JP2015224950A - Medicinal effect evaluation method of crude drug having hypoglycemic action, quality management method using the medicinal effect evaluation method, and manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicinal effect evaluation method of a crude drug and a medicinal effect evaluation method between lots of crude drug which are more inexpensive, simple, has satisfactory repeatability than conventional methods, and to provide a preparation method of an evaluation sample for evaluating the medicinal effect.SOLUTION: A medicinal effect evaluation method between lots of a crude drug having a hypoglycemic action includes: (a) a process of obtaining an extracted fraction for performing medicinal effect evaluation of the crude drug by a method including a process of, after extracting the crude drug by hot water, precipitating obtained hot water extracted fraction by ethanol; (b) a process of making a silkworm to ingest sugar (A) to increase the concentration of sugar (B) in a fat body or in biological fluid of the silkworm; (c) a process of administering the extracted fraction of the crude drug, which was obtained in the process (a), to the silkworm whose concentration of sugar (B) in a fat body or in biological fluid increased, which was obtained in the process (b); and (d) a process of measuring the concentration of sugar (B) in a fat body or in biological fluid of the silkworm to which the extracted fraction of the crude drug was administered.

Description

  The present invention relates to a medicinal effect evaluation method for a herbal medicine having a hypoglycemic action, a quality control method using the medicinal effect evaluation method, and a manufacturing method.

  So far, the present inventors have proposed that a pathological model of silkworm, which is an invertebrate, can be constructed to evaluate the therapeutic effect and toxicity of pharmaceuticals (Patent Document 1, Non-Patent Documents 1 and 2). In addition, the present inventors can evaluate bacterial, fungal infection and antiviral drugs using a silkworm infection model, and have a common aspect in mammals and silkworms in the pharmacokinetics of antibiotics. (Patent Literature 2, Non-Patent Literature 3).

  Silkworms, which are invertebrates, can easily evaluate therapeutic agents using a large number of individuals. In addition, silkworms are cheaper than mammals and can breed a large number of individuals in a narrow space. The inventors have developed a silkworm infection model for bacterial pathogenicity studies, conducted extensive screening, and successfully identified the virulence genes of Staphylococcus aureus. In addition, the silkworm infection model was also useful in the functional analysis of the virulence genes of Staphylococcus aureus (Non-patent Documents 4 and 5). The large-scale screening using a large number of individuals in the silkworm infection model and the functional analysis of bacterial pathogenicity genes were due to the ability to perform in vivo evaluations utilizing the advantages of silkworms.

  Furthermore, silkworms can be easily injected into a body fluid with a quantitative drug using a syringe, which is difficult for small invertebrates such as nematodes and Drosophila. It is also possible to collect a relatively large amount of body fluid and perform biochemical analysis. Taking advantage of these advantages, the inventors have found a condition for making silkworms hyperglycemic, and can evaluate the therapeutic effect of hypoglycemic drugs such as insulin, have a hypoglycemic activity, and are one of herbal medicines prescribed for diabetic patients. The blood sugar level of hyperglycemic silkworms is reduced by administration of the crude extract of Ziochu, and the active ingredient in the antihyperglycemic activity of hyperglycemic silkworm is a polysaccharide containing a large amount of galactose. It was shown to be galactose (Patent Document 3, Non-Patent Document 6).

  Herbal medicines are used for the purpose of improving the constitution without purifying active ingredients from natural products that have been empirically determined to have medicinal effects, are selected according to the disease, and are used for treatment and prevention. Herbal medicine is widely used as a folk medicine not only in Asia but also in Europe and the United States. The efficacy of herbal medicines often varies depending on the production area, harvest time, post-harvest processing adjustment method, storage condition, and the like. This is because the types, contents (ratio), etc. of active ingredients contained in herbal medicines differ between samples (lots).

In quality control of crude drugs, Liquid chromatography mass spectrometry (LC-MS), fingerprint, quantitative analysis of multi-components by single-marker (QAMS), thin layer chromatography bio-autographic assay (TLC-BAA), DNA molecular marker technique Etc. are used. These experimental methods are intended to clarify the types and contents of active ingredients contained in herbal medicines by analyzing secondary metabolites contained in herbal medicines.
However, the active ingredients of most herbal medicines are unidentified, and it is often difficult to perform quality control that guarantees the medicinal properties of herbal medicines by quantitative methods. Establishment of is desired.

  In addition, attempts have been made to test the therapeutic activity of various crude drug lots using mammals such as mice and rats, but there are problems in terms of cost, time, animal welfare, and the like.

  Furthermore, the preparation of active ingredients of herbal medicines for quality control of herbal medicines is complicated and takes time. For example, the preparation of the active ingredient of ziou required 5 steps, and it took about 5 days to prepare the evaluation sample of the active ingredient. Therefore, establishment of a simpler herbal medicine adjustment method is also desired.

JP 2009-047552 A JP 2010-133975 A International Publication No. 2010/004916

Inagaki Y, Matsumoto Y, Kataoka K, Matsuhashi N, Sekimizu K. Evaluation of drug-induced tissue injury by measuring alanine aminotransferase (ALT) activity in silkworm hemolymph. BMC Pharmacol Toxicol. 8; 13 (1): 13 (2012) Hamamoto H, Tonoike A, Narushima K, Horie R, Sekimizu K. Sikworm as a model animal to evaluate drug candidate toxity and metabolism. Comp Biochem Physiol C. 149 (3): 334-9. (2009) Matsumoto Y, Miyazaki S, Fukunaga DH, Shimizu K, Kawamoto S, Sekimizu K. Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans. J Appl Microbiol. 112 (1): 138-46 (2012) Ikuo M, Kaito C, Sekimizu K. The cvfC operon of Staphylococcus aureus contributes to virulence via expression of the thyA gene. Microb Pathog. 49 (1-2): 1-7 (2010) Miyazaki S, Matsumoto Y, Sekimizu K, Kaito C. Evaluation of Staphylococcus aureus virulence factors using a silkworm model. FEMS Microbiol Lett. 326 (2): 116-24 (2012) Matsumoto Y, Sumiya E, Sugita T, Sekimizu K. An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs. PLoS ONE, 6 (3), 2011 Gao H., Wang, Z., Li, Y., and Qian, Z. Overview of the quality standard research of traditional Chinese medicine, Front Med, 2, 195-202. (2011)

This invention is made | formed in view of the said background art, The subject is providing the drug efficacy evaluation method between lots of the crude drug which has a hypoglycemic effect. In particular, it is to provide a method for evaluating drug efficacy using a method for preparing “extracted fraction of herbal medicine having hypoglycemic action for evaluating drug efficacy” which is cheaper, simpler and has better reproducibility than conventional methods.
Another object of the present invention is to provide a quality control method for herbal medicines using a method for evaluating the efficacy between lots of the herbal medicines. Furthermore, it is providing the manufacturing method of a crude drug including the process of performing quality control using the quality control method of this crude drug.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has obtained an anti-glycemic activity between samples (lots) as a result of administering an extract fraction of Gio, a herbal medicine having a hypoglycemic effect, to hyperglycemic silkworms. Therefore, it was found that the efficacy of herbal medicine can be evaluated using hyperglycemic silkworms.

  In addition, the present inventors have found a method for preparing a herbal medicine extract fraction having a hypoglycemic effect for medicinal efficacy evaluation, which requires fewer work steps than the conventional extraction method and has a shorter required time.

That is, the present invention is a drug efficacy evaluation method between lots of herbal medicine having a hypoglycemic action,
(A) a step of obtaining an extract fraction for evaluating the efficacy of the herbal medicine by a method comprising a step of ethanol precipitation of the obtained hot water extract fraction after hot water extraction of the herbal medicine;
(B) increasing the concentration of the sugar (B) in the fat body or body fluid of the silkworm by ingesting the sugar (A) into the silkworm,
(C) a step of administering the extracted fraction of the crude drug obtained in the step (a) to a silkworm having an increased concentration of the sugar (B) in the fat body or body fluid obtained in the step (b),
(D) a step of measuring the concentration of sugar (B) in a fat body or a body fluid of a silkworm to which the extracted fraction of the herbal medicine is administered,
It is intended to provide a method for evaluating drug efficacy characterized by having

  The present invention also provides a quality control method for a herbal medicine having a hypoglycemic action, characterized by using the above-described medicinal efficacy evaluation method.

  The present invention also provides a method for producing a crude drug having a hypoglycemic action, comprising a step of performing quality control using the quality control method.

The present invention has solved the above problems and problems.
With the evaluation method of the present invention, it is possible to evaluate the drug efficacy between herbal medicine samples (lots) by a quantitative method that has been difficult in the past. In addition, the “extraction method for extracting extract fractions for evaluating drug efficacy” used in the drug efficacy evaluation method of the present invention is simpler than the conventional extraction method, and the extraction time can be shortened. Therefore, the present invention has the effect of improving the reproducibility of experiments, standardization of protocols, and versatility of technology.

  In addition, the crude drug quality control method using the medicinal efficacy evaluation method of the present invention, and the crude drug production method including the step of performing quality control using the quality control method are cheaper, simpler and more reproducible than conventional methods. Has the effect of good properties.

In quality control of crude drugs, evaluation of drug efficacy using mammals such as mice and rats can be considered, but mammals such as mice and rats are expensive and cannot be used as a general-purpose evaluation system. Moreover, in evaluating the efficacy of a crude drug, the evaluation method using mammals takes time. In addition, since lots of samples need to be checked, it is necessary to use a large number of samples, which is problematic from the viewpoint of animal welfare.
On the other hand, silkworms are inexpensive, do not require special breeding space, and have few ethical problems. Therefore, it is useful as an in vivo evaluation model for quality control that requires lot checking of many samples.

It is a figure which shows the protocol for the medicinal efficacy evaluation of the raw drug which has a hypoglycemic effect using a hyperglycemic silkworm. (Left) It is the figure which showed the extraction method of the fraction for evaluating the medicinal effect of a geological. (Right) It is the figure which showed the measuring method of the blood glucose level by a hyperglycemic silkworm. It is a graph which shows the blood glucose level of the hyperglycemia silkworm which administered the Geow sample of each lot. From left to right, hyperglycemic silkworms “administer physiological saline (0.9% NaCl)”, “administer Rehmanniae radix extract 1 (Uchida Japanese medicine [Lot.SU312910]”), “Rehmanniae radix extract 2 (NIB- 0155) ”,“ administer Rehmannia radix extract 3 (NIB-0021) ”,“ administer Rehmannia radix extract 4 (NIB-0045) ”,“ administer Rehmannie radix extract 5 ” "radix extract 6 (NIB-0126) is administered" and "human insulin (3.5 mg / mL) is administered". Significant difference test is performed using Student t test, and error bars indicate standard deviation (SEM).

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

[Method of evaluating drug efficacy between lots of crude drugs with hypoglycemic action using hyperglycemic silkworms]
The drug efficacy evaluation method of the present invention is a drug efficacy evaluation method between lots of crude drugs having a hypoglycemic effect,
(A) a step of obtaining an extract fraction for evaluating the efficacy of the herbal medicine by a method comprising a step of ethanol precipitation of the obtained hot water extract fraction after hot water extraction of the herbal medicine;
(B) increasing the concentration of the sugar (B) in the fat body or body fluid of the silkworm by ingesting the sugar (A) into the silkworm,
(C) a step of administering the extracted fraction of the crude drug obtained in the step (a) to a silkworm having an increased concentration of the sugar (B) in the fat body or body fluid obtained in the step (b),
(D) a step of measuring the concentration of sugar (B) in a fat body or a body fluid of a silkworm to which the extracted fraction of the herbal medicine is administered,
It is a medicinal efficacy evaluation method characterized by having.

  The above-described medicinal effect evaluation method may further include other steps as necessary. Hereinafter, steps (a), (b), (c), and (d) will be described in order.

<Process (a)>
In step (a), first, about the crude drug having hypoglycemic action, after extracting with hot water, the obtained hot water extracted fraction is ethanol precipitated to extract a fraction for evaluating the efficacy of the herbal medicine. To do.

  The herbal medicine having a hypoglycemic action used in the present invention is not particularly limited as long as it has a hypoglycemic action. Specific examples include, but are not limited to: ogi, seisei, auren, kacon, calocon, licorice, kengoshi, trash, sanshuyu, jiou, peonies, sakuhakuhi, daiou, eucommia, carrots, bacmonds, peanuts, brux, etc., polygalactose or Herbal medicines containing a polygalactose derivative are preferred, with diou being particularly preferred.

“Polygalactose” refers to a polysaccharide or oligosaccharide containing a large amount of galactose or a galactose derivative as a structural unit.
Here, “galactose or galactose derivative” refers to D-galactose and its derivatives. In addition, α type and β type are included. That is, in the case of D-galactose, [α] _D ²⁰ = + 144 ° α-type and [α] _D ²⁰ = + 52 ° β-type are included.
Further, it may or may not have a branched structure.

The “monosaccharide constituting the polygalactose as a structural unit” is not particularly limited, but is usually bound by a glycosidic bond. The monosaccharide includes both pyranose type and furanose type.
The total number of monosaccharides (number of bonds) is not limited, but the lower limit is preferably 2 or more, more preferably 10 or more, particularly preferably 100 or more, and still more preferably 1000 or more. The upper limit is preferably 10,000 or less, particularly preferably 1000 or less.
The ratio of the number of galactose or galactose derivatives contained in the polygalactose to the “total number of monosaccharides constituting the polygalactose” is not limited, but the number of monosaccharides constituting the polygalactose is 100. In some cases, the number is preferably 10 to 90, more preferably 20 to 80, and particularly preferably 30 to 70.

Examples of the galactose derivative include those in which a substituent is bonded to the galactose ring. Examples of the substituent include an alkyl group such as a methyl group, an ethyl group, and a propyl group; an alkenyl group such as a vinyl group; an aryl group such as a phenyl group; an arylalkyl group such as a benzyl group; an acetyl group (ethanoyl group); Of the alkanoyl group.
Further, as galactose derivatives, galactose hydrates, aldehydes, O-methylated products, O-sulfated products, pharmacologically acceptable salts thereof; acids such as carboxylic acids, phosphoric acids and sulfonic acids are hydroxyl groups And pharmacologically acceptable salts thereof; UDP (uridine diphosphate) galactose which is a metabolic intermediate of galactose, galacturonic acid and the like.

  As monosaccharides constituting polygalactose, in addition to the galactose or galactose derivative described above, preferable examples include hexoses such as glucose, mannose, sorbose, fructose, tagatose, psicose, talose and rhamnose; ribose, arabinose Pentoses such as xylose and lyxose; or monosaccharides such as derivatives thereof.

  In the examples, it was found that the efficacy of the herbal medicine between lots of herbal medicines can be evaluated by using the present invention having a blood glucose lowering action. However, since the hot water extraction method is used, Natural herb medicines can naturally be evaluated between lots of the herbal medicines. Therefore, the “herbal medicine having a hypoglycemic effect” in the present invention is not limited to Geo.

Even between the same herbal medicines, the kind and content (ratio) of active ingredients contained in herbal medicines vary depending on the place of production, harvest time, post-harvest processing adjustment method, storage condition, etc., and the medicinal effects are often different.
Herbal medicines are managed in lots according to differences in production area, harvest time, post-harvest processing adjustment method, packing form, storage state, appearance, and the like. For example, the “Medical Plant Information Database” (http://mpdb.nibio.go.jp/) provided by the Research Center for Medicinal Plant Resources, National Institute of Biomedical Sciences, crude drugs stored at the National Institute of Biomedical Innovation The lot number (management number: NIB-XXX) (4-digit number) of (model crude drug) can be confirmed.

  It is particularly preferable that the extraction solvent is water in terms of easy extraction of ingredients for medicinal efficacy contained in a crude drug having a hypoglycemic action (hereinafter sometimes simply referred to as “herbal medicine”). . Further, a certain amount of water-soluble organic solvent may be contained in water. Furthermore, water includes water-soluble salts such as sodium chloride and potassium chloride; pH adjusting agents or pH buffering agents such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, hydrochloric acid, sulfuric acid, phosphoric acid, and organic acids. A surfactant or the like may be dissolved.

  It is preferable that the component for evaluating the medicinal effect is extracted with hot water in order to increase the extraction efficiency. The fact that the extraction temperature is important was confirmed by the example (Gizhou), so that the condition can be applied to the whole raw material of crude drugs having hypoglycemic action. “Hot water” in the present invention means water at 60 ° C. or higher. It is preferable to extract at 70-170 degreeC, It is more preferable to extract at 80-140 degreeC, It is especially preferable to extract at 90-110 degreeC.

  The extraction time with hot water is not particularly limited as long as the above-described components for medicinal effect evaluation are extracted, and may be appropriately adjusted depending on the hot water treatment apparatus, the amount of hot water, the shape of the raw material, and the like. After hot water extraction, it is preferable to remove components other than active ingredients.

  The ethanol precipitation of the hot water extraction fraction obtained as described above is particularly preferable in that the medicinal efficacy of the herbal medicine can be evaluated at a low cost and can be evaluated with good reproducibility. In addition, monosaccharides such as glucose contained in the hot water extraction fraction can be easily fractionated and removed. The concentration of ethanol is preferably 65 to 100 parts by mass, more preferably 70 to 99 parts by mass, and particularly preferably 75 to 98 parts by mass, assuming that the total liquid used for precipitation is 100 parts by mass in order to increase extraction efficiency.

<Step (b)>
In the step (b), the sugar (A) is ingested by the silkworm to increase the sugar (B) concentration in the fat body or body fluid of the silkworm.

Further, as such larvae, silkworms are particularly preferable from the following points.
(1) It is easy to obtain.
(2) A breeding method has already been established, and there is convenience in breeding.
(3) The properties similar to the internal organs and organs of mammals such as humans have been known to some extent in previous studies.
(4) Genetic lines have been established and genetic homogeneity has been maintained.
(5) Since it is relatively large, slowly moving, and substantially hairless, it is easy to administer drugs, such as being able to inject quantitatively.
(6) Having fat bodies, the fat bodies can be taken out and the contained substances can be quantified.
(7) It is cheaper than mice, rats, etc., and can be bred with a large number of individuals in a small space, and has few ethical problems, so it is easy to perform screening evaluation.
(8) Evaluation can be performed even when there is only a small amount of the test substance.
(9) It is easy to align individuals in the same state, such as aligning ages.
(10) It is possible to collect body fluids and analyze components such as sugars, lipids and enzymes.

  Experiments with mammals must be conducted according to the international principles 3R: Replacement (development of alternative methods), Reduction (reduction of the number of animals), Refinement (reduction of animal pain) (Russell et al The principles of humane experimental technique. London: Methhuen. 238p. 1959). Using silkworm as a substitute animal is consistent with the idea of Relative Replacement in 3R. An evaluation system using silkworms can be a useful alternative experimental system under the limitation of animal experiments using mammals that are problematic from the ethical viewpoint in drug development and quality control.

  The silkworm is preferably large in terms of ease of intake of sugar (A), ease of administration of extracted herbal extracts, ease of collection of body fluids and fat bodies, and the like. Here, the “large silkworm” is a larva having a body length of 1 cm or more, preferably 1.5 cm or more and 15 cm or less, particularly preferably 2 cm or more and 5 cm or less. From the above points, larvae of 4th to 5th instar are preferable, and 5th instar larvae are particularly preferable.

  There is no restriction | limiting in particular as a method of ingesting sugar (A), According to the objective, it can select suitably. Specifically, for example, injection into body fluids, oral ingestion by addition to feed (food), injection into the intestine, etc. are included. Injection into the intestinal tract; oral intake by addition to feed (food) or the like;

  In invertebrates such as silkworms, blood, lymph and intercellular fluid are not clearly distinguished, and the body fluid is also called hemolymph as having both roles. Vertebrates also have a closed vasculature, whereas arthropods and many mollusks have an open vasculature (a vasculature that has no capillaries and no arteries or veins connected).

The sugar (A) is not particularly limited as long as it increases the concentration of sugar (B) in the fat body or body fluid of the silkworm. For example, glucose, heptose, hexose, pentose, tetrose, triose Monosaccharides such as trehalose, maltose, lactose, sucrose, cellobiose, nigerose and sophorose; oligosaccharides such as fructooligosaccharides, galactooligosaccharides, and dairy oligosaccharides; Polysaccharides; deoxy sugars; oxidation products of sugars such as uronic acid; sugar alcohols such as sorbit and arabit, and the like, may increase the concentration of sugar (B).
Among these, glucose, sucrose, oligosaccharide, glycogen or starch is particularly preferable from the viewpoint of increasing the glucose concentration and / or trehalose concentration in the body fluid of silkworm, the point corresponding to human clinical practice, and the like. These may be used alone or in combination of two or more.

The intake of sugar (A) to the silkworm is not particularly limited as long as the sugar (B) in the fat body or body fluid of the silkworm can be sufficiently increased for the evaluation of the medicinal effect, but the feed (food) and sugar (A ) Is preferably 1 part by mass to 20 parts by mass, more preferably 5 parts by mass to 18 parts by mass, and particularly preferably 8 parts by mass to 20 parts by mass.
In addition, the intake time of the sugar (A) that raises the sugar (B) in the fat body or body fluid of the silkworm to a sufficient extent for evaluating the efficacy is not particularly limited, but is preferably 1 minute to 10 days, preferably 5 minutes to One day is more preferable, and 30 minutes to 2 hours is particularly preferable.
The intake rate of the sugar (A) is preferably a value range obtained by dividing the preferable range of the intake amount by the preferable range of the intake time.

In the step (b), it is essential to increase the sugar (B) concentration in the fat body or body fluid of the silkworm. It is preferable to increase or confirm that the sugar (B) concentration in either the fat body or body fluid of the silkworm is increased, and the total concentration of sugar (B) in the fat body and body fluid of the silkworm is increased. It is more preferable to confirm that it has been increased or increased, and it is particularly preferable to confirm that the concentration of sugar (B) in the body fluid has been increased or increased.
It is preferable to damage the silkworm with scissors and take out the liquid therefrom, and measure the concentration of sugar (B) in the liquid.

  As the sugar (B), in particular, the concentration in the fat body or body fluid is increased by ingestion of the sugar (A), and the decrease in the concentration can be measured when the extract of herbal medicine is administered. Although there is no limitation, glucose, trehalose, etc. are mentioned, for example. The sugar (B) may be one kind or a total concentration of two or more kinds. Depending on the quantification method of sugar (B), the total amount of a plurality of types of sugars may be quantified, but in that case, sugar (B) is not limited to one.

  Sugar (A) and sugar (B) may be the same or different. For example, in the case of a silkworm, when sucrose is administered as a sugar (A), the glucose concentration and trehalose concentration in the body fluid of the silkworm immediately increase. The sugar (B) in this case is glucose and trehalose.

  The method for quantifying the sugar (B) is not limited, but the quantification of all saccharides includes the phenol sulfate method, the anthrone sulfate method, the carbazole sulfate method, etc .; the glucose quantification method includes the glucose oxidase method and the like.

The “degree of increase in the concentration of sugar (B)” in “the silkworm having an increased concentration of sugar (B) in the fat body or body fluid” obtained in the step (b) is an extent to which the medicinal efficacy of the crude drug can be evaluated. Is not particularly limited, but is preferably 1.5 to 100 times, more preferably 1.5 to 8 times, and more preferably 1.5 to 3 times is particularly preferable.
If the “degree of increase in the concentration of sugar (B)” is too low, the evaluation of the efficacy of the crude drug may not be confirmed. On the other hand, the upper limit may not be higher than the above range, or may not be necessary. In addition, if an attempt is made to make it higher than necessary, an individual's disorder other than an increase in blood glucose level may occur, and the individual may die in a significant case.

Specifically, the concentration of the sugar (B) in the body fluid is preferably 7 mg / mL (μg / μL) to 200 mg / mL (μg / μL), and preferably 8 mg / mL to 150 mg / mL. More preferably, it is particularly preferably 10 mg / mL to 100 mg / mL. The glucose concentration in the fat body is preferably 10 ng / μg to 1000000 ng / μg, more preferably 100 ng / μg to 100000 ng / μg, and particularly preferably 1000 ng / μg to 20000 ng / μg. .
The measurement method and expression method of the concentration are the same as those in the examples.

  The time from the intake of sugar (A) to the confirmation of the increase in the concentration of sugar (B) in the fat body or body fluid is not particularly limited, but is preferably 1 minute to 1 day, preferably 10 minutes to 15 hours is more preferable, and 30 minutes to 10 hours is particularly preferable.

Silkworm has a mechanism to maintain homeostasis of blood glucose level, can store sugar in fat body corresponding to muscle and human liver, and has ombibixin which is an insulin-like peptide hormone. Downstream is an insulin signaling pathway that includes the MAPK signaling pathway similar to that in humans.
In addition, recombinant human insulin has an action of enhancing the uptake of sugar in the fat body of silkworm through the activation of PI3 kinase. In other words, silkworms are thought to have a mechanism similar to that of human blood glucose control.

<Step (c)>
In the step (c), the extract of the herbal medicine obtained in the step (a) is added to the silkworm having an increased sugar (B) concentration in the fat body or body fluid obtained in the step (b). Let it be administered.

  There is no restriction | limiting in particular as a method of administering the extraction fraction of a crude drug, According to the objective, it can select suitably. Specifically, for example, injection into a body fluid; oral ingestion by addition to feed (food), injection into the intestinal tract; In terms of simplicity and compatibility with humans, injection into the intestine or oral ingestion is preferred. There is no restriction | limiting in particular as dosage of the extract of a crude drug, According to the administration method etc., it can select suitably. It is also preferable to dilute and administer with physiological saline, water or the like.

There is no particular limitation on the timing of administration of the extract of the crude drug, and immediately after the intake of the sugar (A) in step (a), the concentration of the sugar (B) in the fat body or body fluid of the silkworm is almost the maximum value. The concentration of sugar (B) in the body fluid recovers to a normal value, and the concentration of sugar (B) in the fat body or body fluid of the silkworm is almost the maximum value. The point of reaching is preferable in terms of obtaining reproducibility of the experimental results.
Specifically, it is preferably between “immediately after ingesting sugar (A)” and “after 24 hours from ingesting sugar (A)”, and “immediately after ingesting sugar (A)” To “after 6 hours have passed since the sugar (A) has been ingested”, and “just after the sugar (A) has been ingested” is particularly preferable.

<Step (d)>
In the step (d), the concentration of the sugar (B) in the fat body or body fluid of the silkworm to which the herbal extract extract is administered is measured. As for the method of collecting the measurement sample, it is preferable to dissect and collect the fat body, and the body fluid is preferably collected from the cut.

  Examples of the method for measuring the type of sugar (B), the concentration of sugar (B), and the concentration of glucose include the same methods as those described in the step (b). That is, as the sugar (B), the concentration in the fat body or body fluid is increased by the intake of the sugar (A), and the decrease in the concentration can be measured when the test substance is administered. Although there is no limitation, glucose, trehalose, etc. are mentioned, for example. Moreover, sugar (B) is not limited to 1 type, You may measure the total amount of all the sugars (B). However, the saccharide (B) does not include galactose (derivative) or polygalactose.

  For example, in the case of silkworms, glucose and trehalose concentrations in the body fluid of silkworms often increase due to ingestion of sugar (A), and therefore sugar (B) is preferably glucose and / or trehalose. Further, when the galactose (derivative) administered in the step (b) is detected in the body fluid, the saccharide (B) alone is excluded from the amount of galactose (derivative), or the sugar (B) It is preferable to quantify only the glucose in the water.

  As described above, the sugar (B) quantification method includes phenol sulfate method, anthrone sulfate method, carbazole sulfate method and the like for quantification of all saccharides; Can be mentioned.

There is no particular limitation on the time when the concentration of sugar (B) in the fat body or body fluid is measured. Immediately after the extract of herbal medicine is administered in step (c), the sugar ( The selection may be made from the period until the effect of decreasing the concentration in B) is no longer observed.
Specifically, for example, 1 minute to 1 day is preferable, 30 minutes to 10 hours are more preferable, and 60 minutes to 8 hours are particularly preferable from the time when the extract of herbal medicine is administered.

  When measuring the concentration of sugar (B), use a “skin that has increased the concentration of sugar (B) in the fat body or body fluid” as a negative control, in which the extract of herbal medicine is not administered. Is preferred. For the negative control, for example, it is preferable to administer only the same amount of physiological saline. Compared with the negative control, it is preferable to evaluate the medicinal effect of the extracted fraction of herbal medicine depending on how much the concentration of sugar (B) has been reduced.

[Quality control method of herbal medicine with hypoglycemic action using hyperglycemic silkworm]
The quality control method of the present invention is a quality control method for a herbal medicine having a hypoglycemic effect, characterized by using the above-described medicinal effect evaluation method.

  The quality control method of the present invention may use another method and / or include other steps in addition to the above-described drug efficacy evaluation method as necessary.

  The quality control method of the present invention can be used for quality control of raw materials, extracts extracted from raw materials, final products, and the like.

[Method for producing crude drug having hypoglycemic effect using hyperglycemic silkworm]
The manufacturing method of the present invention is a manufacturing method characterized by including a step of quality control using the quality control method.

  The dosage form of the herbal medicine produced by the production method of the present invention is not particularly limited. Examples of the preparation for oral administration include tablets, pills, granules, capsules, powders, liquids, suspensions, Syrups, sublinguals and the like can be mentioned, and preparations for parenteral administration include injections, transdermal absorption agents, inhalants, suppositories and the like.

  In formulating, a pharmaceutically acceptable carrier can be mixed. The type and composition of the carrier can be appropriately determined depending on the administration route and administration method. For example, water, alcohol, edible oil, or the like can be used as the liquid carrier. As the solid carrier, amino acids such as lysine, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, cellulose derivatives such as hydroxylpropylcellulose, and the like can be used.

  Furthermore, in formulation, isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, excipients, binders, disintegrants, diluents, buffers, colorants, wearing Various pharmaceutical additives such as fragrances can be blended. In the case of injections, sterilization is performed together with a suitable carrier to obtain a drug.

[Action]
In the present invention, the action / principle that can evaluate the efficacy between lots of crude drugs having hypoglycemic action with a simple protocol is not clear, and the present invention is not limited to the scope of such action / principle. The following can be considered.
Conventionally, in order to evaluate the efficacy between lots (samples), a multi-stage extraction process has been required. However, the present invention is based on a two-stage process of ethanol precipitation after hot water extraction. It was found that a “fraction for evaluating the efficacy of a crude drug with action” can be easily obtained.
That is, it is considered that a suitable extraction fraction (including polygalactose) for evaluating the medicinal effect of hypoglycemic action can be extracted with hot water and obtained by ethanol precipitation.
Moreover, since monosaccharides, such as glucose, were able to be removed by ethanol precipitation, it was considered that a suitable fraction could be extracted for easy drug efficacy evaluation.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example and a test example, this invention is not limited to specific ranges, such as a following example.

[Evaluation example]
<Type of silkworm, breeding conditions, injection conditions>
Silkworm fertilized eggs (crossbred Hu • Yo × Tukuba • Ne) were purchased from Ehime Yoro Co., Ltd.
The hatched larvae were given artificial feed silk mate 2S (manufactured by Nippon Nosan Kogyo Co., Ltd.) at room temperature to grow to 5th instar larvae. The breeding container used was a square No. 2 petri dish (Eiken Equipment Co., Ltd.) from the eggs to the 2nd instar larvae, and a disposable plastic food pack (Food Pack FD Daifuka, Chuo Chemical Co., Ltd.) was used thereafter. . The breeding temperature was 27 ° C. Unless otherwise specified, larvae on the 1st day of 5th instar, which were fasted after 4th inning sleep, were used in the experiments.
As a sugar-added sample, D-glucose was prepared by mixing artificial feed silk mate 2S (manufactured by Nippon Nosan Kogyo Co., Ltd.) with various weight ratios.
In the silkworm injection experiment, 0.05 mL of a sample was injected into a body segment having a pattern of the fifth body segment of a 5th instar larva. As the syringe barrel (1 mL) and the injection needle (27G × 3/4), those purchased from Terumo Corporation were used.

<Quantification method of blood glucose level>
Body fluid (20 μL) was taken from a cut with scissors on the first proleg and mixed with 9 volumes of 0.6 N perchloric acid to precipitate the protein. Centrifugation was performed at 3,000 rpm for 10 minutes, and the supernatant was used as a body fluid extract.
The total amount of sugar in silkworm body fluid was quantified by the phenol sulfate method (Hodge et al.). 100 μL of bodily fluid extract diluted to an appropriate concentration with distilled water and 100 μL of 5% (w / v) phenol aqueous solution were mixed, 500 μL of concentrated sulfuric acid was added, and the mixture was vigorously stirred and allowed to stand at room temperature for 20 minutes, followed by absorbance at 490 nm. Was measured. A glucose aqueous solution was used as a standard sugar solution.

<Preparation of a new crude crude extract fraction>
MilliQ water was added to 5 g each of ground yellow (Uchida Japanese medicine [Lot. SU312910], NIB-0021, NIB-0045, NIB-0071, NIB-0126, NIB-0155) to 20 mL. After boiling for 30 minutes, the mixture was filtered using filter paper (3MM), and 38 mL of ethanol was added to 2 mL of the filtrate and stirred. Centrifugation was performed at 15,000 rpm (20,400 G) for 3 minutes, the supernatant was discarded, and the precipitate fraction was left overnight in a desiccator (manufactured by ASONE Co., Ltd.) to obtain a crude crude extract fraction.

<Reagent>
Recombinant human insulin was purchased from Sigma and used by dissolving in physiological saline containing 0.1% by mass acetic acid (0.9% by mass NaCl).

Example 1
[Evaluation of efficacy of crude drugs with hypoglycemic action extracted by simple protocol]
Since the hot water extract fraction of Diou, which has a hypoglycemic action, contains abundant monosaccharides such as glucose, the blood sugar level of silkworms rather increases when administered directly to silkworms. So far, using hyperglycemic silkworms, it has been found that the active ingredient in the crudely extracted fraction of Dio is polygalactose (Non-patent Document 6). However, the preparation of the active ingredient of the conventional Geow requires work in 5 stages, and it takes 5 days to prepare the sample (Non-patent Document 6, Table 1 right). Therefore, paying attention to the fact that the active ingredient of Diou is heat-resistant polygalactose, a quick and simple method for preparing the active ingredient in Diou was established.

Table 1 shows the extraction method (left) used in the present invention and the conventional extraction method (right). Conventional extraction methods are described in, for example, Patent Document 3, Non-Patent Document 6, Masaru Kinoshita et al., Pharmaceutical Journal, 1992, Vol. 112, No. 6, p. 393-400 and the like.
Polygalactose can be extracted with hot water due to its chemical properties, and it is expected that monosaccharides such as glucose can be easily fractionated by ethanol precipitation. Therefore, the protocol shown on the left of Table 1 was used.

After obtaining hot water extraction fractions from 5 g of each of the six lots of geows, 0.2 g of crude extraction fractions were obtained in a total of two steps including ethanol precipitation (left in FIG. 1). This was dissolved in physiological saline so that the final concentration would be 1 mg / mL, and used as an extract fraction for a medicinal effect test.
These six lotus extract fractions were injected into the hyperglycemic silkworm obtained by the above method, and after 6 hours, the body fluid of the silkworm was collected and the blood glucose level was measured by the phenol-sulfuric acid method (right in FIG. 1).

Comparative Example 1
[Evaluation of efficacy of herbal medicine with hypoglycemic action extracted by conventional protocol]
Uchida Japanese medicine [Lot. The extract fraction obtained by the conventional extraction method shown in the right of Table 1 above from 200 g of SU312910] was dissolved in physiological saline so that the final concentration would be 1 mg / mL. did. The obtained extracted fraction was injected into the hyperglycemic silkworm obtained by the above method, and the body fluid of the silkworm was collected 6 hours later, and the blood glucose level was measured by the phenol sulfate method (right in FIG. 1).
As a result, Uchida Japanese medicine [Lot. SU312910] was confirmed to have hypoglycemic activity against hyperglycemic silkworms using the conventional extraction method shown in Table 1 right.

The result of Example 1 is shown in FIG.
From the results of Comparative Example 1, Uchida Japanese medicine [Lot. From the SU312910], the blood glucose level of hyperglycemic silkworms to which the extracted fraction obtained using the extraction method in the present invention (the left column in Table 1) was administered was statistically significantly different from that of the saline administration group (P <0.05) decreased (in FIG. 2, Rehmanniae radix extract 1). In addition, it was confirmed that the blood glucose level significantly decreased as a result of administration to hyperglycemic silkworms even in other lot numbers (NIB-0155 and NIB-0021) (Rehmanniae radix extract 2-3 in FIG. 2).
On the other hand, the blood glucose level of hyperglycemic silkworms to which the extract fractions obtained from NIB-0045, NIB-0071, and NIB-0126 were administered showed a statistically significant decrease in comparison with the physiological saline administration group. (P> 0.05) (Rehmanniae radix extract 4-6 in FIG. 2).

  Based on the above results, an experimental scheme was established to obtain a simple rough-cut fraction of geow in a short time with a small amount of starting material. In addition, it was shown that the difference in activity between the lots of Giou obtained by this extraction method can be evaluated by a hypoglycemic assay using hyperglycemic silkworms. From the above results, it is considered that the medicinal effects of herbal medicines having a hypoglycemic effect, such as diau, can be evaluated using hyperglycemic silkworms.

  In quality control of crude drugs, it is important that they are inexpensive, simple and reproducible. In accordance with the examples of the present invention, a new crude extract fraction preparation method yielded a sample with hypoglycemic activity of hyperglycemic silkworms using 5 g of geome, which is about one-fourth the amount of the previous method. . It is considered that polygalactose, which is an active ingredient of diau, was concentrated by this method. In addition, the work process for obtaining the above active ingredients (fractions for evaluating medicinal effects) was shortened from 5 steps to 2 steps, and the required time was shortened from 5 days to 1 day (Table 1). Easier work is considered to improve the reproducibility of experiments, standardization of protocols, and versatility of technology.

  The method using silkworms is considered to be useful not only for evaluating the efficacy of crude drugs but also for determining the presence or absence of toxic substances. The inventors have so far found that silkworms die by administration of various toxic compounds and bacterial toxins, and reported that LD50 values, which are indicators of toxicity to individuals, are in good agreement between silkworms and mammals. (Hossain et al., 2006; Patent Document 1, Non-Patent Document 2). Furthermore, even a dose at which silkworms do not die can be evaluated by monitoring the activity of ALT, a tissue damage marker, in silkworm body fluids (Non-patent Document 1). This result suggests that silkworms can be used to evaluate toxic substances in herbal medicines.

According to the present invention, a simple protocol for evaluating the drug efficacy of a herbal medicine having a hypoglycemic effect was created, and the difference in drug efficacy between the lotus of Giou was evaluated using a hyperglycemic silkworm.
From this, it was found that silkworms are useful as biomonitors for herbal medicine quality control using medicinal efficacy as an index. And it proposes that the medicinal effect of a crude drug can be evaluated using a silkworm, and it is thought that the evaluation technique in vivo using a silkworm can be utilized for the quality control of a crude drug.

  Since the above-described method for quality control of crude drugs is possible by using the method for evaluating the efficacy of the crude drug of the present invention, it is widely used for manufacturing control, quality control, etc. of crude drugs and herbal preparations containing crude drugs. is there.

Claims (5)

A method for evaluating the efficacy between lots of herbal medicines having a hypoglycemic action,
(A) a step of obtaining an extract fraction for evaluating the efficacy of the herbal medicine by a method comprising a step of ethanol precipitation of the obtained hot water extract fraction after hot water extraction of the herbal medicine;
(B) increasing the concentration of the sugar (B) in the fat body or body fluid of the silkworm by ingesting the sugar (A) into the silkworm,
(C) a step of administering the extracted fraction of the crude drug obtained in the step (a) to a silkworm having an increased concentration of the sugar (B) in the fat body or body fluid obtained in the step (b),
(D) a step of measuring the concentration of sugar (B) in a fat body or a body fluid of a silkworm to which the extracted fraction of the herbal medicine is administered,
A method for evaluating drug efficacy, comprising:   The medicinal effect evaluation method according to claim 1, wherein the herbal medicine having a hypoglycemic action contains polygalactose.   The method for evaluating the efficacy of claim 1 or 2, wherein the herbal medicine having a hypoglycemic action is Diou.   A quality control method for a crude drug having a hypoglycemic action, wherein the drug efficacy evaluation method according to any one of claims 1 to 3 is used.   A method for producing a crude drug having a hypoglycemic action, comprising a step of performing quality control using the quality control method according to claim 4.

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