CN114557995A - Application of dihydromyricetin in preparation of medicine for improving and/or treating circadian rhythm disorder and pharmaceutical preparation - Google Patents

Abstract

The invention provides an application of dihydromyricetin in preparing a medicine for improving and/or treating circadian rhythm disorder and a pharmaceutical preparation thereof, which can improve circadian activity/sleep and various physiological metabolism abnormalities caused by circadian rhythm disorder, increase circadian rhythm amplitude, improve sleep, and improve oxidative stress injury and sugar metabolism abnormality caused by circadian rhythm disorder.

Description

Application of dihydromyricetin in preparation of medicine for improving and/or treating circadian rhythm disorder and pharmaceutical preparation

Technical Field

The invention relates to the technical field of biology, in particular to application of dihydromyricetin in preparing a medicine for improving and/or treating circadian rhythm disorder and a pharmaceutical preparation.

Background

The behavior and physiological activity of most organisms have rhythmic fluctuations in a 24-hour period, called circadian rhythms, which are controlled by the body's biological clock. The periodic changes of animal behaviors and physiological biochemistry typically include behavior states, such as: a series of invisible diurnal oscillations in eating, sleeping, and in the functional activities of nerves, metabolism, endocrine, cardiovascular and immune functions. Circadian rhythms include the input pathway, the biological clock itself, and the output pathway. Inputting external environment factors such as light, temperature and other signals to the central biological clock of the brain, and synchronizing the process of the biological clock; the biological clock is a main pacemaker below the hypothalamic visual crossing supranucleus and is assisted by the peripheral biological clock of peripheral tissues and organs; the output pathway is that the biological clock transmits a time signal to peripheral tissues, thereby generating circadian rhythms of physiology and behavior.

The mechanism of circadian rhythm regulation is the cell autonomous transcriptional translational feedback cycle (TTFLs) in mammals, the transcription factors CLOCK and BMAL1 drive the expression of Period (Per1/2) and Cryptochrome (Cry1/2), the protein products of which in turn feedback inhibit CLOCK and BMAL 1. Downstream of these four factors, there are thousands of genes under the control of biological clocks to coordinate tissue-specific metabolic and physiological functional oscillations [3 ].

Drosophila melanogaster has been a model organism, and genes that regulate circadian rhythms are highly conserved among drosophila melanogaster and mammals. The drosophila melanogaster is taken as a research object, so that the generation and running rules of circadian rhythms can be known at the genetic, molecular and neural circuit levels. Among the 289 human disease genes detected, 177 genes are homologous to Drosophila, and Drosophila model becomes the basis for the study of human-related diseases [4 ]. Research has shown that 80% of human genomes have corresponding homologous genes in Drosophila genomes, and due to these characteristics, Drosophila has become the currently accepted ideal model organism for studying circadian rhythms.

In modern society, circadian rhythm disorders, which are increasingly common in living habits and working pressures at night, make people actively or passively exposed to light at night when people should sleep, so that circadian rhythm disorder of activities/sleep and sleep deprivation are caused, and life, study and work of people are influenced. Long-term circadian rhythm disorders are more likely to cause cognitive impairment, immune function decline, endocrine disorders, and a range of cardiovascular diseases, and even affect the treatment effects of cardiovascular diseases. The pineal hormone melatonin is always produced at night, and the duration of its production and secretory attack depends directly on the length of the night. Because melatonin production is closely linked to the light-dark cycle, melatonin plays an important role in circadian rhythms, coordinating behavior and physiology to adapt to changes in the sun, the moon and the four seasons. The mechanisms involved in the antioxidant properties of melatonin are complex, but melatonin deficiency clearly leads to cell damage. This process is also associated with disorders of circadian rhythms. There is increasing evidence that circadian rhythms of physiological and cellular processes are synchronized to contribute to the health of organisms, to suppress cancer and premature aging, etc. The normal circadian rhythm is essential for maintaining a strong antioxidant defense of the body, and circadian rhythm disorders, where oxidative stress and inflammatory reactions occur.

Sugar is the main energy source for all living activities of organisms, and people's preference for sweet taste has already begun in the early stages of life, and does not fall to the level of adults until the love for sugar in the middle of adolescence. In the modern society, sugar has good mouthfeel, is cheap and easy to obtain, so that the sugar is more and more widely applied as an additive in the food industry, people hardly resist the temptation of the delicious foods such as high-sugar milk tea, fruit tea, desserts and the like, and particularly, the edible sweet food is taken as a mode for relieving pressure by young groups. The harm of high sugar diets has become a problem of social concern. Numerous studies have shown that excessive sugar intake increases the risk of caries, leading to obesity, cardiovascular disease, type 2 diabetes (T2DM), metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). Poor sleep duration and quality in children and adolescents is associated with weight gain-induced obesity, decreased insulin sensitivity, hyperglycemia, and generalized cardiometabolic risk factors.

Ampelopsis grossedentata (Ampelopsis grossedentata) belonging to the genus Vitaceae. Distributed in southwest, Shaanxi, Gansu, Hubei, etc. Vine tea is a substitute tea of a minority of people such as the Dong minority, the Miao minority, the Tu family and the like, has a long use history, is used for preventing and treating various diseases, has the effects of resisting inflammation, resisting oxidation, reducing blood sugar and the like, and is listed as a new resource food raw material in China at present. Ampelopsis grossedentata is rich in various flavonoid compounds, wherein Dihydromyricetin (DHM) is a main flavone component of ampelopsis grossedentata, and the content of Dihydromyricetin can be up to more than 40%. Recent studies have shown that dihydromyricetin plays an important role in various pharmacological activities such as antioxidant, anti-inflammatory, antibacterial, anticancer activities, lipid and glucose metabolism regulation activities, and the like. DHM can ameliorate memory impairment caused by acute sleep deprivation, the underlying mechanism of which can be attributed to its ability to reduce oxidative stress and restore synaptic plasticity; studies in terms of longevity have shown that DHM reduces gut dysfunction and prolongs the life of fruit flies, and can also reduce pERK and pAKT signaling, thereby activating FOXO and AOP to modulate longevity; by improving glucose-related metabolism while lowering lipid levels in High Fat Diet (HFD) -fed rats; DHM may improve cognitive dysfunction in type 2 diabetic mice through hypoglycemic effects and activation mechanisms of BDNF protein expression in hippocampus. The DHM can improve the liver injury of mice induced by carbon tetrachloride and provide a potential therapeutic drug for chronic injury.

In summary, circadian rhythm disorders are an abnormal lifestyle to which people are currently experiencing, and people always experience disturbed rhythms actively and passively in work and life, which also poses a great threat to people's health.

Disclosure of Invention

The invention mainly aims to provide application of dihydromyricetin in preparing a medicine for improving and/or treating circadian rhythm disorder and a medicinal preparation, and aims to develop a medicinal preparation for improving circadian rhythm and relieving damage caused by circadian rhythm disorder.

In order to realize the purpose, the invention provides an application of dihydromyricetin in preparing a medicine for improving and/or treating circadian rhythm disorder.

Optionally, the medicament for ameliorating or treating a circadian rhythm disorder comprises one or more pharmaceutically acceptable carriers.

In order to achieve the purpose, the invention provides application of dihydromyricetin in preparing a medicament for improving and/or treating oxidative stress caused by circadian rhythm abnormality.

In order to achieve the purpose, the invention provides application of dihydromyricetin in preparing a medicament for improving and/or treating abnormal glucose metabolism caused by abnormal circadian rhythm.

In order to achieve the above object, the present invention provides a pharmaceutical preparation comprising dihydromyricetin.

The invention utilizes dihydromyricetin as a new medicine for improving or treating circadian rhythm disorder and has good efficacy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other relevant drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a statistical plot of the number of fruit fly diurnal events following dietary intervention with DHM in example 1 of the present invention;

FIG. 2 is a statistical graph showing the number of circadian activities of fruit flies in example 1 of the present invention in the case of normal rhythm and abnormal rhythm;

FIG. 3 is a statistical chart of the effect of DHM addition on Drosophila GSH content in example 2 of the present invention;

FIG. 4 is a statistical graph of the effect of DHM on Drosophila glucose content in example 3 of the present invention;

FIG. 5 is a statistical chart of the effect of DHM on Drosophila longevity in example 4 of the present invention;

FIG. 6 is a statistical graph (one) of the number of fruit fly diurnal activities of DHM on HSD diet in example 5 of the present invention;

FIG. 7 is a statistical plot of the number of fruit fly diurnal activities on HSD diet of example 5 DHM of the present invention (second);

FIG. 8 is a schematic representation of the effect of DHM on HSD drosophila metabolism in example 5 of the present invention;

FIG. 9 is a schematic representation of the effect of DHM on intestinal epithelial cells of Drosophila melanogaster eating a HSD in normal rhythm in example 5 of the present invention;

FIG. 10 is a statistical chart of the effect of DHM on gut epithelial cells of Drosophila melanogaster diet in normal rhythm in example 5 of the present invention;

FIG. 11 is a graph showing the effect of DHM on gut division cells of Drosophila melanogaster diet in normal rhythm in example 5 of the present invention;

FIG. 12 is a statistical graph showing the effect of DHM on gut division phase cells of Drosophila melanogaster diet in normal rhythm in example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments.

It should be noted that those whose specific conditions are not specified in the examples were performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Circadian rhythm disorders are an abnormal lifestyle that people are experiencing today, and people always experience disturbed rhythms actively and passively in work and life, which also poses a great threat to people's health. Therefore, the development of a food for improving the circadian rhythm and relieving the damage caused by the rhythm disorder is of great practical significance, and provides a choice for the health of people.

In view of the above, the present invention provides an application of dihydromyricetin in preparing a medicament for improving or treating circadian rhythm disorder.

Dihydromyricetin (DHM) is a main flavone component of Ampelopsis grossedentata, and the content of DHM can be up to more than 40%.

The invention utilizes dihydromyricetin as a new medicine for improving or treating circadian rhythm disorder and has good drug effect.

Preferably, the drug can be introduced into the body such as muscle, subcutaneous, intradermal, intravenous, mucosal tissue by physical or chemical means such as absorption, injection, permeation, etc.; or mixed or coated with other materials and introduced into body.

Preferably, the medicine for improving or treating circadian rhythm disorder may be prepared in various forms of injection, tablet, pill, powder, granule, capsule, oral liquid, etc. The above-mentioned medicines in various dosage forms can be prepared according to conventional preparation methods in the pharmaceutical field, and other dosage forms can also be adopted, and are not specifically limited herein.

It is to be noted that one or more pharmaceutically acceptable carriers including diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like, which are conventional in the pharmaceutical field, may be added to the drug for improving or treating circadian rhythm disorder, if necessary.

The dihydromyricetin provided by the invention has good application in preparing a medicament for improving and/or treating oxidative stress injury caused by circadian rhythm abnormality.

The dihydromyricetin provided by the invention has good application in preparing medicines for improving or treating abnormal glucose metabolism caused by abnormal circadian rhythm.

The invention provides a pharmaceutical preparation, which comprises dihydromyricetin.

The technical solutions of the present invention are further described in detail with reference to specific examples, which should be understood that the following examples are only illustrative and not limiting.

Example 1: verifying the improving effect of Dihydromyricetin (DHM) on fruit fly circadian rhythm disorder

(1) Material

Wild type Canton-s line black belly male fruit flies were used as experimental subjects (fruit flies were purchased from the SiBCB fruit fly resource library, the center for molecular science and innovation). The basal culture medium of the drosophila melanogaster is purchased from Qingdao high-tech industrial garden Haibo biotechnology, Inc., and the composition of the culture medium is shown in Table 1. Ampelopsis grossedentata extract containing 98% DHM was purchased from Hanzhong Sichuan grape plant and the content was determined by HPLC. DHM standards were purchased from beijing china quality inspection biotechnology limited.

TABLE 1 basal culture medium formulation for Drosophila

(2) Experimental methods

The method comprises the steps of monitoring the activity rule of fruit flies by using a fruit fly behavior monitor (model: YHDAMS, Wuhan Yiming science and technology Limited), putting each group of fruit flies in a fruit fly behavior analysis device, putting a culture medium at one end of a test tube with the diameter of 0.5cm, plugging the other end with cotton after the fruit flies are put in, putting one fruit fly in each test tube, and collecting the activity frequency of each fruit fly by a fruit fly activity monitor (DAM) system every minute. The behavior of the fruit flies was then analyzed in a fruit fly activity monitoring system based on their frequency of activity. The experiment changes the circadian rhythm of the fruit fly by setting different light/dark time lengths.

Description of Activity behavior: the fruit flies penetrate through the middle of the small tube every time, and the infrared ray automatically records the fruit fly movement once; a continuous 5 minute inactivity is considered as Sleep (Sleep), Sleep-1 means that all flies are inactive during the current 5 minute period, and Sleep-0 means that all flies have collected at least one activity during the current 5 minute period.

(3) Experimental grouping processing

Experiments were carried out with the drosophila divided into four groups, and the grouping and treatment methods for each group are shown in table 2.

Setting the illumination time of normal rhythm as 12h light-12 h dark cycle, the instrument automatically operates in 8:00 lamp on, 20: 00 turning off the lamp;

setting the illumination-dark duration of the dysrhythmia group as 19-5 h of dark cycle, namely 8:00 light on, 3 in the morning: 00 turning off the lamp. The rhythm condition of the fruit fly activity is judged by recording the activity times of the fruit flies through the fruit fly infrared monitoring module.

The preparation method of the basal drosophila culture medium comprises the following steps: weighing 20.87g of the mixed culture medium powder in the table, adding 100ml of sterile water, boiling and stirring until a viscous and uniform pasty solution is formed, cooling, adding 0.65ml of propionic acid, stirring uniformly, and subpackaging in sterilized drosophila bottles.

The preparation method of the dry pre-culture medium containing DHM comprises the following steps: to the basal medium, DHM was added to a final concentration of 1 mg/ml.

TABLE 2 Experimental groups

Note: the fruit fly is eclosion male 7-day-old black drosophila melanogaster

(4) Results of the experiment

1. Referring to tables E and F in FIG. 2, under the condition of abnormal rhythm (light-dark: 19-5 h), the fruit flies are in abnormal rhythm state of activity and sleep as the activation times of the fruit flies are increased with the increase of the light time, especially the activity is frequent in the off-light period (night), compared with the fruit flies with normal rhythm (light-dark: 12-12 h).

2. Referring to tables C to D in FIG. 1, tables E and F in FIG. 2, fruit flies at abnormal rhythms (19h-5h) with decreased activity initiation and decreased night activity after dietary intervention with DHM (see FIG. 2: tables E and F), and decreased activity and increased sleep during the light-off period (night) (see FIG. 1: tables C and D).

3. Referring to tables A and B in FIG. 1, in normal rhythms, the addition of DHM to the diet had a minor effect on the fruit fly's circadian activity, with reduced activity frequency only at the circadian time points, with the time points of reduction centered around the light/dark alternating points, and with the reduction in fruit fly activity frequency most pronounced before the end of the light exposure. The sleep tendency in the dark was consistent with that in the control group.

Taken together, it is suggested that DHM dietary intervention may modulate fruit fly circadian rhythm disorders towards normal rhythm.

Example 2: improving effect of DHM on oxidative stress of drosophila melanogaster with abnormal circadian rhythm

(1) Material

Experimental flies from example 1 were used as experimental samples. Phosphate Buffered Saline (PBS) was purchased from shanghai-derived leaf biotechnology limited. The reduced Glutathione (GSH) detection kit is purchased from Beijing Solaibao science and technology Limited.

(2) Experimental method

Determination of drosophila tissue reduced glutathione: taking a certain amount of fruit flies, after carbon dioxide anesthesia, washing fresh tissues for 2 times by PBS (phosphate buffer solution), then weighing 0.1g of fruit fly tissues, and homogenizing the tissues according to a method of the kit; centrifuging at 8000rpm and 4 deg.C for 10 min; taking supernatant, placing in 4 deg.C environment for testing, and storing at-80 deg.C (for 10 days) if the test cannot be completed temporarily, and detecting according to the method of kit.

(3) Results of the experiment

Referring to FIG. 3, under normal rhythm conditions, the addition of 1mg/ml DHM to the medium increased the levels of Drosophila GSH, but there was no significant difference (P > 0.05). Under dysrhythmia (19h-5h), GSH was significantly reduced (P <0.05), while after DHM was added, GSH was significantly increased (P <0.01), returning to normal levels.

In summary, the following steps: the addition of DHM can obviously improve the antioxidant capacity of fruit flies and relieve oxidative stress caused by abnormal circadian rhythm.

Example 3: improvement effect of DHM on sugar metabolism of drosophila with abnormal circadian rhythm

(1) Material

The experimental fruit fly of example 1 was used as the experimental sample. The glucose detection kit is purchased from Beijing Sorbao science and technology Co.

(2) Experimental methods

The drosophila glucose content is detected by a kit detection method. Namely: taking a certain amount of fruit flies, after carbon dioxide anesthesia, weighing 0.1g of tissues, adding 1ml of distilled water, grinding into homogenate, boiling in a boiling water bath for 10 minutes (tightly covering to prevent water loss), cooling, centrifuging at 8000g and 25 ℃ for 10min, and taking supernate for later use. Then glucose content detection is carried out according to a kit method.

(3) Results of the experiment

Referring to FIG. 4, the glucose levels in abnormal fruit flies with circadian rhythms were significantly elevated (P <0.01) compared to normal fruit flies, and the levels of fruit flies were significantly reduced (P <0.01) towards normal after addition of DHM.

In conclusion, DHM can significantly reduce high glucose level caused by abnormal circadian rhythm of fruit flies and improve sugar metabolism of the fruit flies.

Example 4: effect of DHM on improving the longevity of Drosophila

(1) Material

The same as in example 1.

(2) Experimental methods

And (3) placing the 7-10-day-old black-belly male drosophila melanogaster after eclosion in a corresponding culture medium, wherein each group contains 60 drosophila melanogaster/bottle and each group contains 3 flies in parallel, and recording death conditions of the drosophila melanogaster every day. The medium was changed every 4 days and the number of flies dead was recorded daily until all died. The survival rate of the flies (survival rate ═ number of flies surviving per day/total number of flies per bottle 100%) and median survival rate of the flies and the maximum life span of the flies (average of 10% of flies surviving per group) were calculated, respectively.

(3) Results of the experiment

Referring to fig. 5, DHM was added to a basal medium on a normal diet, and then observed for survival in a normal rhythm and an abnormal rhythm, respectively. The results show that the survival rate of the drosophila is improved after 1mg/ml of DHM is added into the basic culture medium. The maximum life of the fruit flies under normal rhythm (12h-12h) is improved by 9 percent compared with the control group, and the median life is improved by 26 percent; under abnormal rhythm (19h-5h), the maximum life span of the fruit fly is improved by 10 percent compared with that of a control group, and the median life span is improved by 13 percent.

In conclusion: DHM can prolong the life of normal and abnormal rhythm drosophila.

Example 5: improvement of circadian Activity of DHM on high-sugar diet Drosophila

(1) Material

The same as in example 1.

(2) Experimental methods

1. Study of circadian rhythm: the study procedure was as in example 1.

The experiment was divided into three groups:

(1) normal diet group (CON): the basal drosophila culture medium is adopted, and the preparation method is the same as that of the embodiment 1.

(2) High sugar diet group (HSD): to the basal medium, 30% sucrose was added.

(3) HSD diet intervention with DHM (HSD + DHM): to HSD medium, 1mg/ml DHM was added.

2. Glucose was measured as in example 3.

3. The triglyceride detection method comprises the following steps: taking a certain amount of drosophila melanogaster, after carbon dioxide anesthesia, weighing 0.1g of tissue, adding 1ml of absolute ethyl alcohol, grinding on ice to homogenate at 2500r/min, centrifuging for 10min, and taking supernatant for testing. The detection is carried out according to the method of the kit, and the kit is built into a product of a bioengineering research institute by Nanjing.

4. Total protein detection method: taking a certain amount of drosophila melanogaster, after carbon dioxide anesthesia, weighing 0.1g of tissue, adding 20 times of normal saline, grinding on ice to homogenate at 2500r/min, centrifuging for 10min, and taking supernatant for testing. The detection is carried out according to the method of the kit, and the kit is a product of Biyuntian biotechnology company.

5. The weight detection method comprises the following steps: the weight of the fruit flies was the average of the weight of 30 fruit flies.

(3) Results of the experiment

1. In terms of improvement of circadian rhythm: referring to Table A in FIG. 6, at normal rhythms (12h-12h light-dark cycle), the light is turned on at 8:00 and 20: when the lamp is 00 turned off, the average activity times of the fruit flies on HSD diet are obviously reduced compared with the fruit flies on normal diet, and the HSD + DHM group is partially improved compared with the HSD group, so that the activity times of the fruit flies in illumination can be increased, which is shown in 8: the average number of activities at 00 lights-on (peak activity) approached the normal diet group, and for 20: 00 the activity when the light is turned off has no effect.

This indicates that HSD diet can reduce the fruit fly activity frequency in day and night compared with normal diet, and the activity is in weakened state, especially the activity is reduced in two peak time of turning on and off the lamp. The addition of DHM can partially improve the activity frequency of fruit flies in HSD diet in the daytime, particularly obviously improve the activity frequency of the fruit flies in the light-on state, and does not influence the activity of the fruit flies at night.

2. Improvements in metabolism

Referring to fig. 8-12, high-glucose diet induced increased glucose and triglycerides in drosophila tissues (P <0.0001), weight loss (P <0.001), decreased total tissue protein (P <0.05), and increased number of intestinal epithelial cell deaths, increased stem cells in the division phase (indicating that high-glucose diet caused stress injury to the drosophila gut).

This indicates that DHM added to a high-sugar diet can significantly reduce the glucose content of drosophila, increase the weight of drosophila, and improve the state of intestinal stem cells. Indicating that DHM can improve intestinal injury and sugar metabolism abnormality caused by HSD diet.

In summary, the present invention provides a use of dihydromyricetin in improving circadian rhythm by using fruit fly as research object, specifically including improving effect of DHM on abnormal circadian rhythm of fruit fly, improving effect of DHM on abnormal circadian rhythm of fruit fly oxidative stress, improving effect of DHM on abnormal circadian rhythm of fruit fly sugar metabolism, improving effect of DHM on life span of fruit fly, and improving effect of DHM on high-sugar diet fruit fly circadian rhythm activity. The invention provides data support for the wide application of the new food raw material vine tea because the content of dihydromyricetin in the vine tea is the highest.

The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.

Claims (5)

1. An application of dihydromyricetin in preparing medicine for improving and/or treating circadian rhythm disorder is disclosed.

2. Use of dihydromyricetin in the manufacture of a medicament for ameliorating and/or treating a circadian rhythm disorder according to claim 1, wherein the medicament for ameliorating or treating a circadian rhythm disorder comprises one or more pharmaceutically acceptable carriers.

3. An application of dihydromyricetin in preparing medicine for improving and/or treating oxidative stress injury caused by circadian rhythm disorder is disclosed.

4. An application of dihydromyricetin in preparing medicine for improving and/or treating abnormal glucose metabolism caused by abnormal circadian rhythm is disclosed.

5. A pharmaceutical preparation characterized by comprising dihydromyricetin.

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