Buckwheat composite extract with broad-spectrum antibacterial activity, preparation method and application

Abstract

The present invention provides a buckwheat composite extract with broad-spectrum antibacterial properties, which comprises, by weight, 20 to 30 parts of buckwheat, 5 to 8 parts of Zanthoxylum bungeanum, 10 to 12 parts of hawthorn leaves, and 5 to 8 parts of sea buckthorn leaves; the extract is a water extract. The present invention also provides a method for preparing the buckwheat composite extract and its application. The buckwheat composite obtained by the present invention has the advantages of a broad antibacterial spectrum and excellent antibacterial properties, has a highly effective antibacterial effect on more than 30 types of bacteria, and has the potential for application as a highly effective antibacterial agent. The present invention solves the shortcomings of buckwheat extract in antibacterial aspects and expands the application value of buckwheat.

Description

Technical Field

[0001] The present invention belongs to the field of antibacterial technology, and in particular relates to a buckwheat composition extract with broad-spectrum antibacterial activity, a preparation method and an application thereof. Background Art

[0002] Buckwheat is a dicotyledonous plant with strong adaptability, can grow in a variety of environments, has a short growth cycle, and can be cultivated in spring, summer, and autumn. Its morphology differs from wheat, with upright stems with longitudinal ridges, triangular or ovate leaves, corymbose flowers, triangular seeds, and a black, hard-shelled bran. Buckwheat is a minor staple food crop used to make noodles, jelly, and other foods.

[0003] Buckwheat cultivation is extremely widespread in my country, and is planted in all directions. The annual sown area in my country is about 1 million square hectares, and the output is about 700,000 tons. Not only is the annual output high, but the export volume also ranks first in the world. As the attention paid to buckwheat continues to increase, people have studied its application value in addition to its edible value. Cao Liangshun et al. reviewed the effective components and pharmacological effects of buckwheat flowers and leaves, and concluded that buckwheat flowers and leaves have application value in the prevention and treatment of cardiovascular diseases, the treatment of diabetes and its complications, and anti-oxidation and anti-fatigue. [1] Zhang Guotao et al. studied the antioxidant properties of buckwheat extracts and obtained flavonoids from them through ethanol extraction. They found that flavonoids in buckwheat extracts are important components that exert antioxidant properties. [2] Guo Lan et al. studied the effects of buckwheat flower and leaf extract on the immune function of tumor-bearing mice and found that it can alleviate the immunosuppression of mice caused by CTX. [3] Wang Jianxing et al. found that fermented extracts of buckwheat flowers and leaves can improve renal damage in spontaneously obese type 2 diabetic db / db mice [4] Li Xiu et al. investigated the safety toxicology of buckwheat bran extract and found that buckwheat bran extract had no obvious teratogenic effect on sperm or chromosomes. [5] .

[0004] In addition to the above-mentioned uses in disease treatment and health care, buckwheat's antibacterial value has also attracted attention. Zhou Yuyan and others studied the in vitro antibacterial activity of golden buckwheat extract and found that it has a good antibacterial effect on porcine Actinobacillus pleuropneumoniae, Streptococcus suis, Salmonella and Staphylococcus aureus. [6] Qiao Hongjie et al. also found that the root extract of golden buckwheat has good in vitro antibacterial activity [7] .

[0005] Although existing studies have shown that buckwheat extract has certain antibacterial effects, its antibacterial spectrum is relatively narrow and has no antibacterial effect on bacteria such as Aspergillus niger, Fusarium spp., Citrus green mold and Penicillium glaucum. [8-9]At the same time, the antibacterial ability of the currently obtained buckwheat extract is not strong. The MIC for most known effective bacteria (such as Escherichia coli and Staphylococcus aureus) is generally not less than 1 mg / ml, which greatly limits its application value as an antibacterial material. In addition, the antibacterial effect of buckwheat extract is also limited by the extraction site and extraction method. Generally speaking, the antibacterial activity of water products is weaker than that of alcohol-extracted products. [6][8] At the same time, the antibacterial effects of extracts from different buckwheat parts also vary greatly. For example, the water extract of buckwheat hull has no antibacterial effect on Staphylococcus aureus.

[10] Therefore, buckwheat extract is still difficult to use as a good antibacterial agent, which makes it necessary to further develop the antibacterial value of buckwheat and promote the use of buckwheat to prepare related high-efficiency antibacterial products.

[0006] To further develop the antibacterial value of buckwheat, researchers have focused on refining antibacterial active ingredients and preparing antibacterial compositions. Dong Xiaoning et al. extracted rutin from tartary buckwheat and found that its MIC against Escherichia coli and Staphylococcus aureus could be reduced to 0.4 mg / ml and 0.2 mg / ml, respectively. The antibacterial effect was superior to that of conventional buckwheat alcohol extracts or water extracts.

[11] Jiang Zhongli et al. found that the alcohol extract of buckwheat, clove and dandelion had better antibacterial effect by cross-breeding.

[12] However, existing research results are still unsatisfactory in expanding the antibacterial spectrum and improving the antibacterial effect.

[0007] In summary, how to develop products with better antibacterial effects based on buckwheat is a technical problem that needs to be solved urgently in this field.

[0008] References for this section:

[0009] [1] Cao Liangshun, Zhang Ying, Zhang Saihang, et al. Current status of research on the active ingredients and pharmacological effects of buckwheat flowers and leaves [J]. Asia-Pacific Traditional Medicine, 2015, 011(004): 64-66.

[0010] [2] Zhang Guotao, Zhang Xiong, Wang Hua, et al. Study on the antioxidant activity of buckwheat extract[J]. Anhui Agricultural Sciences, 2017, 25(25): 97-98+103.

[0011] [3] Guo Lan, Wu Aiping, Zhao Zhiyu, et al. Effects of buckwheat flower and leaf extract on immune function of tumor-bearing mice[J]. Medical Herald, 2013, 32(11): 1421-1424.

[0012] [4] Wang Jianxing, Jiang Yan, Wang Yan, et al. Effects of fermented extract of buckwheat flowers and leaves on renal damage in type 2 diabetic db / db mice[J]. Journal of Jilin University (Medical Edition), 2018, 44(001):95-100, insert 2.

[0013] [5] Li Xiu, Shang Kexin, Lu Jingyu, et al. Safety and toxicological observation of buckwheat bran extract [J]. Shanghai Animal Husbandry and Veterinary Newsletter, 2017, 03(No.140): 36-38.

[0014] [6] Zhou Yuyan, Qiao Hongjie, Li Chunling, et al. Study on the antibacterial activity of golden buckwheat extract in vitro [J]. Journal of Traditional Chinese Veterinary Medicine, 2009, 28(005): 44-46.

[0015] [7]Qiao Hongjie, Li Chunling, Wang Guiping, et al. Study on the antibacterial activity of golden buckwheat root extract in vitro [C]. The First China Veterinary Medicine Conference and the 2008 Academic Annual Meeting of the Animal Pharmacology Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine

[0016] [8] Feng Lisha, Fu Xianlong, Chen Fang, et al. Preliminary study on the antibacterial activity of golden buckwheat extract against plant pathogens [J]. Journal of Sichuan University (Natural Science Edition), 2006, 43(3): 688-691.

[0017] [9] Huang Renshu, Yi Fan. In vitro antibacterial test of (-) epicatechin active substances from golden buckwheat[J]. Jiangsu Agricultural Sciences, 2015, 43(001): 308-310.

[0018]

[10] Park Chunhong, Huo Yue, Li Xiang, et al. Antibacterial activity of buckwheat hull water extract [J]. Journal of Jilin Agricultural University, 2015(04): 482-487.

[0019]

[11] Dong Xiaoning, Zhao Qiang, Yang Ming. Study on the extraction and in vitro antibacterial activity of rutin from tartary buckwheat[J]. Journal of Traditional Chinese Veterinary Medicine, 2010(01):20-22.

[0020]

[12] Jiang Zhongli, Wang Junwei. Antibacterial effect of compound clove, buckwheat and dandelion[J]. Grain and Feed Industry, 2011, 12(008): 39-41. Summary of the Invention

[0021] To address the shortcomings of the prior art, one of the objectives of the present invention is to provide a buckwheat extract composition with broad-spectrum antimicrobial activity. This extract must meet the technical requirements of achieving microgram per milliliter MICs against a wide range of bacteria, possessing a broad antimicrobial spectrum, being easily accessible, and being inexpensive. This approach overcomes the existing limitations of buckwheat extracts, which hinder their use as highly effective antimicrobial agents.

[0022] In order to achieve the above object, the present invention provides the following technical solutions:

[0023] A buckwheat composition extract with broad-spectrum antibacterial activity comprises, by weight, 20 to 30 parts of buckwheat, 5 to 8 parts of zanthoxylum bungeanum, 10 to 12 parts of hawthorn leaves, and 5 to 8 parts of seabuckthorn leaves; the extract is a water-extracted product.

[0024] As mentioned above, buckwheat extract has many shortcomings in antibacterial properties, mainly manifested in the following aspects: (1) The antibacterial activity is not strong, and the MIC against common bacteria is only at the milligram per milliliter level; (2) The antibacterial activity is greatly affected by the extraction site, and the antibacterial spectrum of buckwheat hull is even narrower; (3) The existing technology mainly uses ethanol extraction, which is costly, has great safety risks, and is difficult to scale up.

[0025] Inspired by the discovery by Jiang Zhongli et al. that the alcohol extracts of buckwheat, cloves and dandelions had better antibacterial effects after cross-breeding, [1] The inventors of this patent considered selecting other common plants with antibacterial properties for mutual extraction.

[0026] Zanthoxylum bungeanum is a plant of the genus Zanthoxylum in the Rutaceae family. Its extracts have been found to have antibacterial properties. Ye Yushan and others obtained several compounds from the roots of Zanthoxylum bungeanum through multi-stage extraction and found that these compounds had significant antibacterial effects against Staphylococcus aureus. One of the compounds was also effective in inhibiting methicillin-resistant Staphylococcus aureus. [2] Huang Yiling and others investigated the antibacterial properties of extracts from different parts of Zanthoxylum bungeanum using different extraction methods and found that the antibacterial spectrum of the extracts was also greatly affected by the extraction method and extraction part. Among them, the antibacterial spectrum of the water extract was the narrowest, and it had no antibacterial effect on Escherichia coli, Salmonella and Staphylococcus aureus within the concentration range investigated. [3] Wei Panqiu et al. investigated the antibacterial effect of Zanthoxylum bungeanum extract on common aquatic bacteria, but found that Zanthoxylum bungeanum water extract had no antibacterial effect on common aquatic bacteria. [4] Wang Chunjuan et al. investigated the antibacterial activity of the alcohol extract of Zanthoxylum bungeana and found that it had a good antibacterial effect on standard Staphylococcus aureus and various methicillin-resistant Staphylococcus aureus, but the antibacterial activity of its water extract has not yet been investigated. [5] .

[0027] Based on the above reports showing that the extract of Zanthoxylum bungeanum has a certain antibacterial effect, the inventors of this patent considered matching Zanthoxylum bungeanum with buckwheat and extracting them with water as the extraction reagent, and examining the antibacterial properties of the resulting extracts. In preliminary experiments, we prioritized the effects of matching on the antibacterial properties of two common strains of Escherichia coli and Staphylococcus aureus. Through experiments, we found that matching the two did not produce a corresponding synergistic effect, and the antibacterial performance was not significantly improved. Through a large number of experiments, we examined the antibacterial properties of extracts obtained by matching buckwheat with other plants (such as red peony root, scutellaria baicalensis or wormwood), and similarly found that the antibacterial properties of the resulting extracts against the common bacteria Escherichia coli and Staphylococcus aureus did not change significantly.

[0028] Based on this, we considered pairing buckwheat with at least two other plants and investigating the antibacterial properties of their aqueous extracts. However, after examining a selection of buckwheat, Zanthoxylum bungeanum, Paeonia lactiflora, Scutellaria baicalensis, and Artemisia argyi, we found that extracts from any three of these plants, including buckwheat, exhibited no significant changes in their antibacterial properties against Escherichia coli and Staphylococcus aureus.

[0029] Through continuous exploration, we discovered that extracts derived from a combination of buckwheat, two-sided needles, and hawthorn leaves exhibited significantly enhanced antibacterial properties against Escherichia coli and Staphylococcus aureus. Encouraged by this discovery, we investigated the antibacterial spectrum of the resulting extract. Unfortunately, while the extract exhibited excellent antibacterial activity against some common bacteria, the spectrum remained relatively narrow.

[0030] On this basis, we further added mutually compatible plants and found that adding sea buckthorn leaves to buckwheat, two-sided needles and hawthorn leaves not only did not affect the antibacterial effect against common bacteria, but also expanded the antibacterial spectrum of the extract.

[0031] Gao Yu investigated the in vitro antibacterial activity of 35 Chinese herbal extracts and found that although the antibacterial effect of seabuckthorn on 14 standard bacteria was not higher than 100 mg / mL, it could enhance the antibacterial performance of chloramphenicol and ofloxacin against Pseudomonas aeruginosa. [9] The study found that the presence or absence of synergistic antibacterial effects between extracts of different Chinese herbal medicines, whether macromolecular or micromolecular, significantly impacted the antibacterial activity of these extracts. This suggests that the antibacterial effects of Chinese herbal medicines are multi-targeted, and that the pharmacological mechanisms of Chinese herbal ingredients differ for different strains.

[0032] Gao Yu's discovery not only reveals that Chinese herbal medicine has multi-target characteristics in antibacterial effect, indicating that the antibacterial mechanism of Chinese herbal medicine extracts is relatively complex, but also explains that the combination of water extracts of buckwheat, two-sided needle, hawthorn leaf and sea buckthorn leaf in the present invention produces a synergistic effect based on their specific antibacterial target characteristics, thus having the characteristics of a broad antibacterial spectrum and strong antibacterial effect.

[0033] In addition to the above reports on the antibacterial properties of buckwheat, two-sided needle and sea buckthorn leaf extracts, there are also some reports on the antibacterial properties of hawthorn leaf extracts. Shen Xiaohan et al. investigated the antibacterial activity of hawthorn ethanol extract against Staphylococcus aureus and found that its MIC was 0.625 mg / ml. [6] Xu Ruibo and others also found that the flavonoids in hawthorn leaves have an inhibitory effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. [7] Lin Chunmei et al. investigated the antibacterial effects of hawthorn leaf, fruit and seed extracts and found that the extracts had significant inhibitory effects on Escherichia coli, Staphylococcus aureus and Bacillus subtilis, but had no inhibitory effect on Aspergillus niger. [8] .

[0034] According to the antibacterial results obtained according to the present invention, it can be seen that the composition of the present invention overcomes the shortcomings of the components in the composition in terms of antibacterial spectrum and antibacterial effect, such as solving the antibacterial spectrum defects such as buckwheat extract has no antibacterial activity against Aspergillus niger, Fusarium spp., citrus green mold and glaucogreen Penicillium spp., hawthorn leaves have no inhibitory effect on Aspergillus niger, and the water extract of Zanthoxylum bungeanum has no antibacterial effect on common aquatic bacteria; for example, it solves the defect that the MIC of the extracts of the components in the composition can generally only reach the milligram per milliliter level.

[0035] As a preferred technical solution of the present invention, the buckwheat composition comprises, by weight, 25 parts of buckwheat, 6 parts of two-sided needle, 11 parts of hawthorn leaves and 7 parts of sea buckthorn leaves.

[0036] As shown in one embodiment of the present invention, when the components in the composition are combined by weight in the following manner: 25 parts of buckwheat, 6 parts of dianthus, 11 parts of hawthorn leaves and 7 parts of sea buckthorn leaves, the antibacterial activity of the obtained composition is optimal.

[0037] The buckwheat of the present invention includes bitter buckwheat, golden buckwheat or sweet buckwheat, and can be one or more of buckwheat roots, buckwheat stems, buckwheat leaves and buckwheat husks.

[0038] Compared to the prior art, the present invention demonstrates that, while the type and part of buckwheat used do have some influence on antimicrobial activity, overall, all buckwheat varieties and their parts can be combined with Zanthoxylum bungeanum, hawthorn leaves, and seabuckthorn leaves to enhance their antimicrobial properties and antibacterial spectrum. This overcomes the limitations of buckwheat type and part used in the prior art when using buckwheat extracts as antimicrobial agents, further enhancing the value of buckwheat in the preparation of antimicrobial agents.

[0039] The water extraction product is a liquid phase obtained after extracting the composition with water and removing the residue; the liquid phase includes a filtered filtrate, a concentrated extract or a filtrate freeze-dried powder.

[0040] Another object of the present invention is to provide a method for preparing the buckwheat composition extract, the method comprising the following steps:

[0041] (1) Mix buckwheat, two-sided needle, hawthorn leaf and sea buckthorn leaf according to the weight proportions;

[0042] (2) Add water to the obtained mixture, heat and extract, and take the supernatant after extraction to obtain the product.

[0043] As a preferred technical solution of the present invention, in step (1), the buckwheat, two-sided needle, hawthorn leaf and sea buckthorn leaf are dried and sieved into powder; and / or the extract includes a filtered filtrate, a concentrated extract or a filtrate freeze-dried powder.

[0044] As a preferred technical solution of the present invention, in step (2), during extraction, the material-liquid ratio is 1 g: 15-18 mL; the water temperature is 30-80° C.; and the extraction time is 0.5-2 hours.

[0045] Another object of the present invention is to provide a use of the buckwheat composition extract as an antibacterial agent.

[0046] As a technical solution of the present invention, the buckwheat composition extract is used as an antibacterial agent for food packaging materials.

[0047] Beneficial effects of the present invention:

[0048] The buckwheat composition obtained by the present invention has the advantages of a broad antimicrobial spectrum and excellent antibacterial properties, showing a highly effective antibacterial effect against more than 30 types of bacteria and has the potential to be used as a highly effective antimicrobial agent. This invention overcomes the antimicrobial shortcomings of buckwheat extracts and expands the application value of buckwheat.

[0049] References for this section:

[0050] [1] Jiang Zhongli, Wang Junwei. Antibacterial effect of compound clove, buckwheat and dandelion[J]. Grain and Feed Industry, 2011, 12(008): 39-41.

[0051] [2] Ye Yushan, Liu Jiawei, Liu Xiaoqiang, et al. Study on the antibacterial active ingredients of Zanthoxylum bungeanum root[J]. Chinese Herbal Medicine, 2013, 44(12): 1546-1551.

[0052] [3] Huang Yiling, Feng Jie, Wang Haihua, et al. Study on the antibacterial sites of the roots and stems of Zanthoxylum bungeanum[J]. Pharmacology and Clinic of Traditional Chinese Medicine, 2013, 029(001): 103-105.

[0053] [4] Wei Panqiu, Lu Jingdi, Huang Jie, et al. Inhibitory effects of eight Chinese herbal medicines on common aquatic pathogens [J]. Animal Husbandry and Veterinary Medicine, 2018(11).

[0054] [5] Wang Chunjuan, Zuo Guoying, Han Jun, et al. Screening of in vitro antibacterial activity of 21 traditional Chinese medicines [J]. West China Journal of Pharmaceutical Sciences, 2013, 28(005): 479-482.

[0055] [6] Shen Xiaohan, Song Xiaofeng, Zuo Yanhua, et al. Study on the antibacterial activity of different hawthorn leaf extracts against Staphylococcus aureus [J]. Rural Economy and Science and Technology, 2018(12).

[0056] [7] Xu Ruibo, Yang Wenwen, Wan Beibei. Ultrasonic extraction and in vitro activity study of flavonoids from Hawthorn leaves in Lianyungang[J]. Journal of Jinggangshan University: Natural Science Edition, 2016(6).

[0057] [8] Lin Chunmei, Liu Yunhe, Zhou Mingqian. Study on the antibacterial effect of hawthorn leaf, fruit and seed extracts[J]. Journal of Anhui Agricultural Sciences, 2011, 39(36): 22582-22583.

[0058] [9] Gao Yu. Study on the antibacterial activity of 35 Chinese herbal medicine extracts in vitro[J]. Chinese Ethnomedicine, 2021, Vol.30, No.9. DETAILED DESCRIPTION

[0059] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0060] In the following preliminary experimental examples and embodiments of the present invention, the experimental strain information is as follows:

[0061] Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Aspergillus niger (ATCC16404), Penicillium purpurogenum (ATCC 40687), Aspergillus flavus (ATCC 2003), Fusarium moniliforme (ATCC 2490), Fusarium oxysporum (ATCC 41029), Candida albicans (ATCC 10231), Pseudomonas aeruginosa (ATCC9027), Salmonella typhi (CMCC50071), Serratia marcescens (CMCC 41002), Klebsiella pneumoniae (ATCC 13883), Acinetobacter baumannii (ATCC19606), Alcaligenes faecalis (CMCC 40001), Bacillus subtilis (ATCC 9372), Bacillus subtilis (ATCC6633), Acinetobacter baumannii (ATCC 19606), Listeria monocytogenes (ATCC 19115), Enterobacter cloacae (ATCC 13047), Enterobacter aerogenes (ATCC 13048), Salmonella typhimurium (ATCC 14028), Shigella flexneri (ATCC 12022), Staphylococcus epidermidis (ATCC 12228), Sarcina luteus (ATCC 11001), Bacillus cereus (ATCC 11778), Salmonella (CMCC (B) 50094), Methicillin-resistant Staphylococcus aureus (ATCC 43300), Methicillin-resistant Staphylococcus aureus (ATCC 25923), Bacillus anthracis (CMCC 63001), Salmonella Paratyphi B standard strain (CMCC (B) 50094), Proteus vulgaris (CMCC (B) 49027), Aeromonas hydrophila (ATCC 35654), Citrus green mold (standard bacteria in the inventor's research group's laboratory) and glaucogreen Penicillium (standard bacteria in the inventor's research group's laboratory).

[0062] The tartary buckwheat used in the following preliminary experimental examples and embodiments of the present invention was collected from Liangshan area of ​​Sichuan Province, sweet buckwheat was collected from Inner Mongolia Autonomous Region, two-sided needle was collected from Guangxi Zhuang Autonomous Region, sea buckthorn leaves were purchased from Shanxi Province, hawthorn leaves were purchased from Jiangsu Province, red peony root, scutellaria baicalensis and artemisia argyi were commercially available.

[0063] Preliminary Experimental Example 1

[0064] This preliminary experiment investigated the antibacterial effect of the extract obtained by mixing buckwheat and two-sided needle and extracting with water on Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922).

[0065] This pilot experiment set up 4 experimental groups and 3 control groups, namely:

[0066] Experimental group 1: The whole herb of tartary buckwheat and the whole herb of two-sided needle were mixed in a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1g:15mL. The mixture was extracted at 80℃ for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0067] Experimental group 2: The whole herb of sweet buckwheat and the whole herb of two-sided needle were mixed in a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1g:15mL. The mixture was extracted at 80℃ for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0068] Experimental group 3: Tartary buckwheat stems and leaves and whole herb of Zanthoxylum bungeanum were mixed in a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0069] Experimental group 4: Tartary buckwheat root and whole herb of Zanthoxylum bungeanum were mixed at a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0070] Experimental group 5: Sweet buckwheat hulls and whole herb of Zanthoxylum bungeanum were mixed at a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0071] Control group 1: The whole herb of tartary buckwheat was selected, crushed, and added with water at a solid-liquid ratio of 1 g:15 mL. The extract was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0072] Control group 2: The whole herb of sweet buckwheat was selected, crushed, and added with water at a solid-liquid ratio of 1 g:15 mL. The extract was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0073] Control group 3: The whole herb of Zanthoxylum bungeanum was selected, crushed, and added with water at a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0074] The minimum inhibitory concentrations (MICs) of the extracts obtained from the experimental group and the control group against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were investigated, and the results are shown in Table 1.

[0075] Note: In this preliminary experiment, since it is known that buckwheat extract has poor antibacterial activity, this preliminary experiment only examines whether the addition of Zanthoxylum bungeanum can significantly improve the antibacterial activity. Therefore, when setting the concentration gradient, the value interval is relatively large, namely 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 100 mg / mL, and 150 mg / mL. Therefore, the MIC in Table 1 may not be an accurate value. For example, when the MIC in Table 1 is 50 mg / mL, the accurate MIC may be 50 mg / mL, or it may be between 40 mg / mL and 50 mg / mL; among them, "-" indicates no antibacterial activity (the same applies to the other tables).

[0076] Table 1

[0077]

[0078] Preliminary Experiment Example 2

[0079] This preliminary experiment investigated the antibacterial effects of buckwheat extracts mixed with red peony root, scutellaria baicalensis, and artemisia argyi against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922).

[0080] This pilot experiment set up 4 experimental groups and 3 control groups, namely:

[0081] Experimental group 1: The whole herb of tartary buckwheat and red peony root were mixed at a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0082] Experimental group 2: The whole herb of tartary buckwheat and scutellaria baicalensis were mixed at a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0083] Experimental group 3: The whole herb of tartary buckwheat and mugwort leaves were mixed at a weight ratio of 5:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0084] Control group 1: Red peony root was selected, crushed, and water was added at a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0085] Control group 2: Scutellaria baicalensis was selected, crushed, and water was added at a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0086] Control group 3: Artemisia argyi was selected, crushed, and water was added with a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. The filtrate was concentrated after filtration and freeze-dried to powder to obtain an extract.

[0087] The minimum inhibitory concentrations (MICs) of the extracts obtained from the experimental group and the control group against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were investigated, and the results are shown in Table 2.

[0088] Table 2

[0089]

[0090] Preliminary Experiment Example 3

[0091] This preliminary experimental example investigates the antibacterial effect of extracts obtained by mixing any two selected from red peony root, scutellaria baicalensis, and artemisia argyi with tartary buckwheat and extracting them with water on Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922).

[0092] This pre-experimental example sets up three experimental groups, and the experimental results of the control group of pre-experimental example 2 are used as comparative data.

[0093] Experimental group 1: The whole herb of tartary buckwheat, scutellaria baicalensis, and red peony root were mixed at a weight ratio of 5:1:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0094] Experimental group 2: The whole herb of tartary buckwheat, scutellaria baicalensis and artemisia argyi were mixed in a weight ratio of 5:1:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0095] Experimental group 3: The whole herb of tartary buckwheat, red peony root and artemisia argyi were mixed in a weight ratio of 5:1:1, crushed, and added with water to a solid-liquid ratio of 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to powder to obtain the extract.

[0096] The minimum inhibitory concentration (MIC) of the extracts obtained from the above experimental groups against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) was investigated, and the results are shown in Table 3.

[0097] Table 3

[0098]

[0099] Preliminary Experiment Example 4

[0100] This preliminary experiment first investigated the antibacterial effect of the extract obtained by mixing tartary buckwheat, two-sided needle and hawthorn leaves and extracting them with water on Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922).

[0101] The whole herb of tartary buckwheat, the whole herb of two-sided needle and the leaf of hawthorn were mixed in a weight ratio of 5:1:2, crushed, and water was added to make the material-liquid ratio 1 g:15 mL. The mixture was extracted at 80° C. for 2 hours. After filtering, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0102] The experimental results showed that the MICs of the obtained extracts against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) could reach 50 μg / mL and 20 μg / mL, respectively, indicating that the aqueous extracts of whole herb of buckwheat, whole herb of Zanthoxylum bungeanum and hawthorn leaves have synergistic effects in antibacterial properties against the above two bacteria.

[0103] This preliminary experiment then examined the antibacterial properties of the extract against Aspergillus niger (ATCC 16404), Fusarium moniliforme (ATCC 2490), Fusarium oxysporum (ATCC 41029), Aeromonas hydrophila (ATCC 35654), citrus green mold, and Penicillium glaucum. The extract was found to have no antibacterial effect against Aspergillus niger (ATCC 16404), Aeromonas hydrophila (ATCC 35654), citrus green mold, and Penicillium glaucum, while the MICs against Fusarium moniliforme (ATCC 2490) and Fusarium oxysporum (ATCC 41029) were 100 mg / mL and 150 mg / mL, respectively. This indicates that the extract in this preliminary experiment still fails to effectively expand the antibacterial spectrum and does not address the known shortcomings of buckwheat, hawthorn leaf, and two-sided needle extracts in terms of antibacterial spectrum.

[0104] Example 1

[0105] The whole herb of tartary buckwheat, the whole herb of two-sided needle, the leaves of hawthorn and the leaves of sea buckthorn were mixed in a weight ratio of 5:1:2:1, crushed, and water was added to make the material-liquid ratio 1 g:15 mL. The mixture was extracted at 80°C for 2 hours. After filtering, the filtrate was concentrated and freeze-dried to powder to obtain an extract.

[0106] This example examined the antibacterial activity of the extract against Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922), and found excellent antibacterial effects. Furthermore, the extract was tested against Aspergillus niger (ATCC 16404), Fusarium moniliforme (ATCC 2490), Fusarium oxysporum (ATCC 41029), Aeromonas hydrophila (ATCC 35654), Citrus viridans, and Penicillium glaucum, and similarly found excellent antibacterial activity (see Table 4 for results). Encouraged by these experimental results, this example examined a variety of other experimental bacteria and found that the extract exhibited good antibacterial activity against all tested bacteria (see Table 4 for results). This demonstrates that the extract obtained in this example possesses a broad antibacterial spectrum and excellent antibacterial activity.

[0107] Example 2

[0108] Based on Example 1, this example adjusted the buckwheat species and parts, as well as the ratios with other components. The composition of Example 1 was used as Experimental Group 1, and the following Experimental Groups 2 to 5 were set up:

[0109] Experimental group 2: except that the components, by weight, are 20 parts of buckwheat, 5 parts of two-sided needles, 12 parts of hawthorn leaves and 8 parts of sea buckthorn leaves, the rest are the same as those of experimental group 1.

[0110] Experimental group 3: except that the components, by weight, are 30 parts of buckwheat, 8 parts of two-sided needles, 10 parts of hawthorn leaves and 5 parts of sea buckthorn leaves, the rest are the same as those of experimental group 1.

[0111] Experimental group 4: except that each component is calculated by weight, it contains 25 parts of buckwheat, 6 parts of two-sided needle, 11 parts of hawthorn leaves and 7 parts of sea buckthorn leaves.

[0112] Experimental group 5: except that the material-liquid ratio was 1 g:18 mL, the water temperature was 30°C, and the extraction time was 0.5 h, the rest was the same as that of experimental group 1.

[0113] The MICs of the extracts obtained from Experimental Groups 1 to 5 against each test bacteria are shown in Table 4, where the unit of MIC is μM / mL. In this example, when investigating MIC, a preliminary experiment was conducted to determine the approximate MIC range. Based on this preliminary range, a corresponding concentration gradient was set to ultimately determine the precise MIC value.

[0114] Table 4 Unit: μm / mL

[0115]

[0116] As shown in the antibacterial results in Table 4, the extracts obtained from Experimental Groups 1 to 5 exhibited a broad antibacterial spectrum and high antibacterial activity. Furthermore, we investigated the combination of buckwheat, two-sided needles, hawthorn leaves, and seabuckthorn leaves in the same ratio as in Experimental Group 4. Extraction was performed using 70% ethanol, following the method described in "In Vitro Antibacterial Activity of Golden Buckwheat Extracts" (DOI: 10.13823 / j.cnki.j tcvm.2009.05.045). Antibacterial tests were conducted using the aforementioned test strains. The extracts exhibited antibacterial activity greater than 4 mg / mL (i.e., 4000 μM / mL) against only eight of the test strains. Furthermore, they exhibited antibacterial activity against only 17 strains, with no antibacterial activity against the remaining strains. This indicates that the antibacterial activity of the ethanol extract of the present composition is significantly lower than that of the aqueous extract. This also suggests that the synergistic component in the composition must be a highly water-soluble component, providing guidance for subsequent mechanistic studies.

[0117] Furthermore, based on the protocol of Experimental Group 4, the inventors replaced the whole tartary buckwheat herb with tartary buckwheat roots, husks, and stems and leaves, respectively. They found that the MICs of the resulting extracts against each test bacteria did not vary by more than 30%, indicating that the components of buckwheat that synergize with other components are present in all parts of the buckwheat herb. Based on the protocol of Experimental Group 4, the inventors replaced the whole tartary buckwheat herb with the whole sweet buckwheat herb. They found that the MICs of the resulting extracts against each test bacteria did not vary by more than 20%, indicating that the components that synergize with other components are present in the aqueous extracts of both tartary buckwheat and sweet buckwheat.

Claims

1. A buckwheat composition extract with broad-spectrum antibacterial activity, characterized in that: The buckwheat composition is composed of the following components by weight: 20-30 parts of buckwheat, 5-8 parts of whole herb of Zanthoxylum bungeanum, 10-12 parts of hawthorn leaves, and 5-8 parts of seabuckthorn leaves; the extract is a water-extracted product, which is a liquid phase obtained by extracting the composition with water and removing the residue; the liquid phase includes a filtrate, a concentrated extract, or a lyophilized powder of the filtrate; The buckwheat is any one of the whole herb of tartary buckwheat, tartary buckwheat root, tartary buckwheat husk, tartary buckwheat stem and leaf, and the whole herb of sweet buckwheat.

2. The buckwheat composition extract with broad-spectrum antibacterial activity according to claim 1, characterized in that: The buckwheat composition is composed of the following components in parts by weight: Composition: 25 parts of buckwheat, 6 parts of two-sided needle, 11 parts of hawthorn leaves and 7 parts of sea buckthorn leaves.

3. The method for preparing the buckwheat composition extract with broad-spectrum antibacterial activity according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Mix buckwheat, whole herb of Zanthoxylum bungeanum, hawthorn leaves, and seabuckthorn leaves according to the weight proportions; (2) Add water to the obtained mixture, heat and extract, and take the supernatant after extraction to obtain the product.

4. The preparation method according to claim 3, characterized in that In step (1), the buckwheat, whole herb of Zanthoxylum bungeanum, hawthorn leaves and seabuckthorn leaves are dried and sieved into powder; the extract includes filtered filtrate, concentrated extract or filtrate freeze-dried powder.

5. The preparation method according to claim 3, characterized in that In step (2), during extraction, the material-liquid ratio is 1 g: 15-18 mL; the water temperature is 30-80° C.; and the extraction time is 0.5-2 hours.

6. Use of the buckwheat composition extract according to claim 1 or 2, or the buckwheat composition extract prepared according to the preparation method according to any one of claims 3 to 5, as an antibacterial agent for food packaging materials.

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