Use of valerian lactone A in preparing products containing anti-Escherichia coli

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of valerian lactone A in the preparation of products containing anti-Escherichia coli. The valerian lactone A of the present invention has a good inhibitory effect on Escherichia coli, and its source is usually natural biological valerian, the extraction step is simple, and the acquisition cost is low. Furthermore, valerian lactone A is a natural product with little toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the use of valerian lactone A in the preparation of products containing anti-Escherichia coli. Background Art

[0002] Escherichia coli (E.coli) is a Gram-negative short rod, 0.5×1-3 microns in size. It has flagella around its peritrichous, is motile, and has no spores. It can ferment a variety of sugars to produce acid and gas, and is a normal resident bacteria in the intestines of humans and animals. It enters the intestines of infants with breastfeeding after birth, and stays with humans for life, accounting for almost 1 / 3 of the dry weight of feces.

[0003] The antigenic components of Escherichia coli are complex and can be divided into bacterial antigens (O), flagellar antigens (H) and surface antigens (K). The latter has the ability to resist phagocytosis and complement. According to the difference in bacterial antigens, Escherichia coli can be divided into more than 150 types, of which 16 serotypes are pathogenic Escherichia coli, which often cause epidemic infant diarrhea and adult pleurisy. Escherichia coli is an important material for studying microbial genetics. For example, limited transduction was discovered in Escherichia coli K12 strain in 1954. Lederberg used two nutritional deficiency types of Escherichia coli to conduct experiments, laying the foundation for the study of bacterial conjugation methodology and genetic engineering research.

[0004] Escherichia coli (E.coli) is a representative bacterium of the genus Escherichia. It is generally non-pathogenic and is a common bacterium in the intestines of humans and animals. Under certain conditions, it can cause extraintestinal infections. Some serotypes of strains are highly pathogenic and can cause diarrhea, and are collectively referred to as pathogenic E. coli.

[0005] The bacteria are more heat resistant than other enteric bacilli. Some bacteria can still survive after being heated at 55°C for 60 minutes or at 60°C for 15 minutes. They can survive for weeks to months in natural water and even longer in feces at lower temperatures. Bile salts, brilliant green, etc. have inhibitory effects on E. coli. E. coli is sensitive to sulfonamides, streptomycin, chloramphenicol, etc., but is easily resistant, which is acquired through the transfer of plasmids carrying R factors. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides the use of valerian lactone A in the preparation of products containing anti-Escherichia coli, wherein the valerian lactone A has a structure as shown in the following formula I:

[0007]

[0008] According to an embodiment of the present invention, the products containing anti-Escherichia coli are selected from health foods, medical devices, disinfection products and daily chemical products.

[0009] According to an embodiment of the present invention, the medical devices, disinfectants and daily chemicals are selected from products with antibacterial effects such as mouthwash, toothpaste, aromatherapy volatile liquid, dressing, patch, hand sanitizer, hand gel, soap, spray, gynecological lotion, cosmetics, etc.

[0010] According to an embodiment of the present invention, the spray is a disinfecting spray.

[0011] According to an embodiment of the present invention, when valerian lactone A is used to prepare health foods, medical devices, disinfectants and daily chemicals, it acts as a natural preservative or antibacterial agent to significantly reduce the content of Escherichia coli in the above products.

[0012] According to an embodiment of the present invention, the antibacterial rate of valerian lactone A in the product against Escherichia coli is above 85%, preferably above 88%, and further preferably above 91%.

[0013] Beneficial Effects

[0014] The valerian lactone A of the present invention has a good inhibitory effect on Escherichia coli, and its source is usually natural biological valerian, the extraction step is simple, and the acquisition cost is low. Furthermore, valerian lactone A is a natural product with small toxic and side effects. In summary, valerian lactone A has a significant antibacterial effect, is mild and non-irritating in nature, and is not prone to toxic and side effects. It has a wide range of applications and is expected to develop valerian lactone A into a pure natural plant antibacterial agent, for example, using valerian lactone A to prepare products with antibacterial effects, such as health foods, medical devices, disinfectants, and daily chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the 6- and 24-position H-HMBC fragment correlation spectrum of the compound of the present invention.

[0016] Figure 2 It is the 2 and 23 H-HMBC fragment correlation spectrum of the compound of the present invention.

[0017] Figure 3 It is the CH3-HMBC fragment correlation spectrum at position 20 of the compound of the present invention.

[0018] Figure 4 It is the CH3-HMBC fragment correlation spectrum at position 11 of the compound of the present invention.

[0019] Figure 5 It is the 16-CH3-HMBC fragment correlation spectrum of the compound of the present invention.

[0020] Figure 6 It is the 2, 4, 7 H-HMBC fragment correlation spectrum of the compound of the present invention. DETAILED DESCRIPTION

[0021] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0022] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0023] Example 1 Preparation of valerian lactone A

[0024]

[0025] 1) Extraction: Take 20 kg of valerian root material, grind it and extract it with 50% ethanol (50% ethanol water by weight) by reflux for 2 times, each time for 2 hours, and the amount of ethanol used each time is 120 kg.

[0026] 2) Extraction: Combine the ethanol extracts and concentrate under reduced pressure to a clear paste with a relative density of 1.20, then add n-hexane for extraction and collect the n-hexane extracts.

[0027] 3) Separation: Weigh 700g of 200-300 mesh silica gel, dry-load the column, and use petroleum ether (60-90°C) to elute and balance. Use the above extract as the sample solution to pass through the silica gel chromatography column so that the effective components of the sample solution are completely adsorbed on the silica gel. Then elute with a 99:1 volume ratio of petroleum ether (60-90°C) and ethyl acetate co-solvent, and check the eluted fractions by thin layer chromatography under 254nm ultraviolet light. The developing solvent used in thin layer chromatography is petroleum ether: ethyl acetate (7:1). When the target component appears in the eluted fraction, use a 98:2 volume ratio of petroleum ether (60-90°C) and ethyl acetate co-solvent as the eluent until the target component is no longer detected in the eluted fraction, combine the 98:2 eluted fractions, and concentrate them by rotary evaporator to obtain a crude product;

[0028] The crude product was dissolved in n-hexane and then separated by column chromatography twice according to the above method, and the eluted fraction was concentrated and dried to obtain the pure product. The product was a light yellow oil.

[0029] HPLC detection showed that the purity of the product was 98.8% and the yield was 5.39 g. Since the content of valerian lactone A with a structure as shown in Formula I in the valerian herb was 0.045%, the transfer rate was calculated as follows: 5.39*98.8% / 20000*0.045%=59.2%.

[0030] The detection conditions of the HPLC method are as follows: ODS2 4.6*250mm, 5μm analytical column; column temperature: 30°C; mobile phase: acetonitrile-deionized water (70:30, V / V), flow rate: 0.6ml / min, detection wavelength 268nm, injection volume 20μl.

[0031] The product was detected by UV analysis, and UV(λ MeOH max )=269nm absorption peak, indicating the existence of conjugated system.

[0032] The product was detected by infrared analysis, and the following characteristic absorption peaks appeared in the spectrum: IR (νmax KBr )cm -1 :3443.84,2929.24,2871.71,1735.32,1677.59,1622.83,1453.35,1376.10; The above data show that there are functional groups such as methyl, methylene, carbonyl and olefin in the sample structure.

[0033] The product was detected by mass spectrometry, and the results were: EI-MS (me / z): 291.29, 279.25, 263.31, 234.16, 219.04 (100%), 204.98, 190.95, 178.96, 173.06, 148.92, 144.99, 130.92, 119.11, 107.15, 104.92, 90.99, 76.95, 64.99, 54.94;

[0034] MALDI-TOF MS(me / z): 315.1, 299.1, 273.0, 257.0, 235.0.

[0035] In the spectrum, 219.04 (100%).

[0036] The product was tested by NMR, and the results were:

[0037] 1H-NMR (CDCl3, 500MHz) δppm: 9.28 (s, 1H), 6.35 (d, J=9.5Hz, 1H), 5.35 (dd, J=5.5Hz, J =11.5Hz, 1H), 4.18 (d, J = 12.0Hz, 1H), 4.12 (d, J = 12.0Hz, 1H), 2.88 (m, 1H), 2.84 (dd, 1 H), 2.26 (m, 1H, C14-H), 2.02 (t, 2H, C12-H), 2.00 (s, 3H), 1.28 (s, 3H, C20-CH3), 1.19 (s, 3H, C11-CH3), 1.18 (s, 3H, C11-CH3), 1.03 (s, 3H, C16-CH3), 1.02 (s, 3H, C16-CH3);

[0038] In the spectrum, at 9.28ppm, an H s peak signal is given, which is the H signal of the aldehyde group at position 24; at 6.35ppm, an H d peak signal with a coupling constant of 9.5Hz is given, which is the H signal of the olefin at position 6; at 5.35ppm, an H dd peak signal with coupling constants of 5.5 and 11.5Hz is given, which is the H signal of the olefin at position 2; at 4.18ppm, an H d peak signal with coupling constants of 12.0Hz is given, which is the H signal of position 23; at 4.12ppm, an H d peak signal with coupling constants of 12.0Hz is given, which is the H signal of position 23; at 2.88ppm, an H m peak signal is given, which is the H signal of position 13; at 2.84ppm, an H m peak signal is given, which is the H signal of position 13. At m, a H is given, and the coupling constants are 12.0Hz dd peak signals, which are 4-position H signals; at 2.02ppm, 3 H, s peak signals are given, which are -OCOCH3 group H signals; at 1.28ppm, 3 H, s peak signals are given, which are 20-position CH3 group H signals; at 1.19ppm, 3 H, s peak signals are given, which are 11-position geminal CH3 group H signals; at 1.18ppm, 3 H, s peak signals are given, which are 11-position geminal CH3 group H signals; at 1.03ppm, 3 H, s peak signals are given, which are 16-position geminal CH3 group H signals; at 1.02ppm, 3 H, s peak signals are given, which are 16-position geminal CH3 group H signals;

[0039] 13C-NMR (CDCl3, 125MHz) δppm: 193.80 (d, C-24, aldehyde carbon), 170.89 (s, -OCOCH3, acyl carbon), 175.32 (s, C-22, -OC=O), 155.88 (d, C-6, =CH), 142.64 (s, C-5, =C-), 132.94 (s, C-1, =C-), 130.2 3(d,C-2,=CH), 85.59(s,C-20,-OC-), 60.76(t,C-23,-OCH2), 48.87(d,C-13,-C-COO), 41.37(d,C-19,-CH-), 40.65(t,C-21,-CH2-), 40.43(d,C-14,-CH-), 37.01(d,C-8,-CH- ), 35.03 (d, C-10, -CH2), 29.85 (q, C20-CH3), 29.05 (s, C11), 28.52 (q, C11-CH3), 28.38 (q, C16-CH3), 27.77 (t, C-3, -CH2), 26.52 (t, C-7, -CH2), 25.31, 24.38 (t, C-9, -CH2), 2 3.94(d,C-17,-CH), 23.75(t,C-18,-CH2), 23.31(t,C-4,-CH2), 21.07(t,C-15,-CH2), 20.96(q,C-23-OCOCH3), 20.55(t,C-12,-CH2), 15.86(q,C11-CH3), 15.81(q,C16-CH3);

[0040] The spectrum shows 31 signals, among which 193.80 (d, C-24, aldehyde carbon), 170.89 (s, -OCOCH3, acyl carbon), 175.32 (s, C-22, -OC=O) are very weak signals, which are carbonyl carbon signals; 155.88 (d, C-6, =CH) is a signal containing H protons and 142.64 (s, C-5, =C-) is a quaternary carbon signal, both of which are carbon signals on the double bond of the olefinic bond, and there is a conjugated system with the group; 132.94 (s, C-1, =C-) is a quaternary The carbon signal and 130.23 (d, C-2, =CH) are signals containing H protons, and both are carbon signals on the olefinic double bond; 85.59 (s, C-20, -OC-) is a saturated quaternary carbon signal containing oxygen, 60.76 (t, C-23, -OCH2) is a saturated secondary carbon signal containing oxygen; 48.87 (d, C-13, -C-COO) is a tertiary carbon signal containing an electron-withdrawing group; 41.37 (d, C-19, -CH-), 40.65 (t, C-21, -CH2-), 40. 43 (d, C-14, -CH-), 37.01 (d, C-8, -CH-), 35.03 (d, C-10, -CH2), 29.85 (q, C20-CH3) are primary carbon signals, 29.05 (s, C11), 28.52 (q, C11-CH3) are primary carbon signals, 28.38 (q, C16-CH3) are primary carbon signals, 27.77 (t, C-3, -CH2), 26.52 (t, C-7, -CH2), 25.31, 24. 38 (t, C-9, -CH2), 23.94 (d, C-17, -CH), 23.75 (t, C-18, -CH2), 23.31 (t, C-4, -CH2), 21.07 (t, C-15, -CH2), 20.96 (q, C-23-OCOCH3) are primary carbon signals, 20.55 (t, C-12, -CH2), 15.86 (q, C11-CH3) are primary carbon signals, and 15.81 (q, C16-CH3) are primary carbon signals.

[0041] In the HH cosy spectrum, 6.35 (d, J=9.5 Hz, 1H, C6-H) is correlated with 1.48 (m, 1H), 5.35 (dd, J=5.5 Hz, J=11.5 Hz, 1H, C2-H) is correlated with 2.36, 2.00, 2.88, 2.84 (dd, 1H, C4-H) and 5.35 (dd, J=5.5 Hz, J=11.5 Hz, 1H, C2-H), 2.91 (m, 1H, C13-H) is correlated with 2.26 (m, 1H, C14-H), and 2.02 (t, 2H, C12-H).

[0042] In the HMQC spectrum, 9.28 (s, 1H) and 193.80 (d, C-24, aldehyde carbon), 6.35 (d, J = 9.5 Hz, 1H) and 155.88 (d, C-6, =CH), 5.35 (dd, J = 5.5 Hz, J = 11.5 Hz, 2H) and 130.23 (d, C-2, =CH), 4.18 (d, J = 12.0 Hz, 1H) and 4.12 (d, J = 12.0 Hz, 1H) and 60.76 (t, C-23, -OCH2), 2.91 (m, 1H) and 48.87 (d, C-13, -CH- , C-23-OCOCH3), 1.02 (s, 3H) and 15.81 (q, C16-CH3), 1.03 (s, 3H) and 28.38 (q, C16-CH3), 1.18 (s, 3H) and 15.86 (q, C11-CH3), 1.19 (s, 3H) and 28.52 (q, C11-CH3), 1.28 (s, 3H) and 29.85 (q, C20-CH3), etc.

[0043] In the HMBC spectrum, 9.28 (s, 1H) is correlated with 155.88 (d, C-6, =CH), 142.64 (s, C-5, =C-), 23.31 (t, C-4, -CH2); 6.35 (d, J = 9.5 Hz, 1H) is correlated with 193.80 (d, C-24, aldehyde carbon), 142.64 (s, C-5, =C-), 23.31 (t, C-4, -CH2). From the above, it can be deduced that Figure 1 The fragments shown in . 5.35 (dd, J = 5.5 Hz, J = 11.5 Hz, 1H) is related to 60.76 (t, C-23, -OCH2), 35.03 (t, C-10, -CH2-), 27.77 (t, C-3, -CH2); 4.18 (d, J = 12.0 Hz, 1H) and 4.12 (d, J = 12.0 Hz, 1H) are related to 170.89 (s, -OCOCH3, middle acyl carbon), 130.23 (d, C-2, =CH), 35.03 (t, C-10, -CH2-), and the above results indicate that Figure 2 The fragments shown in the figure are shown in the figure. 2.91 (m, 1H) is related to 26.52 (t, C-12, -CH2-); 1.28 (s, 3H, C20-CH3) is related to 41.37 (d, C-19, -CH-), 40.65 (t, C-21, -CH2-), 85.59 (s, C-20, -OC-), and the above results indicate that Figure 3The fragments shown in . 1.18 (s, 3H, C11-CH3) and 1.19 (s, 3H, C11-CH3) are related to 37.01 (d, C-8, -CH-), 20.55 (t, C-12, -CH2), 29.05 (s, C11), and it is deduced from the above that Figure 4 The fragments shown in . 1.02 (s, 3H, C16-CH3) and 1.03 (s, 3H, C16-CH3) are related to 21.07 (t, C-15, -CH2), 23.94 (d, C-17, -CH), and it is deduced from the above that Figure 5 The fragment shown in . 27.77 (t, C-3, -CH2) is related to 5.35 (dd, J = 5.5 Hz, J = 11.5 Hz, 1H), 2.84 (dd, 1H, C4-H), 1.48 (m, 1H) is related to 142.64 (s, C-5, = C-), 37.01 (d, C-8, -CH-), and combined with the HH cosy spectrum, it is deduced Figure 6 The fragment shown in .

[0044] Based on the above analysis, its molecular formula is C 31 H 46 O5, confirm that the structure of this compound is as shown in formula I. This structure is two sesquiterpenes connected together, one of which is a variant sesquiterpene lactone structure, which conforms to the isoprene rule in terms of biogenic pathway.

[0045] Example 2 Inhibitory effect of valerian lactone A on Escherichia coli

[0046] The antibacterial rate test of the example preparation was carried out with reference to Appendix C "Test methods for bactericidal performance, antibacterial performance and stability of products" of "GB15979-2002 Hygiene Standard for Disposable Sanitary Products".

[0047] (1) Antibacterial types

[0048] Escherichia coli (ATCC 25922).

[0049] (2) Preparation of bacterial solution

[0050] Take a fresh slant culture of the nutrient agar medium of the fourth generation of the strain, wash off the bacterial lawn with 5 mL of 0.03 mol / L phosphate buffer (hereinafter referred to as PBS), dilute it with PBS to a bacterial suspension of the test concentration, make the bacterial suspension evenly, and set aside.

[0051] (3) Operation steps

[0052] ① Take 5 mL of the sample solution (the valerian lactone A prepared in Example 1 is weighed and made into a 1% solution with methanol) and 4 tubes of the control solution (5 mL of sterilized distilled water);

[0053] ② Add 100uL of the above bacterial suspension to each sample solution, mix well, and start timing;

[0054] ③ After the bacterial solution and sample solution have reacted for 20 minutes, take 0.5 mL of the sample solution and drop it into a test tube containing 5 mL of PBS, mix thoroughly, make appropriate dilutions, and then take 2 to 3 dilutions;

[0055] ④Pipette 0.5mL of each dilution onto two plates, pour 15mL of nutrient agar medium cooled to 40-45℃, and rotate the plates to make them fully uniform;

[0056] ⑤ After the agar solidifies, turn the plate over and incubate at 35℃±2℃ for 48h before counting the number of live bacterial colonies;

[0057] ⑥ Repeat the test 3 times and calculate the inhibition rate according to the following formula:

[0058] X=(AB) / A×100%

[0059] Where: X is the inhibition rate; A is the colony count of the control sample; B is the colony count of the test sample.

[0060] (4) Test results

[0061]

[0062] The test results show that the antibacterial rate of the sample liquid on Escherichia coli is 91.7%, and the sample has a significant antibacterial effect on Escherichia coli. Therefore, based on the experimental results, valerian lactone A can be applied to health foods, medical devices, disinfectants and daily chemical products to produce mouthwashes, toothpastes, aromatherapy volatile liquids, dressings, patches, hand sanitizers, hand washing gels, soaps, sprays (such as disinfectant sprays), gynecological washes and other products with antibacterial effects. It can also be added to food and cosmetics as a natural preservative or antibacterial agent.

[0063] Example 3: Use of valerian lactone A in preparing mouthwash

[0064] The following raw materials are mixed and stirred to obtain valerian terpene lactone A antibacterial mouthwash.

[0065] Table 1 Ratio of raw materials of antibacterial mouthwash (mass ratio)

[0066]

[0067]

[0068] Replacing sorbitol in the above table with sorbitol can also produce a mouthwash with an inhibitory effect on Escherichia coli.

[0069] The antibacterial rate test of the mouthwash sample obtained in Example 3 was carried out with reference to Appendix C "Test Methods for Product Bactericidal Performance, Antibacterial Performance and Stability" of "GB15979-2002 Hygiene Standard for Disposable Hygiene Products". The results showed that the antibacterial rate of the mouthwash sample against Escherichia coli was 92.1%.

[0070] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. 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 valerian lactone A in the preparation of products containing anti-Escherichia coli, wherein the valerian lactone A has a structure as shown in the following formula I:

2. The use according to claim 1, characterized in that The products containing anti-Escherichia coli are selected from health food, medical equipment, disinfection products and daily chemical products.

3. The use according to claim 2, characterized in that: The medical devices, disinfection products and daily chemical products are selected from mouthwashes, toothpastes, aromatherapy volatile liquids, dressings, patches, hand sanitizers, hand washing gels, soaps, sprays, gynecological lotions and cosmetics with antibacterial effects.

4. The use according to claim 3, characterized in that: When valerian lactone A is used to prepare health foods, medical devices, disinfectants and daily chemical products, it acts as a natural preservative or antibacterial agent to significantly reduce the content of Escherichia coli in the above products.

5. The use according to claim 3, characterized in that: The spray is a disinfectant spray.

6. The use according to claim 3, characterized in that: The antibacterial rate of the valerian lactone A in the product against Escherichia coli is more than 85%.

7. The use according to claim 6, characterized in that The antibacterial rate of the valerian lactone A in the product against Escherichia coli is more than 88%.

8. The use according to claim 7, characterized in that The antibacterial rate of the valerian lactone A in the product against Escherichia coli is more than 91%.

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