New substances with physiological activity and their applications

Abstract

The present invention relates to a new substance with physiological activity, specifically a new substance produced during microbial mutagenesis and its application, belonging to the field of biotechnology. The new substance with physiological activity is a compound of Formula I, which exhibits significant tyrosinase inhibition and sedative effects. The compound of Formula I can be added to cosmetics as a functional ingredient to prevent melanin production caused by excessive tyrosinase activity, resulting in products with whitening properties. It can also be used as a sedative drug to treat stress, improve sleep, combat anxiety, and relieve irritability.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. 202310168245.4, filed on February 22, 2023, entitled “New Physiologically Active Substances and Applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a new substance with physiological activity, in particular to a new substance produced in the process of microbial mutagenesis and its application, belonging to the field of biotechnology. Background Art

[0004] Kluyveromyces lactis strains are highly safe and designated as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration (FDA). They are important food-grade probiotics in industrial production and have a long history of use in fermented foods. High-quality Kluyveromyces lactis strains selected through artificial mutagenesis can better tolerate adverse environmental influences during processing and storage, maintain high activity, and produce high levels of enzymes during fermentation. Therefore, the selection of high-quality strains through mutagenesis has become a research hotspot.

[0005] Currently, physical, chemical, and biological mutagenesis are the three most common mutagenesis methods. Ultraviolet (UV) mutagenesis, a form of physical mutagenesis, is a common breeding method for inducing and screening superior strains. UV mutagenesis is widely used due to its advantages, including simple equipment, high mutagenesis efficiency, and safe and easy operation. The mutagenic effects of UV on various microorganisms vary significantly depending on the strain. Researchers have used UV mutagenesis to select and breed numerous superior microbial strains, which are now being applied across various industries.

[0006] Due to ultraviolet rays or other physical reasons, some people develop facial pigmentation as they age, affecting their appearance. Therefore, there is a constant demand for skin-whitening products, especially among Asian women. For a long time, most Asian countries, including India, have upheld the idea that "white is beautiful." A World Health Organization (WHO) survey found that at least 40% of women in China, Malaysia, the Philippines, and South Korea use skin-whitening products, with the percentage being even higher in China.

[0007] Traditional whitening ingredients include kojic acid, hydroquinone, and glycolic acid. Although these ingredients have certain whitening effects, kojic acid and hydroquinone are easily decomposed by light and heat, and hydroquinones have a certain impact on the nervous system, causing some neurological symptoms such as dizziness, nausea, and drowsiness. In addition, some people are allergic to hydroquinones, and after topical application of these substances to the skin, a red rash will appear. Glycolic acid is an acidic substance extracted from fruit. After application to the skin, it may irritate the skin and mucous membranes and cause local redness or a burning sensation, especially for sensitive skin.

[0008] Insomnia is a common sleep disorder that can be caused by psychological stress, chronic pain, and medication. Insomnia is often accompanied by varying degrees of depression and anxiety, which can occur simultaneously, leading to decreased mental efficiency, memory loss, slowed reaction times, and autonomic nervous system dysfunction. This can lead to decreased immune function and memory loss, and increase the risk of diabetes, hyperglycemia, coronary heart disease, and Alzheimer's disease. In short, insomnia not only easily leads to organic diseases, but also weakens the immune system and causes greater physical exhaustion. Long-term insomnia is more likely to cause physical and psychological damage, and has become a social problem that seriously affects people's physical and mental health.

[0009] The main medications for insomnia include barbiturates, benzodiazepines, and non-benzodiazepines. These early sedative-hypnotic drugs can easily lead to dependence with long-term use. High doses often cause dizziness, fatigue, distraction, and decreased learning and memory abilities. Sudden discontinuation of long-term use can also lead to adverse reactions such as rebound or withdrawal symptoms. Consequently, researchers are continuously pursuing new sedative-hypnotic drugs with fewer adverse reactions. Summary of the Invention

[0010] Purpose of the invention: To provide a new substance produced during the mutagenesis of Kluyveromyces lactis and its application.

[0011] Technical solution: The applicant accidentally discovered a new substance in the fermentation broth of a UV-induced mutant of Kluyveromyces lactis, which has excellent whitening and sedative activities.

[0012] Specifically, in the first aspect, the present application provides an N-acetylamino alcohol compound, the chemical formula of which is shown in Formula I.

[0013]

[0014] According to the compound naming rules, the chemical name of the compound of formula I is: acetylamino-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-O-glucitol.

[0015] Preferably according to the present invention, the molecular weight of the compound of formula I is 547.51 as analyzed by LC-MS.

[0016] According to the present invention, preferably, the H-NMR spectrum of the compound of formula I is: 1 H NMR (600MHz, DMSO) δ (ppm): 4.615 (d, J=12.6Hz, 1H:OH), 4.414 (d, J=12Hz, 1H:OH), 3.939 (t, 1H), 3.924 (t, 1H:O H), 3.851(d,J=18.6Hz,1H:OH), 3.775(m,1H), 3.764(m,1H:OH), 3.705(m,1H), 3.618(m,1H:OH), 1.975(s,3H).

[0017] C-NMR spectrum of the compound of formula I: 13 C NMR (150MHz, DMSO) δ (ppm): 22.27, 175.09, 82.16, 104.48, 63.20, 63.56, 102.88.

[0018] In a second aspect, the present application provides a method for preparing the compound of formula I, which comprises the step of ultraviolet induction of Kluyveromyces Lactis.

[0019] In one embodiment, the Kluyveromyces lactis is a starting strain, including but not limited to natural strains or genetically modified strains such as Kluyveromyces lactis.

[0020] Preferably, the Kluyveromyces lactis starting strain includes but is not limited to the Kluyveromyces lactis strain with a deposit number of CCTCC NO: M 2022118.

[0021] The strain obtained by UV induction is Kluyveromyces lactis, which was deposited in the China General Microbial Culture Collection (Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences) on January 26, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 29793.

[0022] The method for preparing the compound of formula I preferably further comprises the step of fermenting in a culture medium using Kluyveromyces lactis CGMCC No. 29793 (KL-LNTIIto1-02).

[0023] Preferably, the fermentation medium includes, but is not limited to, lactose and one or more of glucose, fructose, sucrose or maltose.

[0024] Preferably, the culture medium includes but is not limited to glucose, or a mixture of glucose and sucrose.

[0025] Preferably, in the method for preparing the compound of formula I, the culture medium contains but is not limited to metal ions.

[0026] Preferably, the metal ions include but are not limited to dipotassium hydrogen phosphate.

[0027] Preferably, the metal ions include, but are not limited to, one or more of magnesium sulfate, ammonium sulfate and manganese sulfate.

[0028] Preferably, the method for preparing the compound of formula I further comprises the step of recovering the fermentation product.

[0029] The step of recovering the fermentation product includes separation and purification steps. The compound of formula I can be purified by chromatography, anion and cation resin separation, chromatography column separation, and other methods known in the art.

[0030] In a third aspect, the present application also provides the use of the compound of formula I in whitening.

[0031] The applicant unexpectedly discovered that the compound of formula I can inhibit the formation of tyrosinase, thereby achieving the efficacy of whitening and lightening spots. The compound of formula I is safe, non-irritating, environmentally friendly, and stable to light and heat, and can be used for non-diagnostic skin whitening purposes.

[0032] In some embodiments, the use of the compound of formula I in preparing a product having a whitening function is described. The whitening function is the function of inhibiting tyrosinase activity.

[0033] The whitening function is whitening for non-medical purposes, including whitening for cosmetic purposes.

[0034] The use of the compound of formula I in the preparation of a product with whitening function, preferably, the product is a cosmetic with the function of inhibiting tyrosinase activity.

[0035] The present invention further provides a composition comprising a compound of formula I. The composition has the function of inhibiting tyrosinase activity.

[0036] Cosmetic compositions containing a compound of Formula I can be selected from aqueous solutions, oils, emulsions, gels, or pastes, among others. The compound of Formula I can be formulated with any other excipients or combined with other functional ingredients, without particular requirements. Production can be organized according to cosmetic technical specifications to produce the desired cosmetic.

[0037] In a fourth aspect, the present application provides the use of the compound of formula I in the preparation of a sedative drug.

[0038] In a fifth aspect, the present application provides the use of a compound of formula I in the preparation of a drug for improving sleep.

[0039] In a sixth aspect, the present application provides a composition containing a compound of formula I.

[0040] The composition has a sedative or sleep-improving effect.

[0041] The calming effect is reflected in the ability to relieve people's depression and anxiety.

[0042] The composition of the present invention can be prepared into oral dosage forms such as capsules, tablets or granules using conventional formulation techniques.

[0043] The application can be taken by people in a suitable dosage. Preferably, the daily dosage per kg body weight is in the range of 5-15 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 .HPLC spectrum of the reaction solution of Example 1.

[0045] Figure 2 . Figure 1 LC-MS spectrum of the substance with rt=14.094min in the HPLC spectrum.

[0046] Figure 3 . Figure 1 The substance with rt=14.094min in HPLC spectrum 13 C-NMR spectrum.

[0047] Figure 4 . Figure 1 The substance with rt=14.094min in HPLC spectrum 1 H-NMR spectrum.

[0048] Beneficial effects:

[0049] The present invention provides a compound of Formula I. The compound of Formula I exhibits stable performance, safety, and is skin- and environmentally friendly. It exhibits a high ability to inhibit tyrosinase activity and can be added to cosmetics as a functional ingredient to prevent melanin production caused by excessive tyrosinase activity, thereby enabling use in products with whitening properties. The compound of Formula I also exhibits excellent sedative and sleep-improving properties and can be used to improve sleep, combat anxiety, and relieve restlessness. DETAILED DESCRIPTION

[0050] Example 1. Generation of strains

[0051] (1) Preparation of bacterial suspension

[0052] a) Take 4-5 slants of Kluyveromyces lactis HLLWF3 (deposited with the China Center for Type Culture Collection by Shandong Henglu Biotechnology Co., Ltd. on February 16, 2022, with accession number CCTCC NO: M2022118) cultured for 48 hours, wash off the bacterial moss with sterile saline, place in a small triangular flask containing glass beads, and shake for 30 minutes to break up the bacterial clumps;

[0053] b) centrifuging the bacterial suspension (3000 rpm for 15 minutes), discarding the supernatant, and washing the bacteria 2-3 times with sterile saline to prepare a bacterial suspension;

[0054] (2) Flat panel production

[0055] After the starch agar medium is dissolved, pour it into a plate when it cools to about 55℃ and set aside after solidification.

[0056] (3) Ultraviolet treatment

[0057] a) Turn on the UV lamp and preheat for about 20 minutes;

[0058] b) Take one set of 6 cm diameter sterile petri dishes, add 5 mL of the above bacterial suspension, and place a sterile stirring rod into the dish;

[0059] c) Place the plate containing the bacterial suspension on a magnetic stirrer and irradiate with a 15W UV lamp at a distance of 30 cm for 3 minutes.

[0060] d) Under red light, the above mutagenic bacterial suspension was diluted to 10 -1 -10 -6 Bacterial liquid.

[0061] (4) Coating the flat plate

[0062] Take 10 -4 , 10 -5 , 10 -6 Plate three dilutions of the bacterial solution, coating three plates for each dilution. Add 0.1 ml of the diluted bacterial solution to each plate and spread evenly with a sterile glass spreader. Repeat the same procedure with a plate containing the untreated bacterial solution as a control.

[0063] (5) Preservation

[0064] The mutagenized strain was sent to the China Center for Type Culture Collection for preservation. The strain was named: Kluyveromyces lactis, the preservation date was January 26, 2024, and the preservation number was: CGMCC No.29793.

[0065] Example 2. Synthesis of compounds of formula I

[0066] 1. Synthesis of Compounds of Formula I

[0067] The first stage is the bacterial growth period: the starting strain Kluyveromyces lactis (CCTCC NO: M 2022118, control) and the ultraviolet mutagenesis strain CGMCC No. 29793 obtained in Example 1 were taken out respectively, and the cells were cultured with glucose as the carbon source to accumulate the bacterial mass and enzyme amount respectively until the cell growth entered the late logarithmic phase or the stable phase. The strain was streaked and cultured in a solid culture medium. After culturing at 30°C for 2-3 days, a single colony was picked and inoculated into 1.5mL liquid culture medium, and cultured at 30°C and 200rpm overnight. Subsequently, it was inoculated into a 50mL liquid culture medium shake flask at a 2% inoculum size and cultured at 30°C and 200rpm until the OD 600 =1, inoculated into 5 L liquid culture medium (10 L fermentor) at a 2% inoculum size, and cultured at 30°C and 200 rpm for bacterial cell accumulation.

[0068] The second stage was the product synthesis phase: culture was shaken at 30°C and 200 rpm for 40 hours, at which time feeding was initiated (the amount of feeding was recorded). The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, the resulting liquid was disrupted using a high-pressure homogenizer, and then centrifuged to remove protein. The resulting liquid was filtered through a 0.22 μm filter to remove impurities, resulting in the fermentation broth.

[0069] The specific conditions of the culture medium in this embodiment are as follows:

[0070] Solid culture medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar powder, sterilized by high-temperature steam at 115°C for 30 min before use.

[0071] Liquid culture medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 8 g / L lactose, and final concentrations of 5 mM each of dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, and manganese sulfate. Sterilize with high-temperature steam at 115°C for 30 min before use.

[0072] Feed: 2 g / L lactose, 20 g / L glucose, and final concentrations of 5 mM each of dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, and manganese sulfate.

[0073] The test results of the fermentation broth showed that:

[0074] After culturing the starting strain Kluyveromyces lactis (CCTCC NO: M 2022118), no compound of formula I was found to be produced. However, the fermentation broth of the mutagenized strain CGMCC No. 29793 obtained in Example 1 contained compound I at a concentration of 7.7 g / L, and the lactose conversion rate was 48.49%.

[0075] 2. Purification of fermentation broth

[0076] The fermentation broth was decolorized and desalted by ion resin (DuPont UP6150), and then purified by chromatography resin to obtain a purified solution;

[0077] The purified solution was run on a semi-preparative liquid chromatograph to collect the target substance. The detection conditions were as follows: Hanbang semi-preparative liquid chromatograph, COSMOSIL Sugar-D Packed Column (20.0 mm ID × 250 mm), column temperature 30°C, flow rate 10 mL / min, injection volume 1 mL, elution time 30 min, mobile phase 65% acetonitrile / water, isocratic elution, and the substance with a peak elution time of 14 min was collected.

[0078] The material collected on the semi-preparative liquid chromatograph was concentrated by rotary evaporation at 50°C, then re-dissolved with a small amount of pure water, placed at -10°C overnight, filtered, and the filter cake was washed with 60-70% ethanol aqueous solution pre-cooled at 0°C, and dried at 60°C to obtain a solid powder.

[0079] 3. Analysis and identification of the fermentation broth and solid powder obtained above

[0080] (1) HPLC analysis

[0081] Chromatographic conditions: The HPLC analyzer was a 940 ion chromatograph, the chromatographic column was a MetroSep (Carb2 4.0 mm × 250 mm), the column temperature was 40°C, the flow rate was 0.5 mL / min, the injection volume was 20 μL, the elution time was 60 min, the mobile phase was 200 mM NaOH + 2 mM NaAc, the elution was isocratic, and the analysis was performed using an amperometric detector in PAD mode.

[0082] According to the above chromatographic conditions, the fermentation broths obtained in Examples 1-2 were subjected to HPLC analysis.

[0083] The HPLC spectrum of fermentation broth is shown in Figure 1 . Figure 1 It was shown that the peak time rt of the product obtained by fermentation of the UV-mutated strain in Example 1 was 14.094 min.

[0084] The HPLC spectrum of the solid powder showed that the peak time was the same as that in Example 1.

[0085] (2) LC-MS analysis conditions:

[0086] LC-MS mass spectrometry analysis conditions: positive ion mode detection, ion source temperature of 230°C, quadrupole temperature of 180°C, ion energy of 4.0 eV, scan range of 50-2000 m / z, and solvent delay of 5 min.

[0087] According to the above LC-MS mass spectrometry analysis conditions, the substance with a peak time of about 14.094 min in the HPLC spectrum of the fermentation broth of Example 1 was analyzed, and its molecular weight was 547.51, which was consistent with the theoretical molecular weight of the compound of Formula I.

[0088] LC-MS mass spectrum showed that the molecular weight of the substance with peak time rt=14.094 min in the solid HPLC spectrum of the compound of formula I obtained in Example 2 was the same as that in Example 1, 547.51.

[0089] (3) NMR analysis

[0090] H-NMR spectrum detection conditions:

[0091] 1 H and 13 C NMR spectra were recorded on an Agilent DD2-600 spectrometer. 1 H at room temperature 600MHz, 13 C at 150 MHz. The chemical shifts of tetramethylsilane in D2O are expressed in parts per million (ppm). Chemical shifts and coupling constants were calculated from first-order analysis of the spectra.

[0092] The substance with a peak time of about 14.094 min in the HPLC spectrum was subjected to nuclear magnetic resonance analysis.

[0093] H-NMR spectrum: 1 H NMR (600MHz, DMSO) δ (ppm): 4.615 (d, J = 12.6Hz, 1H: OH), 4.414

[0094] (d,J=12Hz,1H:OH), 3.939(t,1H), 3.924(t,1H:OH), 3.851(d,J=18.6Hz,1H:OH) , 3.775(m,1H), 3.764(m,1H:OH), 3.705(m,1H), 3.618(m,1H:OH), 1.975(s,3H).

[0095] C-NMR spectrum: 13C NMR (150MHz, DMSO) δ (ppm): 22.27, 175.09, 82.16, 104.48, 63.20, 63.56, 102.88.

[0096] In summary, after analysis, the substance with a peak time of about 14.094 min in the HPLC spectrum is the compound of formula I, and its chemical formula is as follows:

[0097]

[0098] Example 3-6. Effect of carbon source on fermentation reaction

[0099] The fermentation method described in Example 2 was followed, but glucose in Example 2 was replaced with fructose, sucrose, maltose, or a mixture of glucose and sucrose to investigate the effects of different carbon sources on the fermentation reaction. The results are reported in Table 1.

[0100] Table 1

[0101]

[0102] The data in Table 1 illustrate that: (1) The compound of formula 1 was produced in all fermentation reactions using sucrose, fructose, maltose, and a mixture of glucose and sucrose as carbon sources; among them, the lactose conversion rate was the highest in the reaction using a mixture of glucose and sucrose as carbon source.

[0103] (2) In the reaction using a mixture of glucose and sucrose as the carbon source, the mass ratio of glucose to sucrose in the culture medium was 1:1.

[0104] Examples 7-10. Effects of Metal Ions on Fermentation Reactions

[0105] According to the method described in Example 2 and the fermentation method described in Table 2, the amounts of dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, and manganese sulfate were adjusted to conduct fermentation experiments to investigate the effects of metal ions on the fermentation reaction. The results are recorded in Table 2.

[0106] Table 2

[0107]

[0108] The data in Table 2 illustrate: (1) In the fermentation medium, the medium in Example 7 contains dipotassium hydrogen phosphate, magnesium sulfate and ammonium sulfate, and the conversion rate of lactose in the fermentation reaction is the highest, indicating that the addition of dipotassium hydrogen phosphate, magnesium sulfate and ammonium sulfate can improve the conversion rate of the fermentation reaction.

[0109] (2) When the culture medium contains dipotassium hydrogen phosphate, magnesium sulfate, and ammonium sulfate, the appropriate molar concentration ratio of dipotassium hydrogen phosphate, magnesium sulfate, and ammonium sulfate is 1:1:1.

[0110] Example 11. Tyrosinase activity inhibition experiment

[0111] Materials and reagents are shown in Table 3.

[0112] Table 3. Materials and reagents

[0113] Reagent name Reagent type purity factory Tyrosinase / >1350 u / mg Beijing Coolaibo Technology Co., Ltd. Levodopa / 99％ Shanghai MacLean Biochemical Technology Co., Ltd. Disodium hydrogen phosphate analytically pure / Sinopharm Chemical Reagent Co., Ltd. Citric acid analytically pure / Sinopharm Chemical Reagent Co., Ltd. lactose analytically pure / Macklin's reagent

[0114] Instrument: 752N UV-visible spectrophotometer, Shanghai Youke Instrument Co., Ltd.

[0115] Solution preparation:

[0116] (1) Lactose solution preparation: Prepare a 1 mg / mL aqueous solution according to conventional methods.

[0117] (2) Preparation of solution of compound of formula I: The solid powder obtained in Example 2 was prepared into a 1 mg / mL aqueous solution according to conventional methods.

[0118] (3) Preparation of disodium hydrogen phosphate-citrate buffer:

[0119] a) preparing a 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L citric acid aqueous solution;

[0120] b) Mix 154.5 mL of a sodium hydrogen phosphate aqueous solution and 45.5 mL of a citric acid aqueous solution, and adjust the pH to 6.8 (measure with a pH meter; 0.2 mol / L sodium hydrogen phosphate or 0.1 mol / L citric acid can be used to adjust the pH).

[0121] (4) Preparation of tyrosinase solution: Prepare 100 u / mL tyrosinase solution using the above-mentioned sodium hydrogen phosphate-citrate buffer.

[0122] (5) Preparation of levodopa solution: Prepare 1 mg / mL levodopa solution using the above-mentioned sodium hydrogen phosphate-citrate buffer.

[0123] The specific experimental plan is as follows:

[0124] (1) Take 1 mL of the compound of formula I and 1 mL of lactose, add 0.5 mL of tyrosinase to each, mix well, and bathe in a 37°C water bath for 10 min. Then add 2 mL of levodopa to each, control the reaction time to 5 min, and immediately measure the absorbance T at 475 nm.

[0125] (2) Sample background T0: Take 1 mL of the compound of formula I and 1 mL of lactose, add 0.5 mL of sodium hydrogen phosphate-citrate buffer, mix well, and bathe in a 37°C water bath for 10 min. Then add 2 mL of levodopa and control the reaction time to 5 min. Immediately measure the absorbance at 475 nm.

[0126] (3) Enzyme reaction tube C: Take 1 mL of sodium hydrogen phosphate-citrate buffer, add 0.5 mL of tyrosinase, mix well, and incubate in a 37°C water bath for 10 min. Then add 2 mL of levodopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

[0127] (4) Solvent background C0: Take 1.5 mL of sodium hydrogen phosphate-citrate buffer, incubate in a 37°C water bath for 10 min, add 2 mL of levodopa, control the reaction time to 5 min, and immediately measure the absorbance at 475 nm.

[0128] The inhibition rate of tyrosinase activity was calculated according to the following formula. The results are shown in Table 4.

[0129]

[0130] Table 4. Inhibition rate of tyrosinase activity

[0131]

[0132] Table 4 shows that:

[0133] The compound of formula I has a significant inhibitory effect on tyrosinase activity, indicating that the technical solution of the present invention produces outstanding technical effects.

[0134] Example 12. Study on the Effect of Compound I on the Inhibition Rate of Spontaneous Activity in Rats

[0135] The solid powder of the compound of formula I obtained in Example 2 was taken and a 1 g / L sample solution was prepared with distilled water.

[0136] Animals: 36 Wistar rats, 10 months old, half male and half female, weighing 300±20g, were weighed and recorded, and randomly divided into two groups, 18 rats in each group, namely the control group and the experimental group.

[0137] Experimental methods:

[0138] Before drug administration: the Wistar rats in the control group and the experimental group were placed in the same box respectively, and the number of walking times of the two groups of Wistar rats within 30 minutes was observed and recorded to obtain the spontaneous activity number of the Wistar rats before drug administration.

[0139] Dosage:

[0140] The control group was given distilled water by gavage at 10 mg / kg body weight.

[0141] The experimental group was given the sample solution by gavage at 10 mg / kg body weight.

[0142] After oral administration, the Wistar rats in the control group and the experimental group were placed in the same box respectively, and the number of walking times of the two groups of Wistar rats within 30 minutes was observed and recorded to obtain the spontaneous activity number of the Wistar rats after drug administration.

[0143] The number of spontaneous activities and the inhibition rate of spontaneous activities of Wistar rats within 30 minutes before and after administration are shown in Table 5.

[0144] The spontaneous activity inhibition rate of each Wistar rat was calculated and the average value was taken as the spontaneous activity inhibition rate of each group. The results are recorded in Table 5.

[0145] Table 5

[0146] Before gavage After gavage Spontaneous activity inhibition rate, % experimental group 842.6±84.6 364.2±94.2 56.78 control group 848.8±87.2 836.4±107.4 1.46

[0147] Explanation: (1) Spontaneous activity inhibition rate = 100% × (number of spontaneous activities of rats before administration - number of spontaneous activities of rats after administration) / number of spontaneous activities of rats before administration.

[0148] (2) Spontaneous activity is a physiological characteristic of normal animals. The amount of spontaneous activity often reflects the state of central nervous system excitation or inhibition. Central nervous system depressants such as sedatives and hypnotics can significantly reduce the spontaneous activity of rats. This experiment aims to observe the effect of the compound of formula I on the spontaneous activity of rats to analyze whether the compound of formula I exhibits an excitatory or inhibitory effect.

[0149] The data in Table 5 illustrate that the spontaneous activity inhibition rate of the rats in the test group was significantly higher than that in the control group, indicating that the compound of formula I of the present invention has a certain sedative effect and can be developed as a sedative drug.

[0150] Example 13. Effects of the compound of formula I on sleep time and sleep latency in mice

[0151] This experiment investigated the effect of the compound of formula I on improving the sleep of sleep animals by conducting an experiment on the effect of the compound of formula I on the sleep time and sleep latency of mice induced by sodium pentobarbital.

[0152] Male ICR mice weighing 22-28 g were randomly divided into a control group, a compound of formula I group 1 and a compound of formula I group 2, with 15 mice in each group. The mice were given the drug by gavage, and the administration time was recorded. 20 minutes after the administration, sodium pentobarbital (45 mg / kg) was injected intraperitoneally.

[0153] The experimental groups 1 and 2 were administered with the compound of formula I solution at 10 mg / kg and 20 mg / kg body weight, respectively, by oral gavage. The compound of formula I solution was prepared into solutions of the same volume and then administered by oral gavage.

[0154] Control group: The same volume of distilled water was administered orally.

[0155] The mice were recorded for sleep latency and sleep duration, with the disappearance of the righting reflex as the time of falling asleep and the recovery of the righting reflex as the time of waking. The results are recorded in Table 6.

[0156] Table 6

[0157] Group Sleep latency, min Sleep time, min control group 5.9±0.4 60.7±9.3 Experimental group 1 4.5±0.2 78.1±11.6 Experimental Group 2 3.8±0.2 99.6±9.4

[0158] The experimental results, shown in Table 6, show that oral administration of 10 mg / kg and 20 mg / kg of the compound of Formula I significantly prolonged the total sleep duration of mice induced by sodium pentobarbital (P < 0.05) and significantly shortened the sleep latency of mice in a dose-dependent manner. This suggests that the compound of Formula I has a sleep-inducing effect and can be developed as a drug for improving sleep.

[0159] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be implemented. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. A Kluyveromyces lactis strain, characterized in that The deposit number is CGMCC No.29793.

2. A method for preparing a compound of formula I, characterized in that: The method comprises the steps of fermenting Kluyveromyces lactis strain CGMCC No. 29793 in a culture medium; 3. The preparation method according to claim 2, characterized in that: The method also includes the step of recovering the fermentation product.

4. The preparation method according to claim 2, characterized in that: The culture medium includes lactose and one or more of glucose, fructose, sucrose or maltose.

5. The preparation method according to claim 2, characterized in that: The culture medium includes glucose or a mixture of glucose and sucrose.

6. The preparation method according to claim 2, characterized in that: The culture medium contains metal ions.

7. The preparation method according to claim 6, characterized in that: The metal ion includes dipotassium hydrogen phosphate.

8. The preparation method according to claim 6, characterized in that: The metal ions include one or more of magnesium sulfate, ammonium sulfate and manganese sulfate.

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