Application of camellia pollen or camellia pollen extract and pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia

Abstract

The invention discloses an application of camellia pollen or a camellia pollen extract and a pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia, belonging to the field of biomedicine technology, an application of camellia pollen or a camellia pollen extract in preparing a drug for preventing or treating gastric mucosal intestinal metaplasia, and a pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia, wherein the active ingredient of the pharmaceutical composition is camellia pollen or a camellia pollen extract. The invention has an improving effect on gastric mucosal intestinal metaplasia and can regulate related proteins and serum indicators.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of camellia pollen or a camellia pollen extract and a pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia. Background Art

[0002] Although gastric mucosal intestinal metaplasia is associated with gastric cancer, it is not gastric cancer. Gastric mucosal intestinal metaplasia is not a manifestation of early gastric cancer, but only a risk factor for gastric cancer.

[0003] Intestinal metaplasia of gastric mucosa is the abnormal proliferation of intestinal epithelial mucosa replacing gastric mucosa in the stomach. It is the intermediate stage of the gastric cancer cascade reaction process from chronic gastritis, atrophy to dysplasia and adenocarcinoma, and is a precancerous lesion of the stomach. Metaplastic intestinal epithelium is a highly differentiated epithelium. The most obvious feature is the appearance of goblet cells. In addition, any type of epithelial cells in the intestinal mucosa can appear in the metaplastic epithelium. According to the histological manifestations of intestinal metaplastic mucosa, it can be divided into two types, namely complete intestinal metaplasia and incomplete intestinal metaplasia. It is of great significance to prevent gastric mucosal intestinal metaplasia from turning into gastric cancer.

[0004] Therefore, preventing or treating gastric mucosal intestinal metaplasia has become a research and development hotspot.

[0005] Existing reports have shown that the drugs used to prevent or treat gastric mucosal intestinal metaplasia include the Western medicine folic acid and the Chinese patent medicine Morodan. However, the Western medicine folic acid has certain side effects and the composition of Morodan is relatively complex.

[0006] Therefore, the prior art still needs to be improved and developed.

[0007] The above background technology is for facilitating the understanding of the present invention and is not a known technology disclosed to the general public before the application of the present invention. Summary of the invention

[0008] In view of the above-mentioned defects, the present invention provides a method for preparing a drug for preventing or treating intestinal metaplasia of gastric mucosa by using camellia pollen or a camellia pollen extract, which has an improving effect on intestinal metaplasia of gastric mucosa and can regulate related proteins and serum indicators.

[0009] The technical solution is: application of camellia pollen or camellia pollen extract in the preparation of medicine for preventing or treating gastric mucosal intestinal metaplasia.

[0010] Furthermore, the camellia pollen extract is a camellia pollen water extract or a camellia pollen alcohol extract, and the dosage of the camellia pollen, the camellia pollen water extract or the camellia pollen alcohol extract is 0.9 g / kg / d.

[0011] Furthermore, the camellia pollen water extract is obtained by the following steps:

[0012] The camellia pollen is fully soaked in water, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain a camellia pollen water extract, wherein the weight ratio of camellia pollen to water is 1:3-10.

[0013] Furthermore, the tea pollen alcohol extract is obtained by the following steps:

[0014] The camellia pollen is fully soaked with ethanol, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain the camellia pollen alcohol extract, the weight ratio of camellia pollen to ethanol being 1:1-10.

[0015] Furthermore, the camellia pollen or camellia pollen extract reduces the level of GAS-17 in serum and increases the levels of PG1 and PGE2; and / or

[0016] The camellia pollen or camellia pollen extract reduces serum IL-1β and IL-10 levels.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia.

[0018] The technical solution is: a pharmaceutical composition for preventing or treating gastric mucosal intestinal metaplasia, wherein the active ingredient of the pharmaceutical composition is camellia pollen or camellia pollen extract.

[0019] Furthermore, the camellia pollen extract is a camellia pollen water extract or a camellia pollen alcohol extract, and the dosage of the camellia pollen, the camellia pollen water extract or the camellia pollen alcohol extract is 0.9 g / kg / d.

[0020] Furthermore, the camellia pollen water extract is obtained by the following steps:

[0021] The camellia pollen is fully soaked in water, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain a camellia pollen water extract, wherein the weight ratio of camellia pollen to water is 1:3-10.

[0022] Furthermore, the tea pollen alcohol extract is obtained by the following steps:

[0023] The camellia pollen is fully soaked with ethanol, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain the camellia pollen alcohol extract, the weight ratio of camellia pollen to ethanol being 1:1-10.

[0024] Furthermore, the camellia pollen or camellia pollen extract reduces the level of GAS-17 in serum and increases the levels of PG1 and PGE2; and / or

[0025] The camellia pollen or camellia pollen extract reduces serum IL-1β and IL-10 levels.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] During the research and development, the inventor team found that camellia pollen crude drug, camellia pollen water extract and camellia pollen alcohol extract have an improving effect on gastric mucosal intestinal metaplasia lesions, regulating related proteins and serum indicators, and are significantly better than the current folic acid and morodan treatment methods. Even more surprisingly, they found that the efficacy of camellia pollen crude drug is significantly better than that of camellia pollen water extract and camellia pollen alcohol extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the HE image of the normal control group of the present invention (400x);

[0029] Figure 2 This is the HE image of the control group of the model of the present invention (400x);

[0030] Figure 3 This is the HE image of the folic acid group of the present invention (400x);

[0031] Figure 4 This is the HE image (400x) of the Morodan group of the present invention;

[0032] Figure 5 This is the HE image of the camellia pollen crude drug group of the present invention (400x);

[0033] Figure 6 This is the HE image of the camellia pollen ethanol extract of the present invention (400x);

[0034] Figure 7 This is the HE image of the camellia powder water extract group of the present invention (400x);

[0035] Figure 8 is the AB-PAS image of the normal control group of the present invention (200x);

[0036] Fig. 9 is the AB-PAS image of the control group of the model of the present invention (200x);

[0037] Fig.10 is the AB-PAS diagram of the folic acid group of the present invention (200x);

[0038] Fig.11 It is the AB-PAS diagram of the Morodan group of the present invention (200x);

[0039] Fig.12 This is the AB-PAS diagram of the camellia pollen crude drug group of the present invention (200x);

[0040] Fig.13 This is the AB-PAS graph of the camellia pollen water extract of the present invention (200x);

[0041] Fig.14 This is the AB-PAS diagram (200x) of the camellia pollen ethanol extract group of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be further described below by the following non-limiting embodiments in conjunction with the accompanying drawings. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

[0043] Unless otherwise specified, the following experimental methods are conventional methods, and the experimental materials used are easily available from commercial companies unless otherwise specified.

[0044] In the present invention:

[0045] MNNG, purchased from Tokyo Chemical Industry Co., Ltd., Japan, catalog number: CFFC-M0527-5G;

[0046] Ammonia water was purchased from Sichuan Kebei Biological Co., Ltd., China, catalog number: GB / T631-2007;

[0047] Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.;

[0048] Camellia pollen was purchased from Shanghai Xige Biotechnology Co., Ltd.

[0049] 66 male Sprague-Dawley (SD) rats were purchased from Chengdu Dashuo Biotechnology Co., Ltd., with a body weight of approximately 190 ± 10 g / rat; the rats were housed in the TCM Zang-Fu Disease Laboratory of Chengdu University of Traditional Chinese Medicine. The lighting followed a 12-h light / dark cycle, the room temperature was maintained at 20 ± 2 °C, and the relative humidity was maintained between 50% and 70%.

[0050] In the present invention:

[0051] HE pathological staining of gastric mucosa

[0052] The gastric mucosal tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sliced. The paraffin sections were taken, dewaxed and stained with hematoxylin for 5 minutes, washed and differentiated with hydrochloric acid ethanol for 30 seconds, and rinsed with running water for 1 hour. The sections were then placed in warm water for blueing, counterstained with eosin for 3 minutes, dehydrated with anhydrous ethanol, and fixed with xylene for transparency. The pathological changes of the gastric mucosal epithelium of mice were observed under a microscope at 100X and 200X.

[0053] AB-PAS staining of gastric mucosa

[0054] Gastric mucosal tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sliced. Paraffin sections were taken, dewaxed and stained in Alcian blue solution for 15 minutes, and rinsed with distilled water. Then oxidized in periodic acid solution for 10 minutes, and rinsed with distilled water. Then placed in Schiff reagent for light-proof staining for 30 minutes, and rinsed with running water for 5 minutes. Then counterstained with hematoxylin for 2 minutes, differentiated with hydrochloric acid ethanol for 10 seconds, and rinsed with running water for 10 minutes. Finally, dehydrated with anhydrous ethanol, fixed the specimen after transparentization with xylene, and observed the staining of mouse gastric mucosal epithelium under a microscope.

[0055] Gastric function serum index test

[0056] Rat serum was collected and the levels of GAS-17, PG1 and PGE2 were detected by ELISA.

[0057] Cytokine serum testing

[0058] The serum of rats was collected and the levels of IL-1β, IL-10 and TNF-α were detected by ELISA.

[0059] Statistical processing

[0060] All experimental data were expressed as mean ± standard deviation (X ± S). One-way analysis of variance was used to compare data among groups, and P < 0.05 indicated statistically significant differences.

[0061] Example 1

[0062] Take the same amount of camellia pollen, soak it fully with water or 80% ethanol, extract it twice by boiling reflux, combine the filtrates, concentrate them to dryness, and freeze-dry them to obtain water extract or ethanol extract respectively.

[0063] Among them: the weight ratio of camellia pollen to water is 1:8, and the weight ratio of camellia pollen to 80% ethanol is 1:8. The ethanol is brewing ethanol (i.e. edible ethanol).

[0064] The extracted camellia pollen water extract, camellia pollen ethanol extract and camellia pollen (hereinafter referred to as camellia pollen crude drug) were used in the following Example 2.

[0065] Example 2

[0066] After 1 week of adaptive feeding of rats, 42 rats were randomly divided into 7 groups using a digital random table: normal control group, model control group, model + folic acid group, model + morodan group, model + camellia pollen crude drug group, model + camellia pollen water extract group, model + camellia pollen ethanol extract group, with 6 rats in each subgroup. The rats in the normal group were raised under conventional conditions, and the rats in the model group were given 0.1% ammonia solution to drink freely. In addition, the model rats were given 0.1% ammonia solution to drink freely + 200 μg·mL-1 MNNG solution by gavage, and each group was given the corresponding treatment for a total of 12 weeks, as follows:

[0067] ⑴Blank control group: intragastrically administered with normal saline, 0.1ml / 10g, once a day for 12 consecutive weeks.

[0068] ⑵ Model control group: 0.1% ammonia solution was freely available for drinking + 200 μg·mL-1 MNNG solution was administered orally, 0.1ml / 10g, once a day, for 12 consecutive weeks.

[0069] (3) Model + folic acid group: On the basis of modeling, folic acid treatment was given, 1.8 mg / kg / d, for 12 consecutive weeks.

[0070] (4) Model + Morodan group: Based on the modeling, Morodan was given for treatment at 1.6 mg / kg / d for 12 consecutive weeks.

[0071] ⑸ Model + camellia pollen crude drug group: On the basis of modeling, camellia pollen crude drug treatment was given, 0.9g / kg / d each time, added to normal saline and gavage, for 12 consecutive weeks.

[0072] ⑹ Model + camellia pollen water extract group: Based on the modeling, the camellia pollen water extract group was treated with 0.9g / kg / d for 12 consecutive weeks.

[0073] ⑺Model + camellia pollen ethanol extract group: Based on the modeling, camellia pollen ethanol extract was administered by intragastric administration at 0.9 g / kg / d each time for 12 consecutive weeks.

[0074] In the blank control group, the rats were in good condition, moved quickly, had a good appetite, and had hard and granular feces. In contrast, the rats in the model control group showed a state of lack of vitality, slow movement, loss of appetite, and diarrhea. In the late stage of the experiment, the body weight of the rats in the model control group was significantly reduced compared with the normal group (P<0.01). The above macroscopic manifestations of rats in the folic acid group and the morodan group were partially improved; the above macroscopic phenotypes of rats in the camellia pollen water extract group and the camellia pollen ethanol extract group were also partially improved. The macroscopic manifestations of the model + camellia pollen crude drug treatment group improved most significantly, and the body weight of the rats was significantly higher than that of the model IM rats (P<0.01). The above results show that camellia pollen treatment can restore the body weight of IM rats, and the efficacy of camellia pollen crude drug is significantly better than that of water extraction and alcohol extraction, and the efficacy is significantly better than that of the folic acid group and the morodan group.

[0075] In the twelfth week, the animals were weighed as shown in Table 1.

[0076] Table 1 Weight of each group at week 12

[0077]

[0078] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the folic acid group, ★P<0.05, ★★P<0.01; compared with the morodan group, ☆P<0.05, ☆☆P<0.01; compared with the camellia pollen water extract group, ▲P<0.05, ▲▲P<0.01; compared with the camellia pollen ethanol extract group, △P<0.05, △△P<0.01; unmarked indicates no statistical significance.

[0079] Gastric mucosal HE pathological staining, gastric mucosal AB-PAS staining, gastric function serum index detection, cytokine serum detection and statistical analysis were performed, and the results are as follows:

[0080] ⑴ HE staining was used to observe the pathological changes of gastric mucosa in rats of each group. Figure 1-Figure 7 ,in, Figure 1 HE image of the normal control group (400x). Figure 2 HE image of the model control group (400x). Figure 3 HE image of folic acid group (400x). Figure 4 HE image of the Morodan group (400x). Figure 5 HE image of camellia pollen herbal medicine group (400x). Figure 6 HE image of camellia pollen water extract group (400x), Figure 7This is the HE image of the camellia pollen ethanol extract group (400x). HE staining shows that compared with the normal group, the model group showed intestinal metaplasia changes in the gastric mucosa, inflammatory cell infiltration in the interstitial cells, and fibrosis or fibromuscular hyperplasia in the gastric mucosal lamina propria. After intervention with folic acid and morodan, these abnormal tissue pathological changes improved to a certain extent. Camellia pollen crude drug, camellia pollen water extract, and camellia pollen ethanol extract can improve abnormal lesions of gastric mucosa, among which camellia pollen crude drug is significantly better than water extraction and alcohol extraction. These results show that camellia pollen can effectively improve IM pathological changes in rat gastric mucosa, and the efficacy of camellia pollen crude drug is significantly better than water extraction and alcohol extraction, and the efficacy is significantly better than the folic acid group and morodan group.

[0081] ⑵ AB-PAS staining was used to observe the pathological changes of gastric mucosa in rats of each group, such as Figure 8-Figure 14 ,in, Figure 8 This is the AB-PAS image of the normal control group (200x). Fig. 9 This is the AB-PAS diagram of the model control group (200x). Fig.10 AB-PAS diagram of folic acid group (200x). Fig.11 This is the AB-PAS diagram of the Morodan Formation (200x). Fig.12 AB-PAS diagram of camellia pollen herbal medicine group (200x). Fig.13 AB-PAS image of camellia pollen water extract (200x). Fig.14 This is the AB-PAS image of the camellia pollen ethanol extract group (200x). AB-PAS staining shows that the gastric mucosa of IM rats showed blue or purple intestinal metaplastic lesions. After treatment with camellia pollen crude drug, camellia pollen water extract, and camellia pollen ethanol extract, the blue or purple lesions were significantly reduced, and the therapeutic effect was better than that of the folic acid group and the morodan group. We also found that the use of camellia pollen crude drug was significantly better than that of camellia pollen water extract and camellia pollen ethanol extract. The camellia pollen crude drug group showed a normal staining pattern closest to the blank group.

[0082] ⑶ Gastric function serum index detection is shown in Table 2 below.

[0083] Table 2 Serum indexes of gastric function

[0084]

[0085] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the folic acid group, ★P<0.05, ★★P<0.01; compared with the morodan group, ☆P<0.05, ☆☆P<0.01; compared with the camellia pollen water extract group, ▲P<0.05, ▲▲P<0.01; compared with the camellia pollen alcohol extract group, △P<0.05, △△P<0.01; unmarked indicates no statistical significance.

[0086] The results in Table 2 show that the levels of GAS-17, PG1, and PGE2 in the serum of the model group rats changed significantly compared with the blank group, showing a significant increase in GAS-17, a decrease in PG1, and a decrease in PGE2, indicating intestinal metaplasia of the gastric mucosa. Compared with the model group, the treatment with camellia pollen crude drug, camellia pollen water extract, and camellia pollen alcohol extract reduced the level of GAS-17 in serum, increased the levels of PG1 and PGE2, and was significantly better than the folic acid group and the morodan group. Among them, the effect of camellia pollen crude drug was better than that of camellia pollen water extract and camellia pollen alcohol extract, that is, the camellia pollen crude drug group had the best effect.

[0087] (4) Cytokine serum detection is shown in Table 3.

[0088] Table 3 Cytokine serum detection

[0089]

[0090] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the folic acid group, ★P<0.05, ★★P<0.01; compared with the morodan group, ☆P<0.05, ☆☆P<0.01; compared with the camellia pollen water extract group, ▲P<0.05, ▲▲P<0.01; compared with the camellia pollen alcohol extract group, △P<0.05, △△P<0.01; unmarked indicates no statistical significance.

[0091] The results in Table 3 show that the levels of IL-1β, IL-10, and TNF-α in the serum of the model group rats changed significantly compared with the blank group, showing that IL-1β, IL-10, and TNF-α were significantly increased, indicating the inflammatory response of the gastric mucosa and accelerated intestinal metaplasia. Compared with the model group, the treatment with camellia pollen crude drug, camellia pollen water extract, and camellia pollen alcohol extract reduced the levels of serum IL-1β and IL-10, and the efficacy was better than that of the folic acid group and the morodan group; but it had no regulatory effect on the level of serum TNF-α. Among them, the effect of camellia pollen crude drug was significantly better than that of camellia pollen water extract and camellia pollen alcohol extract, that is, the camellia pollen crude drug group had the best effect.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of camellia pollen or camellia pollen extract in the preparation of a drug for preventing or treating gastric precancerous lesions, characterized in that: The camellia pollen or camellia pollen extract is used to improve intestinal metaplasia of gastric mucosa. The camellia pollen extract is a camellia pollen water extract or a camellia pollen alcohol extract; The camellia pollen water extract is obtained by the following steps: The camellia pollen is fully soaked in water, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain a camellia pollen water extract, with a camellia pollen: water weight ratio of 1:3-10; The tea pollen alcohol extract is obtained by the following steps: The camellia pollen is fully soaked with ethanol, extracted twice by boiling reflux, the filtrates are combined, concentrated to dryness, and freeze-dried to obtain the camellia pollen alcohol extract, the weight ratio of camellia pollen to ethanol being 1:1-10.

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