JP5535514B2 - Test substance evaluation method - Google Patents

Description

  The present invention relates to a test substance evaluation method using nematodes.

  There are a huge number of chemicals today. Among these chemical substances, there are toxic substances such as agricultural chemicals, heavy metals and air pollutants, and useful substances such as drugs, physiologically active substances and dietary supplements.

  In order to evaluate the effects of chemical substances, higher animals such as mice, rats, dogs and monkeys are used. However, using higher animals requires a great deal of labor, time and money. On the other hand, when bacteria and cultured cells are used, higher-order biological phenomena such as morphogenesis and organ formation are not observed, and the metabolic system has a large gap with multicellular organisms.

  For this reason, it is required to use an animal that can be tested more easily than a higher animal, has an animal nervous system and a reproductive organ such as a human, and has similarity to a human. As such an animal, a toxicity test method using the nematode Caenorhabditis elegans has been proposed (see, for example, Patent Documents 1 and 2).

  In the case of substances such as drugs and dietary supplements, it is important to accurately assess the effects of oral intake. However, in any of the methods described in the above-mentioned documents, a test substance is added to the culture medium, and the effect of this on nematodes is evaluated. For this reason, it is not possible to distinguish whether the substance has an effect on nematodes due to ingestion or dermal ingestion. In the case of the method of dissolving in the whole medium, it is necessary to secure a large amount of the test substance. However, it is necessary to conduct a test with a very small amount of a test substance in a natural extract or the like obtained on a trial basis, and it is desirable to ensure that it is taken orally. Furthermore, in the method of adding a test substance to the culture medium, it is assumed that the amount absorbed from the body surface of the nematode differs depending on the molecular weight and chemical properties of the test substance, and it is not easy to grasp the amount transferred into the body.

As a method for providing a test substance evaluation method capable of evaluating the effect of a test substance taken orally on nematodes, a method of encapsulating the test substance in microcapsules and ingesting the nematodes orally is provided. (For example, refer patent document 3).
JP 55-153599 A JP 2005-10110 A JP 2008-170427 A

  In the method described in Patent Document 3, for example, when the test substance is a lipophilic substance, it is microencapsulated using gum arabic, and when the test substance is a hydrophilic substance, phosphatidylcholine, etc. An aqueous solution is encapsulated in a liposome using the phospholipid.

  However, in the case of a lipophilic test substance, the test substance is dissolved in soybean oil and made into microcapsules using gum arabic. For this reason, the amount which can be administered is restrict | limited by the solubility with respect to the soybean oil of a test substance.

  This invention is made | formed in view of the said problem, The objective is to provide the test substance evaluation method which can control the quantity which can administer a lipophilic test substance.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have included lipophilic test substances in a cyclodextrin (hereinafter sometimes referred to as “CD”). It was found that inclusion was possible by concentration. Moreover, it was confirmed that CD inclusion bodies of lipophilic test substances can be taken orally by nematodes. Furthermore, in order for a test substance included in a CD inclusion body taken orally to act on a nematode, the test substance dissociates from the CD in the nematode intestinal lumen or in the cell or body cavity of the nematode. And it is necessary to demonstrate its function. The present inventor has confirmed these facts and found that the effect on the biological function of the lipophilic test substance taken orally can be evaluated, and has completed the present invention.

  The nematode may be Caenorhabditis elegans.

  The effect on the biological function of the test substance can be evaluated by, for example, biological indicators and / or biochemical evaluation.

  When the test substance evaluation method of the present invention is used, the effect of lipophilic test substance taken orally on nematodes can be evaluated by eliminating the influence of transdermal absorption of the lipophilic test substance.

  The present invention is described in detail below.

[Nematode]
Nematodes (Caenorhabditis elegans) used in the test substance evaluation method of the present invention is a kind of soil nematode having a body length of about 1 mm selected from about 1500 known species. This nematode is easy to breed and has a small number of somatic cells of about 1000, and hermaphrodites can grow by self-fertilization. Nematodes have a nervous system, muscles, digestive organs, reproductive organs, and epidermis, and have a basic system as an animal. Its genes are close to humans and belong to the same genuine metazoan as humans. This nematode is used as an experimental animal for molecular biology research of aging and as an infection model of pathogenic microorganisms.

  Such nematodes include, for example, Caenorhabditis elegans Briostol strain N2 hermaphroditic. This nematode can be obtained from, for example, Caenorhabditis Genetics Center (University of Minnesota, St Paul, MN, USA).

Nematodes are raised by partially changing the methods described in “ C. elegans: A PRACTICAL APPROACH” and “Nematode Lab Manual”.

[Test substance]
The test substance that can be evaluated in the present invention is not particularly limited as long as it is orally ingested and its action is evaluated. It is preferable that the test substance is fat-soluble because it is easily included in cyclodextrin. In the present specification, the term “lipophilic” means that the whole molecule contains a fat-soluble group in addition to a fat-soluble substance. When the test substance is a substance in which the whole molecule is fat-soluble, all or part of the substance is included in cyclodextrin. Or when a molecule | numerator contains a fat-soluble group, the part of a fat-soluble group is included by cyclodextrin.

  In the present invention, the “test substance” has adverse effects such as toxicity to humans and animals such as useful substances for humans and animals such as drugs and dietary supplements, poisonous substances, agricultural chemicals, environmental pollution sources, and mutagens. Any of the materials that are given are included. In addition, the “effect on the biological function of the test substance” may be a “positive action” that provides benefits such as extending the life of the nematode, and adversely affects the organism such as toxicity and mutagenesis. It may be a “negative action”. That is, the “action on the biological function of the test substance” in the present invention means some influence caused by giving the test substance to the nematode.

[Cyclodextrin]
Cyclodextrin is a kind of cyclic oligosaccharide in which several molecules of D-glucose are linked by α (1 → 4) glucoside bonds to form a cyclic structure. A combination of 5 or more glucoses is known. The cyclodextrin used for the production of the cyclodextrin inclusion body of the present invention includes α-cyclodextrin (cyclohexaamylose) to which 6 glucoses are bonded, β-cyclodextrin (cycloheptaamylose) to which 7 are bonded, 8 Individually linked natural cyclodextrins such as gamma cyclodextrin (cyclooctaamylose) and chemical modifications such as methylation, hydroxypropylation, acetylation, monochlorotriazinolation, sulfobutylation of these natural cyclodextrins Examples thereof include chemically modified cyclodextrins, modified cyclodextrins such as enzyme-modified cyclodextrins such as maltosylation, and mixtures of two or more thereof. Of these cyclodextrins, α cyclodextrin, β cyclodextrin, and γ cyclodextrin are preferably used, and γ cyclodextrin is particularly preferably used.

  Cyclodextrin has an annular shape like a donut or a bucket with a hole. The inside of the cyclic structure of cyclodextrin has pores that are large enough to include other relatively small molecules. The inner diameters of the holes are about 0.45 to 0.6 nm for the α body, 0.6 to 0.8 nm for the β body, and about 0.8 to 0.95 nm for the γ body. Since there are many hydroxyl groups (hydroxyl groups) at the end of the pores of cyclodextrin, cyclodextrin is soluble in water. On the other hand, the inside of the cavity is hydrophobic because there are oxygen atoms and hydrogen atoms of ether bonds. Accordingly, a hydrophobic organic compound or the like is taken into (included in) the cavity by physical attraction (intermolecular force) without forming a chemical bond. In the present invention, a lipophilic substance is included in cyclodextrin by utilizing this.

  In the present invention, the size of cyclodextrin is important. Nematodes are usually raised on E. coli. Also, nematodes that are large in size (greater than 5 μm and smaller than 10 μm) are difficult to ingest. Therefore, it is preferable that the diameter of the natural aggregate of cyclodextrin generated after the inclusion of the fat-soluble substance is about the same as the size of E. coli (for example, about 1 μm to 5 μm). In addition, after the natural aggregate of cyclodextrin is taken into the intestinal lumen, the aggregate is dispersed into a finer aggregate.

[Evaluation method]
A test substance is included in cyclodextrin, and the dispersed solution is applied to an agar plate for nematode growth. When placed in this state for a while, the solvent is absorbed by the agar and the cyclodextrin remains on the agar plate for nematode growth. Nematodes are raised on this agar plate for a predetermined period. By observing the lifespan of the nematode, the effect on the biological function of the test substance taken orally is evaluated. In addition, after ingesting a test substance such as a drug or dietary supplement, the effect of the drug, dietary supplement, or the like may be evaluated by ingesting a pathogen or the like.

  Further, in the evaluation method of the present invention, the period when the nematode is fed on the agar plate for growing nematodes on which the cyclodextrin is present is changed, thereby accurately evaluating the time when the ingested test substance affects the nematodes. it can.

[Measurement method of intake]
If the cyclodextrin used in the present invention is encapsulated with a fat-soluble fluorescent substance or the like together with a test substance, it is possible to easily measure how much the nematode has ingested the test substance. That is, the nematode feeds cyclodextrin encapsulated with a fluorescent substance or the like. After a certain period of time, the distribution of the fluorescent substance in the nematode body is observed with a fluorescence microscope, the amount of the fluorescent substance recovered by grinding the nematode is measured, and the amount of cyclodextrin intake per unit time is calculated. From this, the oral intake of the cyclodextrin and the test substance to be included can be determined. It is also easy to adjust the dose by adjusting the amount of cyclodextrin applied on the agar plate.

  In the present invention, the test substance evaluation method can be appropriately selected depending on the test substance to be used. For example, evaluation of biological indices such as long and short lifespan of nematodes, movement, number of eggs laying, and mutability, and biochemical indices such as lipid peroxide. For example, the life span of Caenorhabditis elegans Briostol strain N2 hermaphroditic is usually around 25 days. The lifespan can be evaluated by observing the nematode every day and dying the individual who did not move by lightly touching the nematode with an instrument. In addition, in this specification, when reared under a temperature condition of 25 ° C., the larval stage of nematode means 0 to 3 days after hatching, and the adult stage means after 4 days of age.

  According to the method of the present invention, the effect on the biological function of a test substance taken orally can be directly evaluated by eliminating the influence of transdermal absorption of the lipophilic test substance. In particular, oral intake can be measured accurately and its effect can be evaluated. As a result, it is possible to accurately evaluate the influence of substances by oral intake such as drugs and dietary supplements. Further, according to the method of the present invention, the test substance encapsulated in the CD inclusion body taken orally is dissociated from the CD in the nematode intestinal lumen or the like and absorbed from the intestinal lumen into the intestinal cells. The

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this Example.

In the following examples, nematodes, reagents, and culture solutions were as follows.
C. elegans: Caenorhabditis elegans Briostol strain N2 hermaphrodite (C. elegans) was used.
Nematode Growth Medium (NGM plate):
NaCl (Wako Pure Chemical Industries) 1.5 g and Agar (Wako Pure Chemical Industries) 8.5 g were mixed with 485 ml of distilled water, sterilized by autoclave (121 ° C., 15 min), and cooled in a water bath at 60 ° C. for 1 hour. Thereafter, 500 μl of 1M CaCl ₂ , 500 μl of 1M MgSO ₄ , 500 mg / ml of cholesterol (Wako Pure Chemical Industries) adjusted to 5 mg / ml with ethanol (Wako Pure Chemical Industries), 12.5 ml of 1M KPO ₄ were mixed, Dispensing 12 ml each into a petri dish (Kord Products Inc., Bramptom, Ontario, Canada) with a diameter of 60 mm was used.

TRYPTONE SOYA agar plate (TSA):
20 g of TRYPTONE SOYA AGAR (Oxoid) was mixed with 500 ml of distilled water, sterilized with an autoclave (121 ° C., 15 min), cooled, and then dispensed into a 90 mm petri dish (Greiner Bio-One).

M9 buffer:
1.5 g of KH ₂ PO ₄ (Wako Pure Chemical Industries), 3 g of Na ₂ HPO ₄ (Wako Pure Chemical Industries) and 2.5 g of NaCl (Wako Pure Chemical Industries) are mixed with 500 ml of distilled water, and then autoclaved (121 ° C., 15 min. ), And after cooling and adding 500 μl of 1M MgSO ₄ , a mixture was used.

1M sodium azide-M9 solution:
0.65 g of sodium azide (Wako Pure Chemical Industries, Ltd.) was added to 10 ml of M9 buffer and dissolved to prepare a 1M sodium azide-M9 solution. This was used as a mother liquor and diluted with M9 buffer to prepare 50 mM sodium azide-M9 solution and 10 mM sodium azide-M9 solution.

Example 1
(Confirmation of γCD uptake)
Preparation of Fluorescent Substance-γCD 2 g of γCD (CAVAMAX W8 Food: Wacker Chemie AG) was weighed into a mortar, and an appropriate amount of deionized water was added to prepare a paste. 2 mg of fluorescent substance 3, 3′-Dioctadecyloxacarboline perchlorate (DiO) (manufactured by Sigma) dissolved in 1 ml of ethanol was added and mixed until uniform. It was pulverized by drying under reduced pressure to obtain a DiO-γCD inclusion body.

Administration of fluorescent substance-encapsulated γCD to nematodes A bacterial solution in which 10 mg of OP (Escherichia coli OP50 (hereinafter referred to as “OP”)) that is cultured overnight in TSA is suspended in 50 μl of M9 buffer, and fluorescent substance-encapsulated γCD 50 μl of the solution (14.25 μg in terms of DiO) was applied to the NGM plate (14.25 μg / plate). On top of that, several nematodes were transferred using a worm picker and allowed to eat freely for 3 hours.

Preparation of fluorescent microcapsules 3.5 g of distilled water sterilized by an autoclave (121 ° C., 15 min) and 0.75 g of gum arabic were placed in a centrifuge tube and stirred well to dissolve. Thereto, 2.5 mg of fluorescent substance 3, 3′-Dioctadecylcarbocarbineline perchlorate (DiO, Sigma, D4292) 2.5 kg of soybean oil (Wako Pure Chemical Industries) 1 sterilized by filtration with a 0.45 μm pore size disk filter (Toyo Filter Paper, Tokyo) In addition to 0.0 g, add 0.75 g of the supernatant collected while leaving insoluble matter, and treat on ice for 2 minutes with the maximum output of an ultrasonic disperser (UH-50, SMT) to obtain a microparticle with a particle size of 1-5 μm. Capsules were made.

Administration of fluorescent substance-encapsulated γCD and fluorescent substance-containing microcapsules to nematodes OP (Escherichia coli OP50 (hereinafter referred to as “OP”)) that was cultured overnight in TSA was adjusted to 200 mg / ml with M9 buffer 50 μl and 50 μl of fluorescent substance-containing microcapsules were stirred and mixed, and then applied to the NGM plate (due to unknown dissolution amount, but assuming that all DiO was dissolved, about 18 μg / plate). On top of that, several nematodes were transferred using a worm picker and allowed to eat freely for 3 hours.

Confirmation of fluorescent substance inclusion γCD and fluorescent substance-containing microcapsule uptake into nematode 3 ml of 10 mM sodium azide-M9 solution and 0.15 g of Agar (Wako Pure Chemical Industries, Ltd.) were placed in a 15 ml tube and stirred. After that, the tube lid was loosened and dissolved in a microwave oven for several tens of seconds to make a 5% agar solution. The 5% agar solution was quickly dropped on a slide glass (Matsunami) before it hardened, and another flat slide glass was immediately put on the top to make a flat agar pad. When the agar pad solidified, the slide glass on top was slowly removed, and a few drops of 50 mM sodium azide-M9 solution was placed on the agar pad, and a nematode that was scooped with a worm picker was placed on it. A circular micro cover glass (Fisher Scientific) was placed thereon to prepare a nematode sample for microscopic observation.

Results The nematode sample was observed with a fluorescence microscope. FIG. 1 is a photograph of a nematode sample observed with a fluorescence microscope. FIG. 1A shows the case using microcapsules, and FIG. 1B shows the case using γCD. From FIG. 1, it can be seen that the fluorescence intensity is higher when the fluorescent substance inclusion γCD is used, and when the fluorescent substance inclusion γCD is used, a large amount of γCD is ingested under the same conditions.

  From FIG. 1B, it can be seen from the photograph that DiO is absorbed and dispersed from the intestinal lumen into the intestinal cells. On the other hand, in the example of FIG. 1A using microcapsules, it can be seen that DiO stays in the intestinal lumen and is not absorbed into intestinal cells.

(Example 2)
[Confirmation of γCD uptake]
Example 1 was the same as Example 1 except that the dose was reduced to 1/10 (1.425 μg / plate).

Results The nematode sample was observed with a fluorescence microscope. FIG. 2 is a photograph of a nematode sample observed with a fluorescence microscope. From FIG. 2, it can be seen that, in the fluorescent substance inclusion γCD, γCD is ingested even when the fluorescent substance amount is 1.425 μg / plate. That is, it was found that by using γCD, the lipophilic test substance can be taken orally efficiently and reliably.

(Example 3)
(Measurement of the amount of fluorescent substance-containing γCD incorporated into nematodes)
Fluorescent substances - in γCD same manner as in Production Example 1, to prepare a fluorescent substance -γCD clathrate.

The hermaphrodite of the nematode nematode Bristol strain N2 used was subjected to the experiment. Non-pathogenic Escherichia coli Escherichia coli OP50 strain (OP) was used as a food for raising nematodes. A 10 mg wet weight of OP was suspended in 25 μl of M9 buffer and applied to an agar medium (NGM) for nematode growth without addition of peptone. Insect eggs were sprayed on NGM and used in an incubator at 25 ° C. raised to 3 days of age.

Administration of fluorescent substance-containing γCD to nematodes Plates to which DiO was added were prepared under the following three conditions. DiO was added to NGM or applied to the surface so that the DiO concentration was 14.25 μg / plate. When using the fluorescent substance-γCD inclusion body, the fluorescent substance-γCD inclusion body was applied to NGM so that the DiO concentration was 14.25 μg / plate. Fifty of the above three-day-old nematodes were added to each plate and allowed to eat freely for 3 hours.

  50 nematodes each raised for 3 hours under the above conditions were collected and washed. This was physically pulverized using a pestle. The fluorescence intensity of this pulverized liquid was measured (485 nm / 535 nm. 10 s), and the amount of DiO incorporation per nematode was calculated by back calculation. The results are shown in FIG. FIG. 3 is a graph showing DiO uptake (ng) per nematode under each breeding condition.

  From FIG. 3, it can be seen that the nematode raised on the plate (A) to which DiO was added did not ingest DiO. In addition, ingestion of DiO was not observed in nematodes reared on a plate (B) in which DiO was applied to the surface of the agar. On the other hand, in the nematode raised on the plate (C) containing γCD encapsulated with DiO, the intake of DiO (about 0.27 (ng)) was observed.

  From the above results, it was found that when the γCD inclusion body according to the present invention is used, the nematode can be most efficiently incorporated into the test substance when a small amount of the test substance is used. It can be seen that the method using the γCD clathrate according to the present invention is excellent for evaluating a test substance by oral ingestion.

Example 4
[Evaluation of the lifespan of nematodes bred on a medium supplemented with coenzyme Q10 (CoQ10)]
In the present Example, the same thing was used except having used the following culture media.
CoQ10-added agar medium (CoQ10 agar):
75 mg of CoQ10 (manufactured by Sigma) was mixed with 1 ml of ethanol (Wako Pure Chemical Industries), and further mixed with 150 mg of Tween 80, and then mixed with 249 ml of distilled water. Mix with 250 ml of × 2 NGM that has been sterilized by adjusting the concentration to 2 times the usual concentration, and dispense 10 ml each into a petri dish (Kord Products Inc., Bramptom, Ontario, Canada) with a diameter of 60 mm to contain 1500 μg of CoQ10 Plates were prepared. In the same way as in the above experiment, 50 nematodes of 3 days of age were added to each plate, fed with OP, reared daily and confirmed by viability analysis, and compared between CoQ10-added NGM and the control group. investigated.

Agar medium (control agar):
500 ml of liquid NGM before dispensing into a petri dish was prepared. This was used by dispensing 10 ml each into a petri dish (Kord Products Inc., Bramptom, Ontario, Canada) having a diameter of 60 mm.

CoQ10-γCD inclusion bodies:
93.7 g of γCD (CAVAMAX W8 Food: Wacker Chemie AG) and 25.0 g of CoQ10 (manufactured by Sigma) were weighed into a 1000 mL beaker and lightly mixed. 470 mL of deionized water was added to this, and it stirred for 30 minutes at 8,000-12,000 rpm using the homogenizer (ULTRA-TURRAX T25: IKA). The powder was pulverized by spray drying to obtain a CoQ10-γCD inclusion body.

  Moreover, in the following examples, the results obtained by observation were calculated using the Logrank test after calculating the survival rate by Kaplan-Meier method using statistical software Statcel 2 (20) attached to 4 Steps Excel Statistics Second Edition. Differences in survival rates between groups were compared.

Eggs were collected from nematodes in the experimental spawning season, where feeding on CoQ10 agar for the entire period gives the nematode life , and cultured for one day in an incubator at 25 ° C. The OP was cultured in advance at 37 ° C. overnight in TSA until 3 days of age, and then OP was applied on the control agar, CoQ10 agar, and CoQ10-γCD-coated agar to a concentration of 10 mg / plate. The specimens were transferred onto agar and control agar using a worm picker, and then observed every day while being transferred to a plate coated with fresh OP every other day. Individuals that did not move by lightly touching with the picker were considered dead. At this time, the nematodes of the CoQ10 group were bred on CoQ10 agar or CoQ10-γCD-coated agar for the entire breeding period, and the nematodes of the control group were bred on the control agar for the entire breeding period. Also, those that climbed on the petri dish during the observation period and those that went into the agar were omitted from the test population. The results are shown in FIG.

  It can be seen from FIG. 4 that the lifespan of the group (“CoQ10-NGM” in the figure) reared on CoQ10 agar for the entire period is shortened. In addition, the groups reared on CoQ10-γCD-coated agar (“1/10 CoQ10-γCD” and “1/100 CoQ10-γCD” in the figure) had a life span between the control group (“control” in the figure). There was no difference. From this, it was considered that even if nematodes were bred on CoQ10 agar for the entire period, the lifespan of the nematodes was not affected.

(Example 5)
[Evaluation of the lifespan of nematodes reared on a medium supplemented with resveratrol]
In the present Example, the same thing was used except having used the following culture media. Resveratrol is a naturally occurring vegetable polyphenol.
Resveratrol-added agar medium (RES agar):
80 μl of resveratrol solution in which 14.3 mg of resveratrol (manufactured by Sigma) is dissolved in 1 ml of DMSO (Wako Pure Chemical Industries) is added to 100 ml of the above liquid NGM that is dispensed into a petri dish while stirring well, and 50 μM resveratrol is added. Troll-added NGM was prepared, and 10 ml each was dispensed into a petri dish (Kord Products Inc., Bramptom, Ontario, Canada) having a diameter of 60 mm. When 100 μM was prepared, 160 μl was added to 100 ml of NGM to be dispensed in a petri dish with good stirring and adjusted in the same manner.

Agar medium (control agar):
500 ml of liquid NGM before dispensing into a petri dish was prepared. This was used by dispensing 10 ml each into a petri dish (Kord Products Inc., Bramptom, Ontario, Canada) having a diameter of 60 mm.

Resveratrol-γCD inclusion complex:
13.5 g of γCD (CAVAMAX W8 Food: Wacker Chemie AG) was weighed into a 50 mL beaker, and 60 mL of deionized water was added and mixed gently. Resveratrol (product made from Sigma) 1.5g was added to this, and it stirred for 15 minutes at 6,500-9,500 rpm using the homogenizer (ULTRA-TURRAX T25: IKA). The powder was lyophilized to obtain resveratrol-γCD inclusion bodies.

Eggs were collected from nematodes in the experimental spawning season, where feeding on resveratrol agar for the entire period gives the nematode life span , and cultured for one day in an incubator at 25 ° C. After OP was cultured in TSA overnight at 37 ° C. overnight until 3 days of age, OP was adjusted to 10 mg / plate on control agar, resveratrol-agar, and resveratrol-γCD-coated agar. The specimens were transferred onto the VE agar and the control agar applied to each using a worm picker, and then observed every day while moving to a plate with a new OP applied every other day. Death was assumed. At this time, the nematodes of the resveratrol group were reared on resveratrol agar or resveratrol-γCD-coated agar for the entire rearing period, and the nematodes of the control group were kept on the control agar for the entire rearing period. Raised. Also, those that climbed on the petri dish during the observation period and those that went into the agar were omitted from the test population. The results are shown in FIG.

  From FIG. 5, the groups reared on resveratrol agar for the entire period (“50 μMRes” and “100 μMRes” in the figure) and the groups reared on resveratrol-γCD-coated agar (“50Res-γCD” and “ 100 Res-γCD ”) showed no difference in lifespan from the control group (“ control ”in the figure). From this, it was considered that even if nematodes were raised on resveratrol agar for the entire period, the nematode lifespan was not affected.

From the above, it was found that by using γ-cyclodextrin containing a lipophilic test substance, the lipophilic test substance can be evaluated by ingestion of nematodes.

FIG. 1 is a photograph of a nematode sample observed with a fluorescence microscope. FIG. 2 is a photograph of a nematode sample observed with a fluorescence microscope. FIG. 3 is a graph showing DiO uptake (ng) per nematode under each breeding condition. FIG. 4 is a graph comparing the difference in lifespan between the group that was orally administered CoQ10 for the entire period and the control group. FIG. 5 is a graph comparing the difference in lifespan between the group that was orally administered resveratrol for the entire period and the control group.

Claims (3)

  A test substance evaluation method in which a nematode is bred in a medium containing a cyclodextrin clathrate of a fat-soluble test substance, and the effect on the biological function of the test substance taken orally is evaluated.   The test substance evaluation method according to claim 1, wherein the nematode is Caenorhabditis elegans. 3. The test substance evaluation method according to claim 1, wherein the action of the test substance on the biological function is evaluated by a biological index and / or biochemical evaluation.