KR101472085B1 - Methods for zebrafish -Based Screenig Diabetes Therapeutics - Google Patents

Abstract

The present invention relates to a zebrafish-based screening method of a diabetes therapeutic agent and a pharmaceutical composition for preventing or treating diabetes. According to the present invention, a zebrafish-based screening system combining a fluorescent glucose-inducing factor, which is a fluorescent-tagged glucose analogue, with the experimental utility of an animal model of a zebrafish, is established to produce a candidate antidiabetic agent candidate There is an effect that detection can be performed quickly and conveniently. In addition, it is possible to conveniently and rapidly measure the amount of glucose-induced factor intake by measuring the amount of fluorescent glucose-derived factor that is ingested in the zebrafish, and the necessity of an ancillary facility and radioactive purification treatment due to the use of conventional radioactive glucose tracer There is an advantage to remove. In addition, the zebrafish-based system is capable of performing the accompanying toxicity analysis, being simple in housing, capable of ingesting glucose upon contact with zebrafish and fluorescent glucose inducer, thus eliminating the need for advanced techniques such as microinjection, There is an advantage in that input cost and time can be reduced in performing large-scale based screening.

Description

Methods for zebrafish-Based Screenig Diabetes Therapeutics < RTI ID = 0.0 >

The present invention relates to a zebrafish-based screening method of a diabetes therapeutic agent and a composition for treating, ameliorating or alleviating diabetes.

Insulin is the only drug developed to treat inflammatory-onset diabetes (type 1) or postprandial diabetes (type 2) (1). Although many drugs for type 2 diabetes have been developed, these agents are either accompanied by side effects (e.g., increased total mortality risk (2), hypoglycemia (3) and gastrointestinal disease (4)) or require subcutaneous delivery 5). Surgical intervention for diabetes is problematic. For example, pancreas transplantation for type 1 diabetes may be lacking in organ donors, and gastric bypass for type 2 diabetes may result in malnutrition (6). Thus, the discovery of novel compounds (in short, insulin mimetics) that outline the insulin effect is clinically urgently required.

The usual 'bottleneck' in the drug discovery process is the failure of candidate drugs in animal experiments. This is caused by problems related to ingestion, solubility, metabolic stability or toxicity (7, 8). These problems are a major cause of the slowing reported in the discovery of new drugs (9, 10). Screening of large-scale candidate drugs in small-established mammals is possible to address this problem. Small mammals, however, are not suitable for large-scale screening programs due to excessive cost and logistical problems (in short, additional staffing to provide guidance and technical support for the institutions that require housings in specialized facilities). As a result, small-size animal models that can benefit from high fertility and simple housing have been developed as screening tools for drug discovery studies (11). In addition, small organism-based drug screening is useful because drug target identification is unnecessary (9) and can facilitate the discovery of novel active compounds for validation in mammalian experimental models.

In the context of diabetes research, chemical library screening systems for anti-diabetic drugs have been developed based on the measurement of glucose uptake in cells (12, 13). This is accomplished by using two fluorescent-tagged glucose bioprobes (2-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol- In the development of glucose (2-NBDG) and 6-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] -D-glucose (6-NBDG) . This NBDG probe can be used in place of the radioactive glucose tracer and can provide a convenient and fast measurement of glucose flow rate (14). The previous concern about the dynamic behavior of unconventional NBDG probes by the demonstration of the affinity of the NBDG probe to the intracellular glucose transporter (GLUT: glucose transporter) is 300 times higher than that of free glucose (14, 15) It has been relaxed. Moreover, the temperature coefficient (Q10) of 6-NBDG uptake in cells was found to be similar to that of GLUT1 in red blood cells (much higher than Q10 for simple diffusion), indicating that simple diffusion plays a minor role in NBDG uptake (15).

Since animal models were established in the early 1980s, zebrafish ( Danio rerio ) has been developed as an important organism for drug screening (16). Zebrafish has significant advantages over other vertebrate models because it is screenable only in 96-well or 384-well plate formats (17). Moreover, the genome of zebrafish is approximately 80% homologous to the human genome and contains the Drosophila melanogaster (60%), the fruit fly, and the Caenorhabditis nematode elegans (36%) (18-20). In addition, zebrafish and humans share a similar mechanism in the regulation of glucose homeostasis (21, 22).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

We have combined the experimental utility of the NBDG probe, a zebrafish and glucose inducer, to establish a simple vertebrate-based screening system for the discovery of insulin mimetic compounds. As a result, glucose uptake regulators could be detected by fluorescence analysis of NBDG accumulation in homogenized zebrafish larvae and in the eye (relatively high GLUT expression), and these NBDG uptake Were able to inhibit competition for GLUT receptors. In addition, the present inventors confirmed that the system of the present invention is capable of detecting Maslinic acid, which is a compound identified to induce glucose uptake in the present invention, to provide a very simple, Resulting in an economical and effective zebrafish-based screening system.

Accordingly, an object of the present invention is to provide a screening method of a therapeutic agent for diabetes mellitus based on zebrafish.

It is another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diabetes.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a zebrafish-based screening method of a diabetes therapeutic agent comprising the steps of: (a) contacting a test substance with a zebrafish; (b) contacting the result of step (a) with a fluorescent glucose inducing factor; And (c) measuring the amount of the fluorescent glucose-derived factor uptake into the zebrafish through fluorescence measurement; If the test substance increases the uptake of the fluorescent glucose-inducing factor into the zebrafish, it is judged to be a therapeutic agent for diabetes.

We have combined the experimental utility of the NBDG probe, a zebrafish and glucose inducer, to establish a simple vertebrate-based screening system for the discovery of insulin mimetic compounds. As a result, glucose uptake regulators can be detected by fluorescence analysis of NBDG accumulation in homogenized zebrafish larvae and in the eye (a relatively high level of GLUT expression), and such NBDG uptake can be detected by the GLUT receptor It is possible to control the competition for. Further, the present inventors confirmed that the system of the present invention is capable of detecting maslinic acid, which is a compound identified to induce glucose uptake in the prior art, so that the zebrafish-based screening system of the present invention is useful as a novel anti-diabetic Drug candidates can be identified quickly, simply, economically, and effectively.

Accordingly, the present invention provides a screening method for identifying a compound capable of effectively preventing or treating diabetes using a glucose-glucose-inducing factor ingested into zebrafish.

The method of the present invention will be described in detail in each step as follows:

Step (a): Zebrafish and  Contact test material

First, the test substance is brought into contact with the zebrafish.

As used herein, the term " contact " means that a substance and a substance are in contact with each other. The method of bringing the test substance into contact with the test substance in the step (a) includes any case where the test substance can be brought into contact with the test substance. For example, Adding or administering the test substance to a test sample, a test tube, a test tube, a fish water, etc.).

According to one embodiment of the present invention, the zebrafish of the present invention is a zebrafish larva. According to another embodiment of the present invention, the zebrafish larval has an hour post fertilization (24-168 hpf), 24-144 hpf, 24-120 hpf, 48-120 hpf, 48-96 hpf, 60-96 hpf, 60 -84 hpf, 64-80 hpf or 68-76 hpf larvae.

According to one embodiment of the present invention, the test substance is contacted at a concentration of 1-15 mg / mL. According to another embodiment of the present invention, the concentration is 1-13 mg / mL or 1-11 mg / mL. According to some embodiments of the invention, the concentration is 3-15 mg / mL, 5-15 mg / mL or 8-15 mg / mL. According to a particular embodiment of the invention, the concentration is 3-13 mg / mL, 5-13 mg / mL, 8-13 mg / mL. In the present invention, the test substance has no toxic activity within the above concentration range.

Step (b): Fluorescent glucose Inducer and  contact

After carrying out step (a), the result of step (a) is brought into contact with a fluorescent glucose-inducing factor.

The method of contacting the resultant of step (a) with the fluorescent glucose-inducing factor in step (b) includes any case capable of bringing the result of step (a) into contact with the fluorescent glucose inducing factor, the test substance is added or administered to a medium in which the result of step (a) is present (for example, fish water such as E3 water), and the result of step (a) and the fluorescent glucose induction And putting the arguments in a state in which they can be contacted.

According to one embodiment of the present invention, the fluorescent glucose inducer of the present invention is a 2-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol- Glucose (2-NBDG) or 6-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] -D-glucose (6-NBDG). According to another embodiment of the present invention, the fluorescent glucose inducing factor is 2-NBDG.

The 2-NBDG and 6-NBDG of the present invention were developed in 1985 to study Glucose Transporter 1 (GLUT 1) as a fluorescent-tagged glucose analogue, and investigated glucose uptake in bacteria 2-NBDG was developed in 1996 to do so. The fate of 2-NBDG and 6-NBDG is dependent on intracellular uptake. 2-NBDG is rapidly degraded into non-fluorescent products after phosphorylation by hexokinase in the sugar chain pathway, but 6-NBDG is phosphorylated by hexokinase It accumulates in the cytoplasm in a fluorescent form. The chemical formula of 2-NBDG is C ₁₂ H ₁₄ N ₄ O ₈ , the chemical formula of 6-NBDG is C ₁₂ H ₁₄ N ₄ O ₈ , and the chemical structure is shown in FIG.

Since 2-NBDG and 6-NBDG exhibit typical excitation / emission spectra at about 465/540 nm even though they are environmentally sensitive, they can be visualized under a microscope using an optical filter for fluorescein.

According to one embodiment of the present invention, the fluorescent glucose-inducing factor of the present invention is contacted at a concentration of 100 μM-5 mM. According to another embodiment of the present invention, the concentration is 100 μM-3 mM, 100 μM-2.5 mM or 100 μM-2 mM. According to some embodiments of the present invention, the concentration is 200-5 mM, 300 μM-5 mM, 400 μM-5 mM, or 500 μM-5 mM. According to some embodiments of the present invention, the concentration is selected from the group consisting of 150 μM-3 mM, 150 μM-2.5 mM, 200 μM-2.5 mM, 200 μM-2 mM, 300 μM-2 mM, 400 μM- -2 mM, 500 μM-1.5 mM, 500 μM-1 mM, 500 μM-800 μM, or 500 μM-700 μM. According to a particular embodiment of the present invention, the concentration is selected from the group consisting of 500 μM-3 mM, 500 μM-2.5 mM, 550 μM-2.5 mM, 550 μM-2 mM, 550 μM-1.5 mM, 550 μM- -800 μM, 550 μM-700 μM, or 550 μM-650 μM.

According to one embodiment of the present invention, the contact of step (b) of the present invention is carried out for 0.1 to 20 hours. According to another embodiment of the present invention, the contacting of step (b) is carried out for 0.1 to 15 hours or 0.1 to 13 hours. According to some embodiments of the present invention, the contacting of step (b) is conducted for 0.5-20 hours, 0.5-15 hours, or 0.5-13 hours.

Step (c): Zebra fish  Ingestion uptake ) ≪ / RTI > fluorescent glucose Inducible  Measure amount

As a result of performing the step (b), the amount of the fluorescent glucose-derived factor taken into the zebrafish is measured by fluorescence measurement. When the amount of the fluorescent glucose-derived factor measured as a result of the measurement is increased in comparison with the amount of the fluorescent glucose-derived factor taken into the control group not in contact with the test substance, the test substance is judged to be a therapeutic agent for diabetes.

According to one embodiment of the present invention, the fluorescence measurement of the present invention is performed by measuring a fluorescence signal or a fluorescence image.

According to another embodiment of the present invention, the fluorescence signal is a fluorescence signal generated in the homogenized zebrafish. According to some embodiments of the present invention, the fluorescence signal is a fluorescence signal generated from the homogenized zebrafish larval. When fluorescence measurement is performed by the fluorescence signal measurement of the present invention, an increase in the amount of the fluorescent glucose-derived factor taken into the zebrafish indicates an increase in the fluorescence signal emitted from the glucose-derived factor taken into the zebrafish.

According to another embodiment of the present invention, the fluorescence image is a fluorescence image generated in the zebrafish eye. According to some embodiments of the present invention, the fluorescence image is a fluorescence image generated in a zebrafish larval eye. When fluorescence measurement is performed by the fluorescent image measurement of the present invention, an increase in the amount of the fluorescent glucose-derived factor ingested into the zebrafish indicates an increase in fluorescence intensity emitted from the glucose-derived factor into the zebrafish.

According to one embodiment of the present invention, the therapeutic agent for diabetes of the present invention is a therapeutic agent for post-viral diabetes (type 2 diabetes) caused by insulin resistance.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound of formula I or II; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating diabetes comprises:

Formula I

In the above formula (I), R ₁ is H; R ₂ is - (CONH) - [(CH ₂ ) _m -O] _n - (CH ₂ ) _p -NH ₂ wherein m, n and p are each an integer of 1 to 5; R ₃ is H; R ₄ is C ₇ -C ₁₆ aralkyl; The aryl group of the aralkyl may be substituted with halogen; R < ₅ > is H; R ₆ is C ₁ -C ₈ linear or branched alkyl, C ₂ -C ₁₀ Straight chain or branched chain alkenyl, aryl, C ₇ -C ₁₆ aralkyl group (aralkyl), or C ₃ -C ₁₀ cycloalkyl; The aryl may be substituted with halogen;

(II)

In the formula (II), A ₁ -A ₇ are each independently a C ₁ -C ₅ straight chain or branched chain alkyl; And A ₈ is a C ₁ -C ₅ straight or branched chain alkyl alcohol.

The term " C ₇ -C ₁₆ Quot; aralkyl " refers to C ₁ -C ₁₀ substituted by one or more phenyl rings at any position Means alkyl, for example, benzyl, 2-phenylethyl, 3-phenyl (n-prop-1-yl) (Yl), 3-phenyl (n-am (am) -2-yl (yl)), 3,3-diphenylpropyl and the like.

The term " aryl " as used herein refers to a substituted or unsubstituted monocyclic or polycyclic carbon ring which is totally or partially unsaturated, preferably monoaryl or biaryl. The aryl group includes, but is not limited to, a phenyl group, a substituted phenyl group, a naphthyl group, and a substituted naphthyl group.

The term " C ₁ -C ₈ Straight chain or branched chain alkyl "means a straight chain or branched chain alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert- Quot; means an alkyl group of a branched chain.

The term " C ₃ -C ₁₀ Cycloalkyl " means mono-ring or multi-ring saturated hydrocarbons of 3 to 10 carbon atoms and includes, for example, cyclopropyl ring, cyclobutyl ring, cyclobutyl ring, cyclohexyl ring , Or cycloheptyl, and the like. According to one embodiment of the present invention, the cycloalkyl of the present invention is a single ring saturated hydrocarbon of 3-6 carbon atoms, and according to certain embodiments of the present invention, the cycloalkyl of the present invention is cyclohexyl.

As used herein, the term " halogen " used in formula (I) denotes a halogen group element and includes, for example, F, Cl, Br and I. According to one embodiment of the present invention, the halogen of the present invention is F or Cl. According to another embodiment of the present invention, the aryl group of the C ₇ -C ₁₆ aralkyl of R ₄ of the present invention may be substituted with Cl. According to another embodiment of the present invention, the aryl of R < ₆ >

The term alkyl alcohol as used herein in formula II includes, but is not limited to, methanol, ethanol, propanol, butanol, and analogs thereof. According to one embodiment of the present invention, the alkyl alcohol of the present invention is methanol.

According to one embodiment of the present invention, R ₂ of formula (I) of the present invention is - (CONH) - [(CH ₂ ) _m -O] _n - (CH ₂ ) _p -NH ₂ And m, n and p are each an integer 2 according to a specific embodiment of the present invention.

According to one embodiment of the present invention, the compound of formula (I) of the present invention is a compound represented by the following formula (1).

Formula 1

In Formula 1, R < ₅ > is H; R ₆ is C ₁ -C ₈ linear or branched alkyl, C ₂ -C ₁₀ Straight chain or branched chain alkenyl, aryl, C ₇ -C ₁₆ aralkyl group (aralkyl), or C ₃ -C ₁₀ cycloalkyl; The aryl may be substituted with halogen.

According to another embodiment of the present invention, the formula (1) of the present invention is any one of the compounds represented by the following formulas (2-6).

(2)

(3)

Formula 4

Formula 5

6

According to one embodiment of the present invention, the formula (II) of the present invention is a compound represented by the following formula (7).

Formula 7

In Formula 7, A ₁ -A ₇ are each independently C ₁ -C ₅ straight chain or branched chain alkyl; And A ₈ is a C ₁ -C ₅ straight or branched chain alkyl alcohol.

According to another embodiment of the present invention, the formula (II) of the present invention is a compound represented by the following formula (8).

8

In Formula 8, A ₁ -A ₇ are each independently C ₁ -C ₅ straight chain or branched chain alkyl; And A ₈ is a C ₁ -C ₅ straight or branched chain alkyl alcohol.

According to another embodiment of the present invention, the formula (II) of the present invention is a compound represented by the following formula (9).

Formula 9

In Formula 9, A ₁ -A ₇ are each independently C ₁ -C ₅ straight chain or branched chain alkyl; And A ₈ is a C ₁ -C ₅ straight or branched chain alkyl alcohol.

According to a specific embodiment of the present invention, the formula (II) of the present invention is 20 (S) -ginenoside Rh2 represented by the following formula (10).

10

According to one embodiment of the present invention, the test substance screened by the method of the present invention has insulin mimetic activity (e. G., Activity to increase intracellular glucose uptake). According to another embodiment of the present invention, the cell in which the glucose uptake of the present invention is increased is mammalian adipocyte. According to some embodiments of the invention, the mammal of the invention is a mouse.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a zebrafish-based screening method for a diabetes therapeutic agent and a pharmaceutical composition for preventing or treating diabetes.

(b) The present invention establishes a zebrafish-based screening system that combines the fluorescent utility of fluorescent-glucose-tagged glucose analogues with the experimental utility of animal models of zebrafish, So that the substance can be detected quickly and conveniently.

(c) The present invention can conveniently and rapidly measure glucose-derived factor intake by measuring the amount of fluorescent glucose-derived factor that is ingested in its zebrafish, and the use of conventional radioactive glucose tracers to perform ancillary facilities and radioactive purification There is an advantage of eliminating the need for processing.

(d) The zebrafish-based system of the present invention can perform accompanying toxicity analysis, has a simple housing, can ingest glucose when the zebrafish is in contact with a fluorescent glucose inducer, and does not require a high technique such as microinjection , There is an advantage that cost and time of input can be reduced in performing vertebrate-based large-scale screening.

Figure 1 shows the chemical structure of the compounds disclosed in the present invention.
Figure 2 shows a dose-dependent study of 2-NBDG uptake in 72 hpf (hour post fertilization) zebrafish larvae. Figure 2a shows that glucose uptake can be visualized by fluorescence microscopy at 200 mM doses when treated with larvae for 3 hours with increasing doses of 2-NBDG. 2-NBDG fluorescence was significantly increased up to a dose of 1 mM. Figure 2b shows that 2-NBDG uptake in 72 hpf zebrafish larvae occurs in a dose-dependent manner and can be quantified by analysis of dissolved macrophage microplate reader. Figure 2c quantifies the 2-NBDG signal in the zebrafish eye, again showing dose-dependent 2-NBDG uptake from 200 mM.
Figure 3 shows a time-dependent study of 2-NBDG uptake in 72 hpf zebrafish larvae. Figure 3A shows that the 12 hour treatment of 2-NBDG, a fluorescent glucose probe that can be visualized by fluorescence microscopy, results in a time-dependent uptake. Figure 3b shows a fluorescence microplate reader assay of dissolved larvae and indicates that 2-NBDG uptake over a 12 hour period in 72 hpf zebrafish larvae can also be quantified by analysis of dissolved larvae fluorescence microplate reader. Figure 3c shows an image analysis of the 2-NBDG signal in the larval eye and indicates that the time-dependent uptake of the 2-NBDG signal in larval can also be quantified by image analysis of the 2-NBDG signal in the larval eye.
Figure 4 shows the results of treatment of 6-NBDG with larvae for 3 hours, and 6-NBDG uptake can not be visualized in 72 hpf zebrafish larvae by fluorescence microscopy. The 6-NBDG uptake image was obtained using the same microscope setup (exposure time: 500 ms; magnification: 50X; image software used for image acquisition: LAS 4.0 Leica Application Suite) as in the case of image analysis of 2-NBDG uptake Respectively.
Figure 5 shows a comparison of 2-NBDG uptake in 72 hpf and 120 hpf zebrafish larvae. For analysis, emodine, known as a glucose uptake inducer, was treated for 3 hours. Figure 5a shows fluorescence microscopy showing that for 72 hpf larvae, treatment with emodin increased 2-NBDG uptake. On the other hand, in the case of the 120 hpf larvae in Fig. 5B, a high level of 2-NBDG fluorescence appeared in the digestive tract, regardless of the treatment with emodin. Figure 5c shows a fluorescence microplate reader assay of dissolved larvae showing that EMODIN treatment at 72 hpf and 120 hpf zebrafish larval induces 2-NBDG uptake (* = P < 0.05 compared with DMSO treated larvae ). Figure 5d shows an image analysis of the 2-NBDG signal in the zebrafish larval eyes showing that EMODIN treatment induces glucose uptake at 72 hpf and 120 hpf larvae. The magnitude of the eModine-glucose uptake in the 120 hpf larvae was reduced when compared to the 72 hpf larvae (* = P <0.05 compared to DMSO-treated larvae).
Figure 6 shows the effect of hyperglucose supplementation on 2-NBDG uptake. 6A is a microscopic analysis showing that 2-NBDG uptake in 72 hpf zebrafish larvae for 3 hours is reduced by competition with free glucose. Figure 6b shows a decrease in 2-NBDG uptake when incubated with hyperglucose by image analysis of larval eyes (* = P < 0.05 compared to 2-NBDG alone treated larvae).
Figure 7 shows the validation of the zebrafish-based 2-NBDG uptake system of the present invention as a method for identifying glucose uptake regulators. 7A is a schematic diagram showing a process of separating and purifying a compound from C. crenata . Figure 7b shows activity induced fractionation of endothelium derived from Castanea crenata , the chestnut tree represented by the methanol fraction, leading to the best induction of 2-NBDG uptake. Figure 7c to a further purification in methanol fraction represents the eight compounds: UP2.2 (Scoring pole retinoic), UP3.1.1.1 (Marsden acid), UP3.5.3 (new plaque is A _2), UP4.2.3 ( 4-ketononanoic acid), UP5.2.1 (4-hydroxy-5-methoxycinnamic acid), UP3.2 (proxidine), UP3.3 (6,7,8-trimethoxy coumarin) and UP5. 2.3 (3,4,5-trimethoxycinnamic acid). The ability of these compounds to induce 2-NBDG uptake into zebrafish larvae was compared with those treated with 10 / mL of emodin (36), known as a glucose uptake inducer. Procidin and maslinic acid have been found to induce very high levels of glucose uptake. The red line in the graph represents the baseline for selecting 'heat' drugs (ie, the 2-NBDG intake should fall within a 100% increase compared to the untreated larval 2-NBDG intake). Figure 7d shows, for further comparison, that Morinda , an Oriental flowering mulberry longissima (ML-4, ML-6, ML-6, and ML-11) isolated from the roots at a dose of 10 μg / mL. No compounds belonging to the extract of M. longissima were able to induce glucose uptake. Figure 7E shows the chemical structures of the ML compounds (ML-4, ML-6, ML-6 and ML-11).
Figure 8 shows that proxidine (UP3.2), which induces glucose in in vivo , is a novel insulin mimetic compound. The fluorescence microscope of FIG. 8A showed that mammary fat cells could increase 2-NBDG uptake after treatment with 10 μg / mL of maslinic acid (UP3.1.1.1) or 10 μg / mL of proxidine (UP3.2). 10 [mu] M berberine was used as a positive control for induction of glucose uptake. Each compound was treated with adipocytes for 30 minutes. Scale bar = 100 μm. Figure 8b shows that 2-NBDG ingestion of mammalian adipocytes treated with 10 [mu] g / ml of proxidine is sensitive to the induction of insulin resistance, an index of insulin mimetic activity. 100 nM insulin was treated as a positive control and 10 μg / mL ML-6 as a negative control. As described above, 2.5 ng / mL TNF-α was treated for 5 days to induce insulin resistance (37). * = P < 0.05 compared to TNF-treated cells. FIG. 8C shows that the ability of the adipocyte to inhibit 10 mM epinephrine-mediated FFA release indicates that the proxidine is an insulin mimetic compound. 100 nM insulin was used as a positive control and 10 / mL ML-6 was used as a negative control. The increased absorbance at 570 nm indicated that FFA was better retained by the adipocyte-treated adipocytes. Error = SD. * = P <0.05 compared to untreated cells (treated with epinephrine only). Figure 8d illustrates a zebrafish-based approach for the detection of novel anti-diabetic compounds of the present invention.
Figure 9 shows that berberine, known to induce glucose uptake in mammalian tissues, can induce 2-NBDG uptake in 72 hpf zebrafish larvae. Larvae were treated with 10 mM berberine for 1 hour. Figure 9a shows a microscopic image and Figure 9b shows an image analysis of the 2-NBDG signal in the larval eye. Error = SD; * = P <0.05 compared with untreated cells.
10 shows the results of toxicity analysis of the compounds isolated from C. creanata . Figure 10a summarizes the phenotype that occurs in zebrafish larvae treated with the compound. The compound was treated for 72 hours at 8-cell (4/5 hpf). Figure 10b shows a microscope image of the compound treated zebrafish. The empty space in the image indicates that the larvae are dead.
Figure 11 shows the screening results of saponin-based natural material for induction of 2-NBDG uptake into zebrafish larval. Fig. 11A shows that saponin cpp532 (red arrow), which is a 'heat' compound, belonging to a range in which the 2-NBDG intake value is increased by 100% as compared with larvae treated with 2-NBDG alone. The red line in the graph represents the baseline for 'heat' compound screening. Asterisks indicate toxic compounds. Emmodin was used as a positive control. FIG. 11B shows fluorescence microscope images of cpp532-treated zebrafish with increased 2-NBDG uptake compared to larvae treated alone with 2-NBDG. Reference: 500 mm. Fig. 11C shows the chemical structure of the heat compound saponin cpp532 (20 (S) -ginchenoside Rh2).
Figure 12 shows the screening results of triazine-based synthetic compounds for induction of 2-NBDG uptake into zebrafish larval. FIG. 12A is a red arrow showing the 'heat' compound within a range where the 2-NBDG intake value is increased by 100% as compared to the larvae treated with 2-NBDG alone. The red line in the graph represents the baseline for 'heat' compound screening. Asterisks indicate toxic compounds. Emmodin was used as a positive control. Fig. 12b shows the results of the 'heat' compounds PP-Ⅱ-A01, PP-Ⅱ-A02, PP-Ⅱ-A03, PP-Ⅱ-A05, and PP- Ⅱ-A05 with increased 2-NBDG uptake, II-A08 Fluorescence microscope image of processed zebrafish. Reference: 500 mm. Fig. 12c shows the chemical structures of the heat compounds PP-II-A01, PP-II-A02, PP-II-A03, PP-II-A05 and PP-II-A08.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

Plant material

The dried chestnut ( Castanea crenata ) endothelium was purchased from the folk medicine market in Gwangju City, Korea (Mayor of Gwangju). The sample was identified and a voucher specimen was submitted to Chosun University, Korea.

Appliances and reagents

UV spectra were obtained from MeOH using a spectrometer (SHIMADZU CORPORATION, Japan) and optical rotation was obtained in MeOH using a polarimeter (Autopol IV Polarimeter, Rudolph Research Analytical, USA). NMR (nuclear magnetic resonance) spectra were obtained at 300 MHz JEOL FT NMR spectrometer and Varian Inova 500 MHz using TMS (Trimethyl Silane) as an internal standard at KBSI (Korea Basic Science Institute, Kwangju, Korea).

All mass experiments were performed with a Micromass QTOF2 mass spectrometer (Micromass, UK). HP-20, silica gel (63-200 mm particle size, Merck, Germany) and RP-C18 (75 mm particle size, Merck, Germany) were used for column chromatography. Pre-coated TLC was performed using Merck Silica gel 60 F254 and RP-18 F254 plates. HPLC was performed using a UV detector and an Optima Pak C18 column (10x250 mm, 10 mm particle size, RS Tech, Korea) using the Gilson system. HPLC solvents were purchased from Burdick & Jackson (USA).

2-NBDG and non-metabotropic 6-NBDG were purchased from Invitrogen (USA). Berberine and emodin were purchased from Professor Ohwon Oh (College of Pharmacy, Chosun University). Insulin, 2-phenylthiourea, epinephrine and TNF-a derived from small pancreas were purchased from Sigma-Aldrich (USA).

Experimental Method

Extraction and Separation of Compounds from C.Crenata

The dried chestnut endothelium (2.4 kg) was extracted with sonication (5 h for each step) at 50 ° C with MeOH (5 L x 3 times). The methanol solution extract was filtered and concentrated to dryness to obtain a MeOH extract (490 g). The methanol extract was dissolved in water (1 L), and the residue was dissolved in MeOH-H ₂ O (0: 100, 25:75, 50:50, 75:25 and 100: 0, Each 2 L) and finally washed with acetone (2 L) to separate into six fractions. Thereafter, the 100% methanol fraction (UP-MeOH fr, separated by 100% MeOH, 2 L) was purified by silica gel column chromatography using a gradient of n-hexane / acetone (20: 1-0: 60 cm; particle size 63200 [mu] m) to give 6 fractions (UP.1UP.6) according to the TLC profile. Fraction 1 (UP.) Using reverse phase ODS-A column (5.0 x 60 cm; 12 nm, S150 μm particle size) with MeOH-H ₂ O (1: 2, 1: 1.5 and 1: 1, 2.5 L each). 1, 550 mg) was further eluted to separate Compound 1 (Scopoletin, UP2.2) (85 mg).

Reverse phase ODS-A column (5.0 x 60 cm; 12 nm, S150 μm particle size) with MeOH-H ₂ O (40:60, 45:55, 50:50 and 70:30 angles 2.5 L) Compound 2 (Fraxidin, UP-3.2) was isolated (75 mg) in fraction 2 (UP.2).

Fraction 3 was passed through a reverse phase ODS-A column (5.0 x 60 cm; 12 nm, S150 μm particle size) with MeOH-H ₂ O (50:50, 55:45, 60:40 and 100: Chromatography yielded 50 mg of compound 3 (6,7,8-trimethoxycoumarin, UP-3.3), compound 4 (4-hydroxy-5-methoxycinnamic acid -hydroxy-5-methoxycinnamic acid, UP-5.2.1) (10 mg) and four sub-fractions (UP.3.4UP.3.7).

Fraction 5 (UP.5) was also fractionated through RP-C18 column chromatography (4.0 × 60 cm, 150 μm particle size) and eluted with MeOH / H ₂ O (1: ) To obtain five sub-fractions (UP.5.1UP.5.5). Compound 5 (3,4,5 tree body methoxy cinnamic acid (3,4,5-trimethoxycinnamic acid, UP- 5.2.3)) (10.9 mg, t R 22.0 min), Compound 6 (new plaque is A ₂ (Fragransins _{a 2, UP-3.5.3))} (17.7 mg, t R 39.0 min) and compound 7 (Marsden acid (Maslinic acid, UP-3.1.1.1) ) (12.0 mg, t R 45.1 min) to obtain the respective The isotonic solution of 42% MeCN + 0.1% formic acid dissolved in water UV-detected at 205 nm and 254 nm at a flow rate of 2 mL / min over an OptimaPak_C18 column (10 × 250 mm, 10 μm particle size) The sub-fraction 2 (UP.5.2) was purified by a semi-preparative Gilson HPLC system.

Compound 1 (scopollen, UP2.2) : yellow crystals; ¹ H NMR (300 MHz, acetone- d ₆ )? (Ppm):

(1H, s, H-8), 6.11 (1H, s) d, J = 9.3, H- 3), 3.85 (3H, s, 6-OCH 3); ^{13 C NMR (75 MHz, acetone-} d 6) δ (ppm): 161.3 (C-2), 152.0 (C-9), 150.5 (C-7), 146.1 (C-6), 144.6 (C-4 ), 113.2 (C-3) , 113.0 (C-10), 111.9 (C-5), 109.8 (C-8), 56.6 (6-OCH 3).

Compound 2 (Proxidine UP-3.2): yellow powder; ¹ H NMR (300 MHz, acetone- d ₆ )? (Ppm):

7.83 (1H, d, J = 9.6, H-4), 6.98 (1H, s, H-5), 6.18 (1H, d, J = 9.6, H-3), 3.93 (3H, s, 7-OCH _{3), 3.88 (3H, s} , 6-OCH 3); ^{13 C NMR (75 MHz, acetone-} d 6) δ (ppm): 160.8 (C-2), 146.4 (C-6), 144.9 (C-8), 144.4 (C-7), 144.1 (C-4 ), 135.7 (C-9) , 113.3 (C-3), 111.6 (C-10), 105.0 (C-5), 56.7 (6-OCH 3), 61.3 (7-OCH 3)

Compound 3 (6,7,8-trimethoxy coumarin, UP-3.3): Yellow amorphous powder; ^{1 H NMR (300 MHz, acetone-} d 6) δ (ppm): 7.87 (1H, d, J = 9.6, H-4), 7.16 (1H, s, H-5), 6.21 (1H, d, J = 9.6, H-3), 3.93 (3H, s, 7-OCH 3), 3.85 (3H, s, 6-OCH 3), 3.81 (3H, s, 8-OCH 3); ^{13 C NMR (75 MHz, acetone-} d 6) δ (ppm): 160.5 (C-2), 151.2 (C-6), 145.9 (C-9), 143.4 (C-4), 143.1 (C-7 , C-8), 114.8 ( C-3), 113.2 (C-10), 105.8 (C-5), 56.3 (7-OCH 3), 61.4 (6-OCH 3), 61.8 (8-OCH 3)

Compound 4 (4-hydroxy-5-methoxycinnamic acid, UP-5.2.1): amorphous powder; ^{1 H NMR (300 MHz, acetone-} d 6) δ (ppm): 7.58 (1H, d, J = 15.9, H-8), 7.32 (1H, d, J = 1.8, H-3), 7.13 (1H d, J = 8.1, 1.8, H-5), 6.86 (1H, d, J = 8.1, H-6), 6.36 (1H, d, J = 15.8, H- 2-OCH _3); ^{13 C NMR (75 MHz, acetone-} d 6) δ (ppm): 170.6 (COOH), 151.3 (C-2), 148.0 (C-7), 144.9 (C-1), 128.8 (C-4), 119.7 (C-5), 116.8 (C-6), 115.6 (C-8), 111.8 (C-3), 56.3 (2-OCH 3)

Compound 5 (3,4,5 -trimethoxycinnamic acid , UP -5.2.3): amorphous powder; ¹ H NMR (300 MHz, acetone- d ₆ )? (Ppm): 7.67 (1H, d, J = 15.9, H-7), 6.76 (1H, d, J = 15.9 , H-8), 3.8881 (9H, br, s, 1/2/6-OCH 3); ^{13 C NMR (75MHz, acetone- d} 6) δ (ppm): 171.2 (COOH), 150.4 (C-2/6), 148.9 (C-7), 138.3 (C-1), 129.5 (C-4) , 115.6 (C-8), 106.5 (C-3/5), 56.3 (1-OCH 3), 56.2 (2/6-OCH 3)

69.0(c 1.24, CHCl₃); HREIMS m/z 344.1633(calcd for C₂₀H₂₄O₅, 344.1622); ¹H- 및 ¹³C-NMR, 표 1 참조 Compound 6 (new plaque is A _2, UP -3.5.3): colorless oil;

_{69.0 (c 1.24, CHCl 3)} ; HREIMS m / z 344.1633 (calcd for C 20 H 24 O 5, 344.1622); ¹ H- and &lt; ¹³ &gt; C-NMR, see Table 1

Compound 7 ( marcelin acid , UP -3.1.1.1): white amorphous powder; LRIMS m / z 432 [M &lt; + &gt;]; ^{1 H NMR (500 MHz, acetone-} d 6) δ (ppm): 0.90 (1H, m, H-1a), 1.90 (1H, m, H-1b), 3.59 (1H, ddd, J = 3.0, 4.5 (1H, m, H-2), 2.91 (1H, d, J = 10.0, H-3), 0.87 H-6), 1.31 (2H, m, H-7), 1.65 (1H, m, H-9), 1.91-1.93 (1H, m, H-16), 1.05 (m, H-15), 1.59-1.62 (1H, m, H-16a), 2.00-2.03 M, H-21b), 1.53 (lH, m, H-19b), 1.21 S, H3-24), 0.99 (3H, s, H3-25), 0.80 (3H, s, H3-23) (3H, s, H3-26), 1.17 (3H, s, H3-27), 0.94 (3H, s, H3-29), 0.91 (3H, s, H3-30); ^{13 CNMR (125 MHz, acetone- d} 6) δ (ppm): 179.0 (28-COOH), 145.1 (C-13), 123.0 (C-12), 84.0 (C-3), 68.9 (C-2) , 56.2 (C-5), 48.6 (C-9), 47.7 (C-1), 46.9 (C-19), 46.8 39.3 (C-10), 34.5 (C-21), 33.6 (C-30), 33.4 (C-23), 28.5 (C-15), 26.4 (C-27), 24.3 (C-11), 23.9 C-26), 17.5 (C-24), 17.1 (C-25)

Zebra fish  maintain

Zebrafish was maintained according to standard guidelines (39). The zebrafish was preserved and processed according to the guidelines of the Gwangju Institute of Science and Technology. In the wild-type 10 L glass tank cycle of the filtered water and a 14/10 hour light / temperature of 28.5 ℃ cancer zebra fish (Danio rerio ). Artemia Brine (40) twice a day with a combination of salina (Inve Aquaculture Nutrition, USA) and dry food (Amazon Flake, Foosung, Taiwan).

In the zebrafish NBDG  Ingestion evaluation

The zebrafish eggs were incubated in E3 water (5 mM NaCl, 0.17 mM KCl, 033 mM CaCl ₂ and 0.33 mM MgSO ₄ in distilled water) supplemented with 0.2 mM 2-phenylthiourea (decolorizing compound to increase larval transparency) Respectively. 72 hpf larvae were dispensed into 200 μL E3 water at a density of 4 eggs / well in a 96-well plate. The test compound was then added and the larvae were exposed to the test compound for 1 hour. Next, E3 water containing the test compound was removed and replaced with NBDG dissolved in E3 water. The larvae were incubated with NBDG for 3 hours. E3 water containing NBDG was removed and then the larvae were washed with E3 water and then anesthetized with 0.02% tricine dissolved in E3 water. The larvae were then placed on a glass microscope slide in a plastic chamber (1.6 cm diameter and 3 mm depth) containing 3% methylcellulose dissolved in E3 water. NBDG uptake images were captured using a fluorescence microscope (DM2500, Leica, Germany) equipped with a digital camera (DFC425C, Leica, Germany). Images were acquired using Leica Application Suite software (Leica Microsystems, Germany) and Photoshop CS4 (Adobe Systems Incorporated, USA). NBDG uptake into larvae was quantified as Image J software (National Institutes of health, USA).

For homogenization of zebrafish larvae for NBDG uptake measurements using a fluorescent microplate reader (SpectraMAX Gemini XS, Molecular Devices, USA), the larvae were placed in a 1.5 mL Microfuge Tube and incubated with 120 μL Was dissolved in CelLytic Mn solution (Sigma-Aldrich, USA) and then disrupted using watchmaker forceps. The larvae were sonicated using a Vibra-Cell VCX500 (Sonics & Materials, Inc., USA) at 4 ° C with 10 "/ 5" pulses for 10 minutes and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred to a 96-well plate and the NBDG signal was measured (lex = 466 nm, lem = 540 nm) with a fluorescence microplate reader.

Adipocyte culture

3T3-L1 rodent progenitor cells (purchased from Professor Hyun Chul Lee, Yonsei University, Korea) were infected with 10% calf serum (Invitrogen, USA), 50 units / mL penicillin and 50 μg / mL streptomycin (PenStrep, Life Technologies Corporation. , USA) supplemented with DMEM (Dulbecco's Modified Eagle's Medium; Invitrogen, USA). 10% FBS and adipogenic factors cocktail (0.5 mM 3-isobutyl-1-methyl-xanthine, 2 / mL dexamethasone (Sigma-Aldrich, USA), 1 / mL insulin, 50 / mL penicillin and 50 / mL streptomycin) to induce differentiation of 3T3-L1 into adipocytes from post-confluent for 48 hours. After 48 hours, the cells were incubated in fresh DMEM containing 10% FBS, PenStrep and 1 [mu] g / mL insulin. After 8 days, differentiated adipocytes were used in the experiment.

NBDG  Fat Cell-Based Measurement of Intake

NBDG uptake in 3T3-L1 was measured as previously described (38). Adipocytes were plated at a concentration of 5000 cells / well in a 96-well plate culture plate and induced to produce lipids. The culture medium was then replaced with serum-free and low glucose DMEM for 3 hours. The culture medium was then replaced with DMEM medium containing the test compound. After 30 minutes, the medium was replaced with DMEM medium containing 50 [mu] M 2-NBDG. After 1 hour, the adipocytes were washed twice with pre-warmed PBS (phosphate buffered saline) and treated with PBS (pH 10) containing 70 μL of 0.1 M potassium phosphate and 1% Triton X-100 for 10 minutes Respectively. 30 [mu] L DMSO was added and the solution was carefully mixed using a multi-channel pipette. NBDG signals were measured using a fluorescence microplate reader (SpectraMAX Gemini XS, lex = 466 nm, lem = 540 nm, Molecular Devices, USA).

After the PBS washing step, cell images of NBDG uptake were obtained using a fluorescence microscope (IX81, Olympus, Japan) and Metamorph software (version 7.5, Molecular Devices, USA).

Free fatty acid release Assay

The epinephrine-stimulated free fatty acid (FFA) released from adipocytes was measured according to the conventionally disclosed method 41 using a free fatty acid quantitation kit (Biovision, USA). The conventionally disclosed method is as follows: Confluent 3T3-L1 adipose precursor cells in 24-well plates were incubated in serum-free culture medium for 3 hours. Then, adipocytes were treated with 10 [mu] M epinephrine dissolved in the salt to induce FFA release. The drug of interest was added 10 minutes before the treatment of epinephrine. Cell lysates were obtained and 50 mL organic phase was used for analysis.

Zebra fish  Based toxicity test

Our protocol for toxicity analysis was based on conventional literature (35). After scattering, a zebrafish embryo was dispensed into 200 μL E3 water at 1 al / well in a 96-well plate. The compound was added to the 8-cephem (5/4 hpf) over 72 h. E3 water containing each compound was replenished daily. The compound was identified every 24 hours after addition of the compound using a stereo microscope (Zeiss Stemi 2000-C, Edmund Optics Inc., USA) and the following developmental and anatomical parameters were observed: 1) somite development, 2) tail detachment, 3) otic vesicles, otoliths and eye development, 4) heart rate, 5) circulation, 6) delayed hatching, 7) presence of skeletal anomalies, 8) Body position, 9) swimming ability. In order to confirm the Initial Mortality Rate (IMR), 50 untreated larvae were incubated under the same conditions. After evaluation of developmental and anatomical parameters, the larvae were anesthetized by incubation in E3 water containing 0.02% tricine for 10 minutes and plated in plastic chambers (1.6 cm diameter and 3 mm diameter containing 3% methylcellulose dissolved in E3 mm depth) were placed on a glass microscope slide. Images of compound-treated larvae were photographed with a differential interference contrast (DIC) microscope (DM2500, Leica, Germany) equipped with a digital camera (DFC425C, Leica, Wetzlar, Germany). Images were acquired using Leica Application Suite software (Leica Microsystems, Germany) and Photoshop CS4 (Adobe Systems Incorporated, USA).

Statistical analysis

Student's t-test (Microsoft Excel, 2010 version) was used for the comparison of experimental groups. A P value of 0.05 or less was considered significant. Unless otherwise noted, all results are the mean of three separate experiments, and error bars are the standard deviation of the mean.

Experiment result

Zebra fish  Larvae are 2- NBDG Can be taken in a dose-dependent manner.

The chemical structure of the compounds used in the present invention is shown in Fig. The first experiment of the present invention to establish a fluorescence-based screening system based on glucose uptake in zebrafish larvae was to ensure that accurate measurement of 2-NBDG uptake was possible. We chose 72 hpf larvae because they performed GLUT transporter expression and glucose metabolism at the time of development in the prior art (21, 23). Caenorhabditis elegans) to the 2-NBDG intake study 200 mM dose of the conventional in vivo it was used with 24. Thus, we incubated zebrafish with the following doses of 2-NBDG: 0, 100, 200, 600, 1 and 2 mM. As shown in Figure 2A, 2-NBDG uptake was visualized by fluorescence microscopy at a dose of 200 [mu] M and the fluorescence signal was maintained until the dose was increased by 2 mM. The next step of the invention was to develop a method capable of quantifying glucose uptake in 72 hpf larvae. Previous studies have shown that it is possible to measure intracellular 2-NBDG uptake using fluorescent microplate reader analysis of cell lysates (14). The results of the present invention show that 2-NBDG uptake can be detected in homogenized zebrafish larvae using a fluorescence microplate reader (FIG. 2B). A noticeable increase in the 2-NBDG fluorescence signal was detected at a dose of 600 [mu] M, which is similar to the results of the microanalysis of the present invention of 2-NBDG uptake. At 72 hpf, zebrafish larval eyes are known as the abundant expression sources of other members of the GLUT family of transporters (23). From Fig. 2a, it was observed that larval eyes showed a dose-dependent increase in 2-NBDG uptake. The shape of the eye in the 72 hpf larvae is necessary for image analysis of fluorescence intensity. Using Image J software (National Institutes of Health), we were able to quantitate dose-dependent increases in 2-NBDG uptake (FIG. 2B).

The zebrafish  2- NBDG  A time-dependent increase in intake.

The NBDG probe showed binding to the GLUT receptor with a much higher affinity than D-glucose, but the NBDG delivery via GLUT is much slower (15). In addition, 2-NBDG is phosphorylated by hexokinase after cell ingestion and is then rapidly degraded to a non-fluorescent material by the glycolytic pathway (14). Thus, we measured the time-dependent changes in 2-NBDG signal after 2-NBDG was ingested into 72 hpf larvae. We chose 600 μM 2-NBDG at doses that can visualize 2-NBDG uptake without saturation of the NBDG signal (FIG. 2a). We were able to visualize 2-NBDG uptake at 0.5 hour after treatment and observed an increase in 2-NBDG uptake by 8 hours after treatment (Fig. 3a). In addition, microplate reader analysis showed that the 2-NBDG signal increased rapidly after 30 minutes, still after 1 hour of treatment, and began to increase again after 4 hours of treatment. 2-NBDG lightness was maintained at 8-12 hours after treatment (Fig. 3B). We employed an image J software to quantitate the time-dependent increase of 2-NBDG uptake (Fig. 3c), adopting a similar approach to the 2-NBDG dose dependent uptake assay in the zebrafish larval. 2-NBDG uptake in the eyes was detected after 1 hour of treatment and showed a linear pattern of more time-dependent uptake compared to microplate reader analysis of the homogenized larval (compare Figs. 3b and 3c).

Zebra fish  To observe glucose uptake in larvae, 6- NBDG 2- NBDG .

2-NBDG and 6-NBDG were used to measure glucose uptake in cells (14). We tested 6-NBDG uptake in 72 hpf zebrafish larvae. However, when using the same image capture parameters (DM2500, Leica, 500 ms exposure) as in the experiment for 2-NBDG uptake, the signal of 6-NBDG was much weaker. The 6-NBDG signal was scarcely observed at the 1 mM dose (Figure 4). Therefore, we selected 2-NBDG to visualize glucose uptake in larvae.

2- NBDG  In the observation of ingestion, 72 hpf Zebrafish  Larger than 120 hpf Zebrafish  It is preferable to larvae.

We chose 72 hpf initially for 2-NBDG uptake analysis in zebrafish larvae because we have been evaluating glucose transduction expression and mammalian-like glucose homeostatic mechanisms at this stage of development (21, 23) . However, the present inventors also tested 2-NBDG uptake at 120 hpf (Fig. 5a-d) to determine whether a later time point is more desirable. For this test, the present inventors compared the effect of treatment with emodin (6-methyl-1,3,8-trihydroxy anthraquinone 3) on the intake of 2-NBDG. Emmodin is a plant-derived laxative resin capable of inducing glucose uptake and improving diabetes in animal models (24). Emmodine treatment for 72 hpf and 120 hpf larvae induced plate reader analysis of homogenized larval and 2-NBDG uptake as observed in the 2-NBDG fluorescence signal in the eyes (Figures 5c and 5d). However, the imodin-induced 2-NBDG uptake in 120 hpf larval eyes was less than 72 hpf larvae.

Zebra fish In larvae  2- NBDG  Ingestion is caused by competition with free glucose To be reduced  .

An important test to demonstrate that fluorescent-labeled glucose ingestion depends on glucose transport activity is competition with excess glass glucose (14). 72 hpf zebrafish larvae were incubated with 600 [mu] M 2-NBDG and 30 mM D-glucose for 3 hours (Figure 6). Image analysis of 2-NBDG uptake into larval eyes shows that incubation with free glucose reduces 2-NBDG uptake, suggesting that the glucose transport mechanism is an important factor for 2-NBDG uptake into larvae.

At the zebra fish  2- NBDG  The measurement of intake New  Insulin mimetic compounds are identified.

A method to determine the glucose intake control zebra fish of the present invention to validate the base 2 NBDG-intake system, the inventors of Japanese chestnut Castanea Activity guided fractionation of endothelium derived from crenata was adopted. Figure 7a shows the overall experimental design for compounds isolated from C. crenata . Three fractions (methanol, ethyl acetate and hexane) were tested for the effect of 2-NBDG ingestion into zebrafish larvae (Figure 7b). For the zebrafish-based assay of the present invention, a 100% increase in 2-NBDG uptake was selected as a baseline for 'heat' drug selection. The methanol fraction was found to induce 2-NBDG uptake to the best, and the methanol fraction was further purified to obtain the following eight compounds: UP2.2 (scopoletin), UP3.1.1.1 (muslinic acid), UP3. 5.3 (pragramin A ₂ , UP4.2.3 (4-ketononanoic acid), UP5.2.1 (4-hydroxy-5-methoxycinnamic acid), UP3.2 (proxidine) 7, 8-trimethoxycoumarin) and UP5.2.3 (3,4, 5-trimethoxycinnamic acid). Figure 7c shows the results for 2-NBDG uptake in zebrafish larvae as inducers of glucose uptake (24) and 2-NBDG, which are known to be positive control. In addition, for further comparisons, Morinda Four compounds isolated from the roots of longissima were also tested (Figure 7d). Morinda Longissima was chosen because it has not been reported to have conventional anti-diabetic activity. From the methanol fraction of C. crenata, we found proxydin and maslinic acid that increased the 2-NBDG uptake by more than 100%, but no compound of M. longissima induced glucose uptake (FIG. 7d).

The present inventors have shown that using the screening system of the present invention, the proxidine can induce glucose uptake in the mammalian adipocyte system. Fat cells are the primary site of action for insulin-mediated glucose uptake (14) and treatment with proxidine (UP3.2) induces 2-NBDG uptake (Figure 8a). Berberine 12 (26), known as the inducer of glucose uptake, was used as a positive control (verifying the ability of berberine to induce glucose uptake in the present zebrafish system (FIG. 9)). Insulin mimetics replace the insulin effect in cell physiology and represent an ideal candidate for the development of anti-diabetic agents (13). Therefore, the present inventors have examined insulin mimetic activity of proxidine (UP3.2) (FIGS. 8B and 8C). Procidin-induced glucose uptake in adipocytes was sensitive to TNF-a, a test for insulin resistance and insulin mimetic activity (Fig. 8b) (27). A second test for insulin-inducing activity is the ability to inhibit the release of epinephrine-stimulated free fatty acids from adipocytes (28). Pre-treatment of adipocytes with proxidine reduced the release of free fatty acids induced by epinephrine (Figure 8c).

Another very useful feature of zebrafish-based assays is that they can be easily tested for toxicity in developed larvae (25). Toxicity analysis of the present invention suggested that 10 mg / mL for this compound is a suitable dose (selected by the toxicity data in FIG. 10).

New  For insulin mimic drug candidate discovery Zebra fish - Based Screening

We screened saponin-based natural substances (10 μg / mL dose for 3 hours) for induction of 2-NBDG uptake into zebrafish larvae. Compared with larvae that only treated with 2-NBDG alone, they were selected as 'heat' compounds when the 2-NBDG intake value was within the range of 100% increase. As a result, saponin cpp532 (20 (S) -ginsenoside Rh2) was identified as a 'heat' compound.

We also screened a triazine-based synthetic compound (20 μM dose for 3 hours) for induction of 2-NBDG uptake into zebrafish larvae. The compounds PP-II-A01, PP-II-A02, PP-II-A03, PP-II-A05 and PP-II-A08 were identified as "heat" compounds.

Review

In the present invention, we have established the establishment of a novel system for rapid and convenient detection of anti-diabetic agent candidates active in vertebrate animals. The present inventors have combined the experimental utility of zebrafish animal models with the fluorescent-tagged glucose analogue 2-NBDG (the radioactive glucose tracer requires specialized laboratory equipment and radioactivity purification and does not allow temporal / spatial resolution 14)). The protocol of the present invention can significantly accelerate the drug discovery approach in diabetes research.

The results of the present invention indicate that the appropriate concentration of 2-NBDG to observe the variability of glucose transport in zebrafish larvae is 600 [mu] M (Fig. 2b). Search the database PubMed.Gov (US National Library of Medicine National Institutes of health) is Drosophila indicating no study of 2-NBDG uptake in melanogaster . The protocol of the present invention for the detection of compounds that regulate glucose uptake depends on the simple addition of 2-NBDG to fish water. The simplicity and simplicity of the protocol length of the present invention eliminates the need for microinjection of 2-NBDG. Zebrafish suggests distinct advantages over initial testing of anti-diabetic drug candidates beyond the mouse (eg, a relatively simple housing and a small amount of test compound and 2-NBDG).

Analysis of the 2-NBDG uptake in the zebrafish larvae of the present invention showed that the stabilizer was started to reach a relatively early intake and then increased again (Figs. 3b and 3c). This time course may reflect the intracellular fate of 2-NBDG, which undergoes rapid phosphorylation by hexokinase and subsequent degradation into corresponding non-fluorescent substances (14). That is, after a fast initial uptake, 2-NBDG is degraded by the process and several hours after treatment, the process can saturate in the cell, which may allow for additional accumulation of 2-NBDG. We therefore selected 3-hour 2-NBDG treatment for the protocol of the present invention because the 2-NBDG signal is easily detected by the efficacy of the anti-diabetic drug candidate but still has additional cell accumulation capability Because.

The present invention demonstrates the ability to measure 2-NBDG uptake in zebrafish larvae by either of two methods: analysis of fluorescence plate reader in homogenized larval or image analysis of 2-NBDG uptake in larval eyes. The latter is better suited for analysis of large numbers of treated larvae because the assay is relatively fast and characterized by GLUT expression (72 hpf larva expresses a range of zebrafish GLUTs such as zglut1b , zglut6 and zglut12 (23) Furthermore, the data of the present invention show that both emodine and berberine, known as compounds that induce glucose uptake, can induce 2-NBDG uptake in larval eyes (Figs. 5d and 9).

In the present invention we compared emodin-induced 2-NBDG uptake at two developmental times (72 hpf and 96 hpf) (FIG. 5). 72 hpf was found to be more appropriate because of the greater difference in the 2-NBDG uptake of emmodin-induced NBDG to the control than 96 hpf. A complicated factor in the use of 96 hpf zebrafish is that at this stage of development the digestive tract becomes functional and zebrafish can swallow foreign substances (29). Therefore, microplate reader-based analysis of 2-NBDG uptake in 120 hpf larvae can be complicated by the 2-NBDG solution digestibility of zebrafish. This can be explained by the fact that the 2-NBDG signal observed by fluorescence microscopy is brighter than in the digestive tract (Fig. 5B).

An important consideration when evaluating glucose probes is the demonstration that the cellular glucose transport mechanism is involved in probe ingestion (14). The data of the present invention show that 2-NBDG uptake in zebrafish larvae can be inhibited by excessive extracellular glucose (Fig. 6). The test is used to validate novel glucose probes such as Cy5.5-D-glucosamine, IRDye 800CW 2-DG and CyNE 2-DG (14). 2-NBDG was commercially available, relatively inexpensive, and characterized by several studies, so it was selected as a glucose probe. The present invention has for the first time demonstrated that 2-NBDG can be used to detect anti-diabetic compounds that are altered in zebrafish larvae.

We used activity-guided fractionation from plant material to demonstrate the utility of our experimental system for the detection of novel regulators of glucose uptake (Figure 7). Zebra fish-based glucose uptake assay system of the present invention Castanea The methanol fraction of endothelium derived from crenata has shown to contain compounds that promote glucose uptake. In addition, we showed that most of the potential glucose uptake-inducing compounds in this fraction were proxidine and maslinic acid. Previous studies have shown that marlesine is an anti-diabetic compound (30). Other compounds of C. crenata that have not been reported as anti-diabetic compounds or glucose uptake inducers (pragramin A ₂ , 4-ketononanoic acid, 4-hydroxy-5-methoxycinnamic acid, 6,7,8 -Trimethoxycoumarin and 3,4,5-trimethoxycinnamic acid) were not 'heat' compounds in the zebrafish assay of the present invention. Scopolletin has been reported as an activator of AKT phosphorylation in the hepatocyte lineage, which can potentially lead to glucose uptake. However, no potential anti-diabetic activity against scopollin has been described (31).

In the present invention, the present inventors examined the efficacy of the proxidine as a novel insulin mimetic drug (Fig. 8). The present inventors have found that the discovery of the proxidine as an insulin mimetic drug active in in vivo is the best potential of the zebrafish test for novel anti-diabetic drug discovery for the novel fluorescent-based novel anti-diabetic drug discovery of the present invention We believe that we are proving our surname.

In short, the present inventors have developed a first assay for the discovery of active anti-diabetic compounds in vertebrates (Fig. 8d). Since the major protein components of the insulin signaling system of zebrafish express structural and functional similarities with mammals (21, 32), the protocol of the present invention utilizes zebrafish. Moreover, the zebrafish-based system of the present invention enables the accompanying toxicology analysis to accurately predict the drug response of mammals.

According to the knowledge of the present inventors, other small organism-based tests for the development of anti-diabetic medicaments have been used for the treatment of silkworm, Bombyx mori ) and the nematode Caenorhabditis elegans (33, 34). Thus, the zebrafish-based screening system, a vertebrate-based approach of the present invention, represents a major technological advance. Furthermore, the method using the glucose probe of the present invention, 2-NBDG, enables simple, low-cost and high-speed glucose flow visualization in zebrafish larvae. This can greatly facilitate the drug discovery approach for diabetes research.

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While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

A zebrafish-based screening method of a diabetes therapeutic agent comprising the steps of:
(a) contacting a test substance with a zebrafish ( Danio rerio );
(b) contacting the result of step (a) with a fluorescent glucose inducing factor; And
(c) measuring the amount of the fluorescent glucose-derived factor uptake into the zebrafish through fluorescence measurement; If the test substance increases the uptake of the fluorescent glucose-inducing factor into the zebrafish, it is judged to be a therapeutic agent for diabetes.
2. The method of claim 1, wherein the zebrafish is a zebrafish larva.
3. The method of claim 2, wherein the zebrafish larvae are 24-168 hpf (hour post fertilization) larvae.
The method of claim 1, wherein the test substance is contacted at a concentration of 1-15 mg / mL.
2. The method of claim 1, wherein the fluorescent glucose inducing factor is selected from the group consisting of 2-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol- ) Or 6-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] -D-glucose (6-NBDG).
6. The method of claim 5, wherein the fluorescent glucose inducing factor is selected from the group consisting of 2-deoxy-2- [(7-nitro-2,1,3-benzoxadiazol- ). &Lt; / RTI &gt;
The method according to claim 1, wherein the fluorescent glucose-inducing factor is contacted at a concentration of 100 μM-5 mM.
The method of claim 1, wherein the contacting of step (b) is performed for 0.1-20 hours.
2. The method of claim 1, wherein the fluorescence measurement is performed through a fluorescence signal measurement or a fluorescence image measurement.
10. The method of claim 9, wherein the fluorescence signal is a fluorescence signal generated in a homogenized zebrafish.
10. The method of claim 9, wherein the fluorescence image is a fluorescence image generated in a zebrafish eye.
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