠均发生慢性萎缩性胃炎及轻度肠化病理状态，塞来昔布干预组和荷叶碱干预组可以有效治疗大鼠慢性萎缩性胃炎和肠化病理状态，其中塞来昔布和荷叶碱高剂量组效果最好，有胃部恢复正常状态的大鼠，其余大部分减弱到轻度非萎缩性胃炎病理状态。对病理判读结果进行统计作图，所获得的结果被显示在图1中。
光镜下观察，正常组大鼠胃粘膜表面光滑，结构层次完整，上皮细胞排列紧密，无明显炎症细胞浸润和糜烂溃疡形成，见图2A显示的正常组。慢性萎缩性胃炎模型组可见胃粘膜固有层内见淋巴细胞、浆细胞、嗜酸性细胞浸润和大量杯状细胞，多数炎症细胞聚集，肠上皮化生，局部灶性区域出现增生，见图2B显示的模型组。塞来昔布组(图2C)和荷叶碱高剂量组(见图2E)较模型组慢性萎缩性胃炎病理状态明显减轻，有恢复正常状态的大鼠。大部分大鼠的固有层内见少许淋巴细胞、浆细胞和嗜酸性细胞浸润，肠上皮化生消失。荷叶碱低剂量组(见图2D)胃组织固有层内有少量淋巴细胞和浆细胞浸润，病理状态减轻到慢性非萎缩胃炎状态，未见溃疡形成。
实施例2：荷叶提取物具有治疗慢性萎缩伴异型增生胃炎大鼠的作用
实验方案：实验动物来自北京斯贝福生物技术有限公司。选择无特定 病原体级(Specific pathogen free，SPF)200±20g SD大鼠24只，雌雄各半，随机分为4组：正常对照组、慢性萎缩性胃炎(chronic atrophic gastritis，CAG)模型组、荷叶提取物两个剂量干预组，每组6只，如表2所示。荷叶提取物购自河北晨光生物科技集团邯郸有限公司，规格：1kg，荷叶碱含量0.4％以上。脱氧胆酸钠购自美国Amresco公司，规格100g。模型组和荷叶提取物干预组采用大鼠给予脱氧胆酸钠溶液、氨水和乙醇，诱发大鼠发生慢性萎缩性胃炎。20mmol/L的脱氧胆酸钠每日灌胃及60％乙醇，剂量6ml/kg，每周两次空腹灌胃，0.1％氨水自由饮用，连续造模12周，造模大鼠胃粘膜出现萎缩并伴有不同程度的肠上皮化生或异型增生。在造模结束后，荷叶提取物干预组分别给予高剂量(含荷叶碱20mg/kg)、低剂量(含荷叶碱10mg/kg)灌胃给药，一天一次。实验期间观察大鼠体重和摄食量的改变。
治疗性给药4周后，大鼠摄食量和体重较造模阶段增加，毛色恢复光亮，活动能力恢复等表型恢复正常状态。给药结束阶段，将大鼠椎处死后，迅速取出胃组织，并进行病理切片准备。将获取的胃组织沿胃大弯剪开，用生理盐水冲洗，滤纸吸干，用福尔马林溶液(10％甲醛溶液)固定。包埋切片，逐级酒精脱水，然后二甲苯透明。浸蜡，进行石蜡包埋。染色阶段，将切片放入二甲苯溶液中浸泡，每次10分钟，共3次；随后放入无水乙醇浸泡和自来水中漂洗。最后用苏木精(染细胞核)和伊红(染细胞浆)染色，用中性树胶封片。最后在光镜下观察大鼠胃部组织病理变化。
表2荷叶提取物治疗慢性萎缩性胃炎实验分组
Figure PCTCN2019104997-appb-000004
实验结果：
1.光镜下观察胃组织病理切片
根据病理切片判读结果统计，相比正常组大鼠，造模阶段大鼠发生慢性萎缩性胃炎中度肠化或异型增生病理状态，荷叶碱提取物组可以有效治疗大鼠慢性萎缩性胃炎异型增生病理状态，其中荷叶提取物高剂量组效果最好，有胃部恢复正常状态的大鼠，其余减弱到轻度肠化病理状态。对病理判读结果进行统计作图，所获得的结果见图3。
光镜下观察，正常组大鼠胃粘膜表面光滑，纹理清晰，上皮细胞排列紧密，无明显糜烂溃疡形成，见图4A。慢性萎缩性胃炎模型组可见局灶间质出血，大量杯装细胞聚集，腺体中度异型增生，见图4B。荷叶提取物低剂量组部分大鼠腺体异型增生消失，有少量淋巴细胞聚集和嗜酸性粒细胞浸润。见图4C。荷叶提取物高剂量组较模型组病理状态明显减轻，有恢复正常状态的大鼠。部分大鼠有少许嗜酸性粒细胞浸润，腺体异型增生消失，中度肠化减轻为轻度肠化或消失，见图4D。
实施例3：荷叶碱及荷叶提取物对胃黏膜损伤有保护作用
实验方案：实验动物来自北京斯贝福生物技术有限公司，选择无特定病原体动物(Specific pathogen free，SPF)级200±20g SD大鼠30只，雄性，随机分为5组：正常对照组、胃粘膜损伤模型组、荷叶碱低剂量组、荷叶碱高剂量组和荷叶提取物组，如表3所示。荷叶碱低剂量组、荷叶碱高剂量组和荷叶提取物组连续灌胃14天，每天一次。荷叶碱购自成都瑞芬思生物科技有限公司，规格5g，纯度98％以上。荷叶提取物购自河北晨光生物科技集团邯郸有限公司，规格：1kg，荷叶碱含量0.4％以上。荷叶碱低剂量组、荷叶碱高剂量组和荷叶提取物组大鼠连续给药结束1h后，对照组和模型组的大鼠严格禁食24小时(不禁水)，此期间亦禁止给予药物。模型组、荷叶碱低剂量组、荷叶碱高剂量组和荷叶提取物组大鼠灌胃给予无水乙醇1.0mL/只；造模结束1h后，大鼠腹主动脉采血处死，切腹取胃，向胃内注入10％甲醛溶液，固定20min，然后沿胃大弯曲部剪开，洗净胃内容物，在解剖显微镜下进行大体观察，并测量损伤长度(mm)。
表3保护胃黏膜损伤实验分组
Figure PCTCN2019104997-appb-000005
实验结果：
在正常组和模型组解剖中发现，模型组大鼠出现不同程度胃黏膜损伤。给予荷叶碱和荷叶提取物组大鼠胃黏膜损伤较模型组有改善、大鼠活动能力增多，表明荷叶碱和荷叶提取物能够保护胃黏膜损伤。
解剖后镜下观察，结果如图5A－5E所示。正常组无充血和出血，见图5A。模型组胃黏膜局部高度充血和出血、粘膜下层水肿，见图5B。荷叶碱低剂量组胃黏膜局部充血和出血减轻，见图5C。荷叶碱高剂量组胃黏膜无充血和出血，见图5D。荷叶提取物组胃黏膜充血和粘膜下层水肿减轻，见图5E。
实施例4：荷叶碱及荷叶提取物能够抑制胃肠道干细胞异常增殖
实验方案：利用葡聚糖硫酸钠(Dextran sulfate sodiu，DSS)诱导果蝇胃肠道干细胞异常增殖模拟肿瘤发生过程。实验用果蝇为esg-GFP，购自清华果蝇中心。GFP(esg-Gal4驱动表达的绿色荧光蛋白)可以特异性标记肠道干细胞和成肠细胞。配置含20％蔗糖，3％DSS(平均分子量40kDa)和0.5％二甲基亚砜(DMSO)的溶液，将该溶液与果蝇食物混合，放置半干后用于喂食果蝇造模。造模期间不同组给予荷叶碱和荷叶提取物。荷叶碱购自成都瑞芬思生物科技有限公司，规格5g，纯度98％以上。荷叶提取物购自河北晨光生物科技集团邯郸有限公司，规格：100g，荷叶碱含量0.4％以上。葡聚糖硫酸钠购自北京中科科奥生物科技有限公司，品牌mp，规格50g。二甲基亚砜购自美国Amresco公司，规格500ml。
实验分组：
1)荷叶碱实验分组
模型对照组：培养基+0.5％DMSO+3％DSS
荷叶碱低剂量组：培养基+0.5％DMSO+3％DSS+荷叶碱100μM
荷叶碱高剂量组：培养基+0.5％DMSO+3％DSS+荷叶碱200μM
2)荷叶提取物实验分组
模型对照组：培养基+0.5％DMSO+3％DSS
荷叶提取物低剂量组：培养基+0.5％DMSO+3％DSS+荷叶提取物(含100μM荷叶碱)
荷叶提取物中剂量组：培养基+0.5％DMSO+3％DSS+荷叶提取物(含200μM荷叶碱)
荷叶提取物高剂量组：培养基+0.5％DMSO+3％DSS+荷叶提取物(含300μM荷叶碱)
每个组3个平行管。收集一天内孵化的处女蝇，在25℃用正常培养基饲养3天，果蝇转移到模型组、荷叶碱低剂量组、荷叶碱高剂量组、荷叶提取物低剂量组、荷叶提取物中剂量组和荷叶提取物高剂量组培养基中，每组约20只，在25℃继续培养3-5天。实验结束时，解剖出胃肠道，观察GFP变化。
实验结果：
显微镜下统计结果显示，荷叶碱和荷叶提取物组能够有效抑制果蝇胃肠道干细胞异常增殖和胃肠道粘膜溃烂。荷叶碱实验中，荷叶碱低剂量组和高剂量组减少胃肠道干细胞异常增殖比例，部分果蝇胃肠道干细胞增殖恢复正常，如图6所示。荷叶提取物实验中，荷叶提取物随剂量升高不同程度地减轻果蝇胃肠道干细胞异常增殖，部分果蝇胃肠道干细胞增殖恢复正常，胃肠道粘膜溃烂消失，如图7所示。果蝇胃肠道显微镜下解剖结果，如图8A-8G所示。荷叶碱实验DSS模型组果蝇胃肠道干细胞出现重度异常增殖，见图8A。荷叶碱低剂量组果蝇胃肠道干细胞异常增殖减轻，如图8B所示。荷叶碱高剂量组果蝇胃肠道干细胞增殖恢复正常(见图8C)。荷叶提取物实验DSS模型组果蝇胃肠道粘膜溃烂、干细 胞重度异常增殖，见图8D。荷叶提取物低剂量组果蝇胃肠道干细胞异常增殖减轻(见图8E)。荷叶提取物中剂量组果蝇胃肠道粘膜溃烂消失，胃肠道干细胞异常增殖减轻，见图8F。荷叶提取物高剂量组果蝇胃肠道黏膜溃烂消失，胃肠道干细胞增殖恢复正常，如图8G所示。
实施例5：荷叶碱对胃炎癌转化细胞模型有显著抑制作用
本实施例中采用的化学诱变剂N-甲基N-硝基亚硝基胍(MNNG)诱导人胃上皮细胞(GES-1)形成的胃炎癌转化模型按文献[21]的报道。荷叶碱购自成都瑞芬思生物科技有限公司，规格1g，纯度98％以上。MNNG购自北京百盈利创生物科技有限公司，品牌梯希爱，规格5g。GES-1细胞购自上海栩冉生物科技有限公司。磷酸盐缓冲生理盐水(PBS)购自美国Amresco公司，规格10L。二甲基亚砜(DMSO)购自美国Amresco公司，规格500ml。胎牛血清(FBS)购自美国Amresco公司，规格100ml。3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(MTT)购自上海碧云天生物技术有限公司，规格500mg。实验方案：
将GES-1细胞种到96孔板中(8000个细胞/孔，留一列空白孔，边缘孔弃用并加等体积PBS减少蒸发)，次日将旧液吸出，加入含10％FBS不含双抗培养基，加入2×10 -5的MNNG诱导刺激，避光培养24h，，弃除含MNNG的培养液。加药组设置7个荷叶碱浓度梯度(0.1μM,1μM,10μM,100μM,200μM,500μM,1000μM)，每个梯度3个重复孔；每孔加入已配好的荷叶碱作用48h，将旧液吸出，加入含0.5mg/ml MTT的完全培养基(含10％FBS，总体积100μl/孔)，37℃孵育4h，将旧液吸出，加入100μl/DMSO，水平震荡仪混匀10min后，酶标仪检测吸光度。
实验结果：
与正常对照组比较，MNNG刺激的GES-1细胞活力显著增强，荷叶碱具有显著抑制MNNG-GES-1细胞活力的作用(P<0.05)，如图9所示。对实验结果进行数据处理，测得荷叶碱对胃炎癌转化细胞的半数抑制浓度(IC50)为92.72μmol/L，如表4、图10所示。体现了荷叶碱对胃炎癌转化细胞有抑制作用。
表4：荷叶碱对胃炎癌转化细胞的生长抑制作用
Figure PCTCN2019104997-appb-000006
实施例6：荷叶提取物改善慢性萎缩性胃炎或胃癌前病变患者症状和客观指标
实验方案：本实验针对慢性萎缩性胃炎伴肠化或异型增生患者，给予荷叶提取物治疗，通过调查患者相关血液指标和临床症状改善情况评估其疗效。荷叶提取物由河北晨光生物科技集团邯郸有限公司提供。依据新悉尼标准，纳入病理诊断为慢性萎缩性胃炎或伴肠上皮化生、异型增生的患者。受试者在填写知情同意书后，每天饮用荷叶提取物1袋(10g/袋)，每周反馈一次慢性萎缩性胃炎症状改善情况，饮用2个月后到医院复查并化验血液相关指标(血清胃泌素17)。血清胃泌素17在胃癌发生、发展中有促进作用，当血清胃泌素17水平升高提示存在胃癌发生风险。血清胃泌素17在胃癌发生、发展中有促进作用，当血清胃泌素17水平升高提示存在胃癌发生风险[25]。本实验中症状统计的受试患者共10人。受试患者总体情况为：男性3人，女性7人；年龄为45-50岁2人，50-55岁1人，60-65岁4人，65-70岁3人；受试前诊断为慢性萎缩性胃炎3人、慢性萎缩性胃炎伴肠化3人、慢性萎缩性胃炎伴肠化及异型增生4人。其中，化验血液指标患者2名，受试患者1年龄为60岁，性别女，受试前诊断为慢性萎缩性胃炎伴有中度肠化及轻度异型增生；受试患者2年龄为52岁，性别女，受试前诊断为慢性萎缩性胃炎。
实验结果：如表5所示，饮用荷叶提取物前，患者1血液化验指标血清胃泌素为25.84pmol/L偏高，饮用荷叶提取物2个月后该指标恢复为正常范围。患者2血液化验指标血清胃泌素17pmol/L偏高，饮用荷叶提取物2个月后该指标恢复为正常范围(如表5所示)。如图11所示，通过统计受试患者在饮用荷叶提取物前后的症状情况，发现荷叶提取物能够缓解慢性萎缩性胃炎相关症状，如胃痛改善率为100％，嗳气吞酸改善率为87.5％，胃胀改善率为83.3％。上述实验结果表明，荷叶提取物能够有效改善慢性萎缩性胃炎伴肠化或异型增生患者的症状和客观指标。
表5.荷叶提取物对萎缩性胃炎或胃癌前病变患者血液指标改善情况
  血清胃泌素17(pmol/L)
患者1(饮用前) 25.84(偏高)
患者1(饮用后) 3.71
患者2(饮用前) 16.25(偏高)
患者2(饮用后) 1.99
以上各实施例的实验结果综合表明：荷叶碱、荷叶提取物可以有效地治疗慢性萎缩性胃炎和胃癌前病变、防止和/或阻断胃炎癌转化；而且，荷叶碱、荷叶提取物安全性好，能够保护胃黏膜损伤。由此可见，荷叶碱、荷叶提取物可以作为有效治疗慢性萎缩性胃炎、治疗胃癌前病变和/或阻断胃炎癌发生的药物、保健品和/或食品；荷叶碱、荷叶提取物可以作为有效治疗慢性萎缩性胃炎、治疗胃癌前病变和/或阻断胃炎癌发生的药物、保健品和/或食品的有效组成部分。
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Claims (7)

  1. 一种物质作为制备治疗慢性萎缩性胃炎的产品的应用，其中：
    所述产品是从下列中选出的一种：
    药品，
    保健品，以及
    食品
    所述物质是从下列中选出的一种：
    荷叶碱，
    荷叶碱的衍生物，
    荷叶提取物，
    以及
    包含从荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种的混合物。
  2. 根据权利要求1所述的应用，其中，荷叶碱属于阿朴啡型生物碱类化合物，分子式为C 19H 21NO 2，分子量为295.38g/mol；结构式为：
    Figure PCTCN2019104997-appb-100001
  3. 一种物质作为制备阻断胃炎癌转化发生的产品的应用，其中：
    所述产品是从下列中选出的一种：
    药品，
    保健品，以及
    食品，
    所述物质是从下列中选出的一种：
    荷叶碱，
    荷叶碱的衍生物，
    荷叶提取物，
    以及
    包含从荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种的混合物。
  4. 根据权利要求3所述的应用，其中，荷叶碱属于阿朴啡型生物碱类化合物，分子式为C 19H 21NO 2，分子量为295.38g/mol；结构式为：
    Figure PCTCN2019104997-appb-100002
  5. 一种产品，其特征在于其以从下列物质中选出的一种作为活性成分：
    荷叶碱，
    荷叶碱的衍生物，
    荷叶提取物，
    以及
    包含从荷叶碱、荷叶碱的衍生物、荷叶提取物组成的物质中选出的至少两种的混合物，并含有其他辅料，其中所述产品为选自药品、保健品、食品的一种。
  6. 根据权利要求5所述的产品，其中，所述产品用于治疗慢性萎缩性胃炎、保护胃黏膜、阻断胃炎癌转化、治疗胃癌前病变、阻止胃癌的发生中的至少一种。
  7. 根据权利要求5-6之一所述的产品，其中，所述药品、保健品、食品的剂型是从片剂、胶囊、丸剂、针剂、缓释剂、控释剂、粉剂、饮料等剂型中选出的一种。
PCT/CN2019/104997 2018-09-13 2019-09-09 荷叶碱和荷叶提取物作为制备治疗萎缩性胃炎和/或阻断胃炎癌转化发生药物的应用 Ceased WO2020052526A1 (zh)

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