Anti-Helicobacter pylori agent, composition for inhibiting Helicobacter pylori, and methods for producing the same

Abstract

To provide an anti-Helicobacter pylori agent and a composition for suppressing Helicobacter pylori has distillate, which is at least one selected from pine distillate and Japanese Yew (Taxus cuspidata) which has conventionally been discarded as a byproduct, as an active ingredient, and is ingestible daily due to less adverse effect because of plant-derived distillate, and a method for producing them.SOLUTION: An anti-helicobacter pylori agent has distillate, which is at least one selected from the group consisting of pine distillate and Japanese Yew distillate, as an active ingredient.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an anti-Helicobacter pylori agent, a composition for inhibiting Helicobacter pylori, and methods for producing them. [Background technology]

[0002] Helicobacter pylori (H. pylori) is a gram-negative bacillus that survives in the human stomach and has a neutral pH optimum of 7.0. It uses flagella, protected by a myelin sheath, to penetrate the gastric submucosa and secrete virulence factors, such as proteases and effector molecules CagA (cytotoxin associated gene A) and VacA (vacuolating toxin A), which damage the gastric mucosa and cause acute gastritis. H. pylori infection can progress to chronic gastritis and peptic ulcers (gastric ulcers and duodenal ulcers), and is also known to increase the risk of gastric cancer.

[0003] The typical treatment for eradicating Helicobacter pylori involves taking a combination of drugs for about a week, but this is known to have side effects such as dehydration and diarrhea.

[0004] Therefore, from the perspective of reducing side effects, several proposals have been made regarding the control of H. pylori using food ingredients.

[0005] Regarding the antibacterial effects of foodstuffs on Helicobacter pylori, Non-Patent Document 1 discloses a report on broccoli sprouts, Non-Patent Document 2 discloses a report on coffee, and Non-Patent Document 3 discloses a report on plum extract. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Akinori Y., Fahey JW., Atsushi F., Mari N., Souta I., Songhua Z., Masafumi T., Hideo S., Ichinosuke H., Masayuki Y. (2009).Dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in Helicobacter pylori-infected mice and humans.Cancer Prevention Research,2,353-360. [Non-patent document 2] Yuki O., Yukata Y., Kayo Y., Kazunori H., Katsumi K., Naoaki I. (2003). The antibacterial effects of coffee on Escherichia coli and Helicobacter pylori. J. Clin. Biochem. Nutr., 34, 85-87. [Non-patent document 3] Kimie F., Miyuki H., Mari F., Intetsu K., Kotaro O., Yoshiaki W. (2002). Anti-Helicobacter pylori effects of bainiku-ekisu (concentrate of Japanese apricot juice). Nihon Shokakibyo Gakkai Zasshi., 99, 379-385. Summary of the Invention [Problem to be solved by the invention]

[0007] Helicobacter pylori increases the risk of chronic gastritis and gastric ulcers, which are precursors to stomach cancer, and the infection rate of Helicobacter pylori is known to be particularly high in Japan. Therefore, there is still a need for substances with anti-H. pylori activity, and in particular, there has been a long-awaited development of new anti-H. pylori agents of natural origin that can be taken daily.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide an anti-Helicobacter pylori agent and a composition for inhibiting Helicobacter pylori, which contain as an active ingredient at least one distillate selected from the group consisting of pine distillate and yew distillate, which have traditionally been discarded as by-products, and which, because they are plant-derived, have few side effects and can be taken on a daily basis, as well as methods for producing them. [Means for solving the problem]

[0009] In order to achieve the above object, an anti-Helicobacter pylori agent according to a first aspect of the present invention comprises: The active ingredient is at least one distillate selected from the group consisting of pine distillate and yew distillate.

[0010] For example, the pine is at least one selected from the group consisting of Picea glehnii, Abies sachalinensis, Larch and Pumila dwarf.

[0011] For example, the pine is Picea glehnii.

[0012] A composition for inhibiting Helicobacter pylori according to a second aspect of the present invention comprises: The active ingredient is at least one distillate selected from the group consisting of pine distillate and yew distillate.

[0013] For example, the pine is at least one selected from the group consisting of Picea glehnii, Abies sachalinensis, Larch and Pumila dwarf.

[0014] For example, the pine is Picea glehnii.

[0015] A method for producing an anti-Helicobacter pylori agent according to a third aspect of the present invention comprises: The method includes a step of obtaining a distillate by distilling at least one plant selected from the group consisting of pine and yew.

[0016] A method for producing a composition for inhibiting Helicobacter pylori according to a fourth aspect of the present invention comprises: The method includes a step of obtaining a distillate by distilling at least one plant selected from the group consisting of pine and yew. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide an anti-Helicobacter pylori agent and a composition for inhibiting Helicobacter pylori, which contain as an active ingredient at least one distillate selected from the group consisting of pine distillate and yew distillate, which have traditionally been discarded as by-products, and which, because they are plant-derived, have few side effects and can be taken on a daily basis, as well as methods for producing the same. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of the method for restoring and culturing H. pylori in this example. [Figure 2] FIG. 1 is a schematic diagram of the microplate antibacterial test method in this example. [Figure 3] FIG. 1 is a graph showing the anti-Helicobacter pylori activity of the distilled solution of red spruce in this example. [Figure 4] FIG. 1 is a graph showing the anti-Helicobacter pylori activity of pine distillate and yew distillate in this example. [Figure 5] FIG. 1 is a graph showing the anti-Helicobacter pylori activity of each molecular weight fraction of the distilled solution of Picea glehnii in this example. DETAILED DESCRIPTION OF THE INVENTION

[0019] (1. Anti-Helicobacter pylori agent) The anti-Helicobacter pylori agent according to the present invention contains, as an active ingredient, at least one distillate selected from the group consisting of pine distillate and yew distillate.

[0020] The pine used in the present invention can be any tree belonging to the Pinaceae family, without any particular limitation. For example, trees of the Picea genus of the Pinaceae family include Picea glehnii and Picea abies. Trees of the Abies genus of the Pinaceae family include Abies sachalinensis, Fir, Abies salicina, Abies mariesii, Abies veitchii, Abies veitchii, Balsam fir, Abies mittimarinus, White fir, Amabilis fir, Abies sachalinensis, California red fir, Grand fir, Noble fir, and the like. Trees of the Pinaceae genus Pinus include Pinus densiflora, Pinus grandiflorus, Pinus strobus, Pumila dwarf pine, and the like. Trees of the Larix genus of the Pinaceae family include Larix larch, and the like. Trees of the Tsuga genus of the Pinaceae family include Tsuga sega. Two or more different types of pines may be used in combination.

[0021] The pine used in the present invention is preferably at least one species selected from the group consisting of Picea glehnii, Abies sachalinensis, Larch, and Pumicea pumila, and more preferably Picea glehnii is used.

[0022] The yew (scientific name: Taxus cuspidata) used in the present invention can be any tree belonging to the genus Taxus in the family Taxaceae, without any particular limitation. In the present invention, yew also includes ver. Cuspidata (f. luteobaccata) and ver. Nana (Aurescens).

[0023] Regarding the parts of pine and yew to be used, any parts of pine and yew can be used, such as branches, leaves, trunks, roots, flowers, and fruits, but preferably, branches and leaves of pine and yew are used.

[0024] Pine distillate is a distillate obtained by distilling pine, but is not pine essential oil (pine oil). Pine essential oil and pine distillate are obtained by distilling pine. Pine essential oil is typically used in cosmetics, fragrances, and the like, while pine distillate is treated as a by-product and not utilized. However, in the present invention, pine distillate, which would previously have been discarded, is used. Examples of distillation methods include steam distillation, atmospheric distillation, low-pressure distillation, reduced-pressure distillation, molecular distillation, simple distillation, rectification, continuous distillation, and batch distillation, with steam distillation being preferred. More specifically, steam distillation can be performed by placing pine in a still, adding water, and heating the still to generate vapors from pine components, which are then condensed by passing through a cooling tube or the like to obtain pine essential oil (upper layer) and pine distillate (lower layer).

[0025] The yew distillate is a distillate obtained by distilling yew as described above. The details of the distillation method are the same as those described above.

[0026] The method for producing pine distillate (steam distillation) will be explained using Picea glehnii as an example. Picea glehnii branches and leaves are cut with scissors and crushed, then a predetermined amount of water is added and the mixture is placed in a known steam distillation apparatus and heated for several hours to generate steam from the Picea glehnii components. The steam is then condensed by passing it through a cooling tube or the like, yielding pine essential oil (upper layer) and pine distillate (lower layer). The pine distillate can be separated using a known method to obtain Picea glehnii distillate.

[0027] The anti-Helicobacter pylori agent according to the present invention contains, as an active ingredient, at least one distillate selected from the group consisting of pine distillate and yew distillate. The anti-Helicobacter pylori agent according to the present invention may, for example, contain only pine distillate, a mixture of pine distillate and yew distillate, or only yew distillate.

[0028] The anti-Helicobacter pylori agent according to the present invention has anti-Helicobacter pylori activity and is therefore effective in inhibiting, killing, or eliminating Helicobacter pylori, and is useful for preventing or treating Helicobacter pylori-associated diseases (e.g., gastric ulcer, chronic gastritis, acute gastritis, duodenal ulcer, gastric cancer, gastric MALT lymphoma, etc.). In this specification, "preventing" a disease includes inhibiting or delaying the onset of the disease and inhibiting its recurrence, while "treating" a disease includes not only completely curing the disease but also alleviating symptoms and inhibiting its progression. The anti-Helicobacter pylori agent according to the present invention contains, as an active ingredient, at least one distillate selected from the group consisting of pine distillate and yew distillate, both of which are derived from plants. This agent has few side effects, can be taken daily, and can effectively inhibit, kill, or eliminate Helicobacter pylori.

[0029] The anti-Helicobacter pylori agent according to the present invention is administered to a subject requiring inhibition, sterilization, or elimination of Helicobacter pylori, for example, a mammal such as a primate including a human, a chimpanzee, and a rhesus monkey, a companion animal including a dog and a cat, a livestock animal including a pig, a cow, a goat, a horse, and a sheep, or a rodent including a mouse, a rat, a hamster, and a guinea pig. A preferred subject is a human.

[0030] The method of administration of the anti-Helicobacter pylori agent of the present invention can be appropriately selected from oral administration, topical administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration, etc. The dosage form may also be any, and an appropriate drug delivery system (DDS) may be used. These dosage forms are produced by formulating the active ingredient using conventional methods. Furthermore, various pharmaceutically acceptable pharmaceutical substances can be blended as needed for the formulation. Pharmaceutical substances can be appropriately selected depending on the dosage form of the formulation, and examples include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, flavoring agents, sweeteners, solubilizers, etc.

[0031] The anti-Helicobacter pylori agent according to the present invention is preferably used as an oral preparation (a solid preparation such as a tablet, capsule, granule, fine granule, powder, chewable tablet, or troche, or a liquid preparation such as a liquid or syrup). By taking the anti-Helicobacter pylori agent according to the present invention as an oral preparation on a daily basis, it is possible to effectively inhibit, kill, or eliminate Helicobacter pylori.

[0032] The dosage and frequency of administration of the anti-Helicobacter pylori agent according to the present invention can be appropriately determined by a person skilled in the art depending on the state of infection with Helicobacter pylori, the state of Helicobacter pylori-related disease, the patient's health condition, age, body weight, administration route, administration form, etc., so that an effective amount is administered to the patient.

[0033] (2. Composition for inhibiting Helicobacter pylori) The composition for inhibiting Helicobacter pylori according to the present invention contains, as an active ingredient, at least one distillate selected from the group consisting of pine distillate and yew distillate. Details of the pine, yew, distillate, etc. are the same as those described above.

[0034] The pine used in the present invention can be any tree belonging to the Pinaceae family, and examples thereof include the pine species mentioned above. Preferably, at least one species is selected from the group consisting of Picea glehnii, Abies sachalinensis, Larch, and Pumila spp., and more preferably Picea glehnii is used. The yew used in the present invention is the same as above. The parts of each plant used are the same as above.

[0035] The composition for inhibiting Helicobacter pylori according to the present invention has anti-Helicobacter pylori activity and is therefore effective in inhibiting, killing, and eliminating Helicobacter pylori, and is useful for preventing or treating Helicobacter pylori-associated diseases (e.g., gastric ulcer, chronic gastritis, acute gastritis, duodenal ulcer, gastric cancer, gastric MALT lymphoma, etc.). The target subjects, dosage, and frequency of administration are the same as those described above.

[0036] The composition for inhibiting Helicobacter pylori according to the present invention may be administered by any method, as described above, but oral ingestion via food or beverage is preferred. Specific examples of food or beverage include health foods or supplementary foods, including beverages, commonly known as soft drinks, energy drinks, health foods, foods for specified health uses, functional foods, functionally active foods, nutritional supplements, supplements, feed, and feed additives. Daily oral ingestion of the composition for inhibiting Helicobacter pylori according to the present invention can effectively inhibit, kill, and eliminate Helicobacter pylori.

[0037] (3. Method for producing anti-Helicobacter pylori agent or composition for inhibiting Helicobacter pylori) The method for producing an anti-Helicobacter pylori agent or a composition for inhibiting Helicobacter pylori of the present invention includes a step of obtaining a distillate by distilling at least one plant selected from the group consisting of pine and yew. Details of the anti-Helicobacter pylori agent or the composition for inhibiting Helicobacter pylori are the same as those described above.

[0038] The pine used in the present invention is not particularly limited as long as it is a tree belonging to the family Pinaceae, and examples thereof include the above-mentioned pine species. Preferably, at least one species is selected from the group consisting of Picea glehnii, Abies sachalinensis, Larch, and Pumila spp., and more preferably, Picea glehnii is used.

[0039] The yew (scientific name: Taxus cuspidata) used in the present invention can be any tree belonging to the genus Taxus in the family Taxaceae, without any particular limitation. In the present invention, yew also includes ver. Cuspidata (f. luteobaccata) and ver. Nana (Aurescens).

[0040] Regarding the parts of pine and yew to be used, any parts of pine and yew can be used, such as branches, leaves, trunks, roots, flowers, and fruits, but preferably, branches and leaves of pine and yew are used.

[0041] In the present invention, the distillation method may be, for example, steam distillation, atmospheric distillation, low-pressure distillation, vacuum distillation, molecular distillation, simple distillation, rectification, continuous distillation, batch distillation, etc., with steam distillation being preferred. For example, when pine is used, pine essential oil and pine distillate are obtained by distilling the pine. However, in the present invention, essential oil is not used, and the water-soluble pine distillate is used as the anti-H. pylori agent or the composition for inhibiting H. pylori (similarly, yew essential oil is not used, but yew distillate is used). More specifically, the steam distillation method may be, for example, a method in which pine is placed in a still, water is added, and the mixture is heated to generate steam by vaporizing the pine components, and the steam is condensed by passing it through a cooling tube or the like to obtain pine essential oil (upper layer) and pine distillate (lower layer).

[0042] The production method of the present invention will be explained using a method (steam distillation) using Picea glehnii as an example. Picea glehnii branches and leaves are cut with scissors and crushed, then a predetermined amount of water is added, and the mixture is placed in a known steam distillation apparatus and heated for several hours to generate steam from the Picea glehnii components. The steam is then condensed by passing it through a cooling tube or the like, yielding an essential oil (upper layer) and a distillate (lower layer). The distillate can be separated using a known method to obtain a Picea glehnii distillate, which can be used as an anti-Helicobacter pylori agent or a composition for inhibiting Helicobacter pylori.

[0043] The present invention also includes an anti-Helicobacter pylori agent or a composition for inhibiting Helicobacter pylori obtained by the above-mentioned production method.

[0044] (4. Summary) As described above, the anti-Helicobacter pylori agent and composition for inhibiting Helicobacter pylori according to the present invention can be produced at low cost because they contain, as an active ingredient, at least one distillate selected from the group consisting of pine distillate and yew distillate, which have traditionally been discarded as by-products. Because these distillates are derived from plants, unlike chemicals, they have reduced side effects, can be taken daily, and can effectively inhibit, kill, or eliminate Helicobacter pylori. Furthermore, in light of the growing interest in natural materials in recent years, these distillates can be suitably used by users who need to inhibit, kill, or eliminate Helicobacter pylori, particularly as oral preparations or foods and beverages that can be taken daily. [Example]

[0045] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0046] The anti-Helicobacter pylori activity of pine distillate and yew distillate was investigated.

[0047] (sample) (Preparation of distillate) Branches and leaves collected from Picea glehnii, Pumicea pumila, Abies sachalinensis, Larch, and Yew trees planted in Ebetsu City, Hokkaido, were crushed in a mill (IFM-C20G, Iwatani). 400 mL of water was added to 600 g of crushed branches and leaves, and the mixture was distilled for 2 hours using a steam distillation apparatus (Pure Stiller, Huanghe). The distillate obtained was used as the sample.

[0048] (Molecular weight fraction of red spruce distillate) The distillate of Picea glehnii was ultrafiltered (Amicon Ultra, Merck) to separate three molecular weight fractions: ≥10 kDa, 3–10 kDa, and ≤3 kDa. The antibacterial activity of the aqueous layer after hexane extraction of each fraction was also evaluated.

[0049] (H. pylori strains) Helicobacter pylori strain 12093 (RIKEN Bank) was used, frozen at -80°C. Operations were performed in a safety cabinet (SDV-ECIIB, Hitachi) to prevent contamination, and sterile filter tips were used for dispensing the H. pylori solution.

[0050] (Medium preparation) (Preparation of Nutrient Broth-Horse Serum (NBhs) medium) 10.0 g of Nutrient Broth (CM0067, Oxoid) was added to 400 mL of the distillate and dispersed uniformly. The mixture was then sterilized and dissolved in an autoclave (BS-245, Tomy) at 120°C for 20 minutes. After rapid cooling under running water, 20 mL of horse serum (H1138, Sigma) was added under sterile conditions and stirred to prepare H. pylori growth medium (NBhs medium). 30 mL of NBhs medium was dispensed into 50 mL sterile tubes (91050, BMBio) and stored at 4°C.

[0051] (Preparation of SCDLP broth medium) 7.6 g of SCDLP bouillon medium (E-MC72, Eiken) was dissolved in 200 mL of water, and then sterilized in an autoclave at 120°C for 20 minutes.

[0052] (Restoration culture of H. pylori) 0.5 mL of thawed H. pylori stock solution was added to 25 mL of NBhs medium and cultured at 37°C for 72 hours under microaerobic conditions using Anaeropack Microaerobic (A-28) and a standard square jar (Mitsubishi Chemical) (Figure 1).

[0053] (bacteria quantity measurement) The bacterial count was measured by the McFarland turbidimetric method (McFarland J. (1907). An instrument for estimating the number of bacteria. JAMA, 49, 1176-1178). McFarland turbidity standard solutions were prepared by mixing 1% (w / w) barium chloride solution and 1% (w / w) sulfuric acid solution at different ratios. The bacterial count of the bacterial solution restored and cultured in NBhs medium was determined using the following procedure. After dispensing 200 μL of each of the turbidity standard solution and the restored culture into a 96-well flat-bottom plate (655101, Greiner), the turbidity at 650 nm was measured using a microplate reader (Emax, Molecular Devices), and the bacterial count was calculated from the calibration curve. For H. pylori, the bacterial count was measured at the specified number (2.0 × 10 8 If the cell count was less than 1000 cfu / mL, the cells were cultured for an additional 24 to 48 hours.

[0054] (Antibacterial test) The antibacterial test was performed by microplate turbidimetry (Fig. 2).

[0055] (positive control) A 10 mg / mL benzalkonium chloride stock solution was prepared by diluting 10% (w / w) Osvan (Nippon Suido) 10 times with water. At the time of use, this stock solution was further diluted 2,000 times with NBhs medium to prepare a 5 μg / mL benzalkonium chloride solution.

[0056] (Measurement procedure for anti-Helicobacter pylori activity) After vortex-mixing NBhs medium and each sample in a 2 mL tube, a series of 2.5- to 40-fold dilutions were prepared using NBhs medium. Using a multichannel micropipette, 50 μL of each 2.5- to 40-fold diluted sample was dispensed into columns 4 to 12 of a sterile 96-well flat-bottom plate (22216019, Costar) using NBhs medium as a control in column 2, 5 μg / mL benzalkonium chloride as a positive control in column 3, and NBhs medium in columns A to F. Then, 2.0 × 10 bacterial counts were prepared using NBhs medium. 850 μL of H. pylori solution adjusted to cfu / mL was dispensed. Instead of bacterial solution, 50 μL of NBhs medium was dispensed into rows G and H to serve as a blank to correct for sample coloration. After 72 hours of incubation at 37°C under microaerobic conditions using Anaeropack microaerobic and Anaeropack standard square jars, the plates were agitated ultrasonically (Ultrasonic Steri-Cleaner, Leo) for 5 minutes and then with a plate mixer (MS3 digital, Ika) for 1 minute, and the turbidity at 650 nm was measured using a microplate reader.

[0057] (Statistical processing) Using the control as the standard, a significance test was performed at the 1% or 5% level using the Dunnett test (R, The R project for statistical computing).

[0058] (result) The results of the antibacterial test of the distilled liquid of red glehnese spruce against H. pylori are shown in Figure 3. In Figure 3, "Collected 210806" represents the distilled liquid obtained from red glehnese spruce collected on August 6, 2021, "Collected 210811" represents the distilled liquid obtained from red glehnese spruce collected on August 11, 2021, and "Collected 210814" represents the distilled liquid obtained from red glehnese spruce collected on August 14, 2021. All three distilled liquid samples obtained from the branches and leaves of red glehnese spruce collected on different days exhibited concentration-dependent anti-H. pylori activity (Figure 3).

[0059] The results of the antibacterial test against H. pylori for the distillates of four other pine species (pine pumila, todomatsu, and larch) and yew are shown in Figure 4. As with the distillate of red spruce, all distillates showed a tendency to show concentration-dependent anti-H. pylori activity (Figure 4).

[0060] The results of the antibacterial test against H. pylori of molecular weight fractions of the distilled liquid of red spruce are shown in Figure 5. A comparison of molecular weight fractions obtained by ultrafiltration of the distilled liquid of red spruce showed a nearly constant anti-H. pylori activity regardless of molecular weight size (Figure 5). In order to investigate the involvement of lipophilic aroma components, the aqueous layer after hexane extraction was also measured, and the antibacterial activity of all fractions tended to be weaker than that of the original distilled liquid (Figure 5).

[0061] From the above, it was confirmed that the pine distillate and yew distillate of this example have strong anti-H. pylori activity. The pine distillate and yew distillate of this example are effective in inhibiting, killing, and eliminating H. pylori, and are expected to be applied to safe and reliable medicines, foods, and beverages of natural origin.

Claims

1. An anti-Helicobacter pylori agent containing as its active ingredient the distillate of the branches and leaves of Picea glehnii.

2. A composition for inhibiting Helicobacter pylori, the active ingredient of which is the distillate of the branches and leaves of Picea glehnii.

3. A method for producing an anti-Helicobacter pylori agent, comprising the step of obtaining a distillate by distilling the branches and leaves of red spruce, and using the obtained distillate as an active ingredient.

4. A method for producing a composition for inhibiting Helicobacter pylori, comprising the step of obtaining a distillate by distilling the branches and leaves of red spruce, and using the obtained distillate as an active ingredient.

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