JP7682014B2 - Agent for preventing or improving diabetic neuropathy - Google Patents

Description

The present invention relates to an agent for preventing or improving diabetic neuropathy.

The number of diabetes patients in Japan is increasing with changes in lifestyle and social environment. Once diabetes develops, it is difficult to cure, and if left untreated, it can lead to complications such as retinopathy, nephropathy, and neuropathy. Diabetic neuropathy is the most common complication of diabetes, and manifests as repeated constipation and diarrhea, erectile dysfunction, dizziness, abnormal sweating, and other autonomic neuropathy, as well as sensory neuropathy, which presents numbness and abnormal sensations. These symptoms are thought to be the result of abnormalities in various parts of the body caused by the loss of peripheral nerve fibers and decreased nerve function. In many cases, symptoms begin with subjective symptoms such as numbness and pain in the feet, and gradually spread to the central nerves. If appropriate treatment is not given, the loss of sensory nerve fibers makes it impossible to feel pain or heat. As a result, foot ulcers form, and if left untreated, this can lead to gangrene, and in the worst case, lower limb amputation, significantly reducing the patient's quality of life (QOL).

The cause of diabetic neuropathy is believed to be metabolic abnormalities in peripheral nerve tissue caused by persistent hyperglycemia, and reduced blood flow due to thickening and narrowing of small blood vessels. Risk factors involved in the onset and progression of diabetic neuropathy include poor glycemic control, duration of diabetes, hypertension, dyslipidemia, smoking, and alcohol consumption. Of these, the most important factor is poor glycemic control, and it is said that neuropathy occurs frequently in cases of poor glycemic control. However, even if poor glycemic control is a risk factor for onset and progression, it has not been necessarily established whether strict glycemic control can suppress onset and progression.

To date, no cure for diabetic neuropathy has been established, and the main treatments are symptomatic treatments to alleviate pain symptoms and treatments to slow the progression of neuropathy, such as with aldose reductase inhibitors (e.g., Epalrestat).

On the other hand, the fruit of the fig (Ficus carica L.) is widely used as food, and the dried fruit and leaves are called figs and fig leaves, respectively, and are used as herbal medicines. It has also been reported that fig extracts have a fat decomposition promoting effect (Patent Document 1) and an anti-influenza virus effect (Patent Document 2), and that fig leaf extracts have a blood sugar lowering effect (Patent Document 3).

However, it is not known that figs are effective in preventing or improving diabetic neuropathy.

JP 2009-242432 A JP 2004-059463 A JP 2009-108025 A

The present invention relates to providing an agent for preventing or improving diabetic neuropathy, which improves the symptoms of diabetic neuropathy.

The inventors have searched for natural materials and found that fig fruit extract has the effect of suppressing diabetes-induced neurological disorders such as decreased nerve conduction velocity, decreased sensory function, decreased balance function, and myelin disorder.

That is, the present invention relates to the following 1) and 2).
1) An agent for preventing or improving diabetic neuropathy, comprising fig or an extract thereof as an active ingredient.
2) A food for preventing or improving diabetic neuropathy, containing fig extract as an active ingredient.

According to the present invention, it is possible to provide a medicine, quasi-drug, food, or supplement for preventing or improving diabetic neuropathy. According to the present invention, it is possible to promote the recovery of nerve function damaged by diabetes, and therefore it is possible to provide a new means for improving QOL.

The effect of fig extract intake on improving decreased nerve conduction velocity. The effect of fig extract intake on improving sensory dysfunction. The effect of fig extract intake on improving impaired balance function. The effect of fig extract intake on improving thinning of myelin diameter. The effect of fig extract intake on improving decreased nerve conduction velocity. The effect of fig extract intake on improving impaired balance function. Participant recruitment and selection process Effect of fig consumption on blood sugar levels Effect of fig intake on nerve function (nerve conduction study) Effect of fig intake on sensory function (plantar sensation test) Effects of fig intake on neurological and sensory functions (perception threshold test)

In the present invention, "fig" refers to Ficus carica L. of the Moraceae family.
The part of the fig used for extraction may be, for example, the stem, shoot, bud, wood, bark, lichen, root, rhizome, corm, tuber, seed, fruit, etc. or a combination thereof, but it is preferred to use the fruit, and more preferably to use dried fruit.

The method for producing the fig extract used in the present invention is not particularly limited, and the extract can be obtained by extracting the above-mentioned plant parts using a known method. In the present invention, extracts produced by solvent extraction using various extraction solvents are preferably used.

As the solvent for extraction, either polar or non-polar solvents can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as toluene; halogenated hydrocarbons such as dichloromethane, chloroform, and dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as fats and oils, waxes, and other oils; and mixtures thereof. Suitable solvents include water, alcohols, and alcohol-water mixtures, and more preferred are alcohol-water mixtures. Ethanol is preferred as the alcohol.
Furthermore, the alcohol-water mixture can be used by mixing in any ratio, but is preferably a mixture having an alcohol ratio of 50 to 99.9% (v/v % at 20° C.), more preferably a mixture having an alcohol ratio of 75 to 99.8%, and even more preferably a mixture having an alcohol ratio of 90 to 99.7%.

The amount of the extraction solvent used is not particularly limited as long as sufficient extraction efficiency is obtained. For example, the amount is preferably 2 to 60 times by mass, more preferably 3 to 30 times by mass, and most preferably 5 to 15 times by mass relative to the amount of the dried figs.
The extraction conditions are not particularly limited as long as sufficient extraction efficiency is obtained. The extraction temperature is preferably 0°C or higher and below the boiling point of the solvent used, more preferably room temperature, but if the extraction temperature is higher, extraction can be completed in a shorter time. The extraction period (time) is preferably 10 minutes to 1 day when the extraction is performed by heating to 40°C or higher, for example, 12 to 24 hours at 40 to 50°C, 2 to 12 hours at 50 to 60°C, and 10 minutes to 2 hours at 60 to 70°C. In addition, when extraction is performed at 40°C or lower, 1 to 30 days is preferable, more preferably 7 to 21 days. For example, 1 to 7 days at 40 to 30°C, 7 to 21 days at 30 to 20°C, and 21 to 30 days at 20 to 10°C are included. More preferably, it is 7 to 14 days at room temperature.

The extraction method is not particularly limited, but may be any of the usual methods such as solid-liquid extraction, liquid-liquid extraction, soaking, decoction, percolation, reflux extraction, pressurized heating extraction, distillation, and supercritical extraction.

The fig extract of the present invention may be a crude product, so long as it meets, for example, food and pharmaceutical acceptable standards and exerts the effects of the present invention. If necessary, it may be subjected to treatments such as removal of inactive contaminants, deodorization, decolorization, etc., by known techniques such as liquid-liquid distribution, solid-liquid distribution, activated carbon treatment, ion exchange resin treatment, etc.
Furthermore, the concentration or proportion of a specific component (fraction) may be increased by appropriately combining known separation and purification methods (for example, removing a fraction having a molecular weight of 10 kD or more by ultrafiltration membrane separation). Purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane separation, high performance liquid chromatography, column chromatography, etc.

In the present invention, the above extract can be used as it is, or it can be used after diluting, concentrating or freeze-drying the extract and preparing it into a powder or paste. It can also be freeze-dried and dissolved or diluted at the time of use with a solvent normally used in extraction, such as water, ethanol, propylene glycol, a water-ethanol mixture, a water-propylene glycol mixture, or a water-1,3-butylene glycol mixture. It can also be used after being encapsulated in a vesicle such as a liposome or a microcapsule.

As shown in the Examples below, when fig extract is added to diabetic neuropathy model animals and raised, neuropathy such as decreased nerve conduction velocity, decreased sensory function, decreased balance function, and myelin disorder is suppressed without lowering blood sugar levels.
Therefore, fig or an extract thereof can be an agent for preventing or improving diabetic neuropathy, and can be used to manufacture an agent for preventing or improving diabetic neuropathy. That is, the fig or an extract thereof of the present invention can be used for preventing or improving diabetic neuropathy. Here, the use may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical procedures, i.e., a concept that does not include a method of surgery, treatment, or diagnosis of humans, more specifically, a concept that does not include a method of surgery, treatment, or diagnosis performed on humans by a physician or a person under the instruction of a physician.

In the present invention, the term "diabetic neuropathy" refers to neuropathy that appears after the onset of diabetes. "Diabetic neuropathy" is divided into distal symmetric polyneuropathy and focal mononeuropathy, and the diabetic neuropathy of the present invention includes both of them.
Distal symmetric polyneuropathy is divided into sensory/motor neuropathy and autonomic neuropathy. In sensory/motor neuropathy, a decrease in nerve conduction velocity is observed in nerve conduction tests early on in the disease, and sensory abnormalities such as spontaneous pain, numbness, paresthesias, and hypoesthesia appear in the distal lower limbs, as well as motor abnormalities such as muscle weakness, muscle atrophy, and impaired balance. As the symptoms ascend, symptoms also appear in the distal upper limbs. Eye and facial movements are also impaired.
Autonomic nervous system disorders can present a variety of symptoms, including pupillary dysfunction, orthostatic hypotension, cardiac nerve disorders (sudden death, painless myocardial infarction), abnormal sweating, gastrointestinal motility disorders (constipation, diarrhea), bladder dysfunction, and erectile dysfunction.
Focal mononeuropathy includes cranial neuropathy (especially external ophthalmoplegia), neuropathy of the trunk and limbs, and diabetic amyotrophy (lumbosacral root plexus neuropathy).

In the present invention, "improvement" of diabetic neuropathy includes "treatment.""Treatment" refers to alleviating the symptoms of diabetic neuropathy, or preventing or delaying the progression (worsening) of the symptoms.
Furthermore, "prevention" of diabetic neuropathy refers to preventing, suppressing or delaying the onset of neuropathy after the onset of diabetes, or reducing the risk of onset of neuropathy.

The agent for preventing or ameliorating diabetic neuropathy of the present invention can itself be a drug, quasi-drug, or food that exhibits the effect of improving diabetic neuropathy, or can be a material or preparation to be added to these.
In addition, the above-mentioned foods (also referred to as "foods for preventing or improving diabetic neuropathy") include not only general foods and beverages, but also foods labeled as such, functional foods, foods for patients, foods for specified health uses, foods with functional claims, and supplements, as necessary.

The above-mentioned pharmaceutical products (including quasi-drug products) containing the fig or its extract of the present invention can be administered in any dosage form, but oral administration is preferred. When administering, the active ingredient can be mixed with a solid or liquid pharmaceutical non-toxic carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical formulation.

Examples of such preparations include solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, suspensions, and emulsions; and lyophilized preparations. These preparations can be prepared by conventional methods for preparations by appropriately adding conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients.

The food containing the fig or its extract of the present invention may take the form of soft drinks, tea drinks, coffee drinks, fruit juice drinks, carbonated drinks, jellies, wafers, biscuits, bread, noodles, sausages, and other foods and nutritional foods, as well as nutritional supplement compositions in the same form as the oral preparations described above (solid preparations such as tablets, capsules, and lozenges). Among these, tablets are preferred.
Foods of various forms can be prepared using the fig or an extract thereof of the present invention alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

The content of fig or its extract in the above-mentioned medicines (including quasi-drugs) and foods varies depending on the form of use, but is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and preferably 10% by mass or less, more preferably 5% by mass or less, and also preferably 0.001 to 10% by mass, more preferably 0.01 to 5% by mass, calculated as the dried matter (for example, dried matter dried in the sun for 1 to 1.5 weeks).

The dosage or intake of the above medicines (including quasi-drugs) and foods may vary according to the condition, body weight, sex, age, or other factors of the subject, but in the case of oral administration or intake, the amount of fig per adult (body weight 60 kg) per day, calculated on a dry matter basis, is preferably 10 g or more, more preferably 30 g or more, and preferably 50 g or less, more preferably 45 g or less, and preferably 10 to 50 g, more preferably 30 to 45 g. Also, the amount of fig extract per day, calculated on a dry matter basis, is preferably 1 g or more, more preferably 4 g or more, and preferably 30 g or less, more preferably 10 g or less, and preferably 1 to 30 g, more preferably 4 to 10 g.

Target subjects for taking or administering the agent for preventing or improving diabetic neuropathy of the present invention include diabetic patients who have developed the above-mentioned sensory/motor neuropathy, autonomic neuropathy, or focal mononeuropathy, or diabetic patients or people with high blood sugar levels, who wish to prevent, suppress, or delay the onset of the neuropathy.

In relation to the above-described embodiment, the present invention further discloses the following aspects.
<1> An agent for preventing or improving diabetic neuropathy, comprising fig or an extract thereof as an active ingredient.
<2> A food for preventing or improving diabetic neuropathy containing fig extract as an active ingredient.

<3> Use of fig or an extract thereof for producing an agent for preventing or improving diabetic neuropathy.
<4> Use of fig extract for producing a food for preventing or improving diabetic neuropathy.

<5> Fig or an extract thereof for preventing or improving diabetic neuropathy.
<6> Non-therapeutic use of fig or an extract thereof for preventing or ameliorating diabetic neuropathy.

<7> A method for preventing or improving diabetic neuropathy, comprising ingesting or administering fig or an extract thereof to a subject in need thereof.

<8> In <1> to <7>, the fig is preferably a fig fruit.
<9> In <1> to <7>, the extract is preferably an alcohol-water mixed solution, more preferably an extract with a 50 to 99.9 V/V % alcohol-water mixed solution.
<10> In <1> to <7>, the diabetic neuropathy is one or more selected from sensory abnormalities such as spontaneous pain in the distal lower limbs, numbness, paresthesias, and hypoesthesia; motor function abnormalities such as muscle weakness, muscle atrophy, and impaired balance; eye movement or facial movement disorders; pupillary dysfunction; orthostatic hypotension; cardiac nerve disorders (sudden death, painless myocardial infarction); sweating disorders; gastrointestinal motility disorders (constipation, diarrhea); bladder dysfunction; erectile dysfunction; cranial nerve disorders (particularly external ophthalmoplegia); trunk and limb neuropathy; and diabetic muscular atrophy (lumbosacral root plexus neuropathy).
<11> In any of the agents or foods of <1> to <4>, the content of fig or an extract thereof in the medicine (including quasi-drugs) or food is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and preferably 10% by mass or less, more preferably 5% by mass or less, or is 0.001 to 10% by mass, more preferably 0.01 to 5% by mass, calculated on a dry matter basis.
<12> In <1> to <11>, the amount of fig or its extract, or the amount of medicine (including quasi-drugs) or food containing the same, administered or ingested orally, is preferably 10 g or more, more preferably 30 g or more, and preferably 50 g or less, more preferably 45 g or less, or preferably 10 to 50 g, more preferably 30 to 45 g, of fig per day per adult (body weight 60 kg), calculated on a dry matter basis. Also, the amount of fig extract is preferably 1 g or more, more preferably 4 g or more, and preferably 30 g or less, more preferably 10 g or less, or preferably 1 to 30 g, more preferably 4 to 10 g, calculated on a dry matter basis.

Production Example 1 Preparation of Fig Extract 500 g of dried fruit (sun-dried fruit) of fig (Moraceae) [Ficus carica L.] was cut into 1/4 size using scissors, and then 5 L of 99.5 V/V% (20°C) ethanol was added and the mixture was immersed at room temperature for 8 days under static conditions. After that, the mixture was separated from the extraction residue by filtration, and the extract was concentrated under reduced pressure. Further, this concentrate was diluted with ion-exchanged water and then freeze-dried. As a result, 99 g of fig extract was obtained.

Example 1: Effect of Fig Extract on a Diabetic Neuropathy Model Induced by Streptozotocin Administration 1) Streptozotocin (150 mg/kg, diluted with citrate buffer pH 4.0) or citrate buffer (pH 4.0) was intraperitoneally administered to 3-week-old C57BL/6J mice, and 4 days after administration, blood was sampled from the tail vein and blood glucose levels were measured. Mice with blood glucose levels of 260 mg/dL or higher were selected and divided into groups (solvent-administered control diet group, streptozotocin-administered control diet group, streptozotocin-administered fig extract diet group, streptozotocin-administered epalrestat (0.04%) diet group; n=8-10 for each group) so that there would be no significant difference in blood glucose levels, and the mice were raised on each diet. The composition of the diets is shown in Table 1.
The "fig extract" used was the extract prepared in Production Example 1.

2) One and three weeks after feeding, blood was collected from the tail and blood glucose levels were measured at regular intervals.

3) Balance function was measured 5 weeks after food loading using a beam test. In the beam test, a convex rod was placed parallel to the ground at a height of 100 cm above the ground, and the mouse was made to walk from the start point to the goal point, and the time required was recorded. The total length of the passage was 50 cm, and the width was designed to be 11 mm at the start point and 6 mm at the goal point. Measurements were taken twice and the average was calculated. Note that the maximum time was set at 20 seconds, and if the mouse was unable to reach the goal by that time, it was recorded as 20 seconds.

4) Six weeks after the food challenge, sensory function was measured using the Von Frey test. In the Von Frey test, a 1.4 g filament was pressed against the mouse's hind paw and the escape response was observed. Ten trials were performed, and the frequency of escape responses was recorded.

5) Five weeks after the dietary loading, the motor nerve conduction velocity (MNCV) of the tibial nerve was measured under anesthesia using an electromyography-evoked potential tester (MEB-9402MB).

6) The diameter of myelin in the nerve was evaluated by calculating the g-ratio. The peroneal nerve after 7 weeks of food loading was pre-fixed with 2.5% glutaraldehyde and post-fixed with 1% osmic acid. After embedding in Epon resin, semi-thin (1.5 μm) sections were prepared and stained with 0.5% toluidine blue.
Statistics were performed using the Kruskal-Wallis test for the Von Frey test, and the Dunnett test for the other tests. The significance level was set at P<0.05.

7) Results a) Blood glucose levels one week after dietary loading are shown in Table 2.
Streptozotocin administration caused a significant increase in blood glucose levels. Furthermore, after 3 weeks of dietary loading, blood glucose levels were 600 mg/dL or higher in all streptozotocin-administered animals. However, no change in blood glucose levels was observed after the ingestion of epalrestat or fig extract diet.

b) Streptozotocin administration significantly decreased nerve conduction velocity in the tibial nerve, a motor nerve. On the other hand, the decrease in nerve conduction velocity caused by streptozotocin administration was significantly improved by the ingestion of epalrestat and fig extract diets (Figure 1).
c) In the Von Frey test, the sensory function was impaired by streptozotocin administration, whereas the sensory function was significantly improved by the ingestion of epalrestat and fig extract diets (Figure 2).
d) A decrease in balance function due to streptozotocin administration was observed in the beam test. However, the decrease in balance function due to streptozotocin administration was significantly improved by the ingestion of the fig extract diet (Figure 3).
e) Histological analysis revealed that streptozotocin administration caused a decrease in myelin diameter. On the other hand, the intake of epalrestat and fig extract diets significantly improved the decrease in myelin diameter caused by streptozotocin administration (Figure 4).

Example 2 Effect of Fig Extract on Diabetic Neuropathy Model in db/db Mice 1) Five-week-old db/db mice (which exhibit hyperphagia and hyperglycemia due to a mutation in the leptin receptor and are used as a type II diabetes model mouse) were divided into three groups (db/db-control diet group, db/db-epalrestat diet group, and db/db-fig extract diet group) so that blood glucose levels were equal (n=10 per group) and were raised on each diet (the composition of the diet was the same as in Table 1 above). As a control group, db/m mice (heterozygotes of db/db mice) were raised on the control diet (db/m-control diet group).

2) Five weeks after feeding, blood was collected from the tail and blood glucose levels were measured at regular intervals.

3) Balance function was measured four weeks after food loading using a beam test. In the beam test, a convex bar was placed parallel to the ground at a height of 100 cm above the ground, and the mouse was made to walk from the start point to the goal point, and the time required was recorded. The total length of the passage was 50 cm, and the width was designed to be 11 mm at the start point and 6 mm at the goal point. Measurements were taken twice and the average was calculated. Note that the maximum time was set to 100 seconds, and if the mouse was unable to reach the goal, it was recorded as 100 seconds.

4) Four weeks after the dietary loading, the motor nerve conduction velocity (MNCV) of the tibial nerve was measured under anesthesia using an electromyographic evoked potential tester (MEB-9402MB). Statistics were performed using the Dunnett test, with a significance level of P<0.05.

5) Results a) Blood glucose levels after 5 weeks of dietary loading are shown in Table 3.
No changes in blood glucose levels were observed with the epalrestat diet or fig extract compared to the db/db-control diet group.

b) Nerve conduction velocity was significantly decreased in the db/db-control group compared to the db/m-control group, whereas nerve conduction velocity was significantly improved in the fig extract group compared to the db/db-control group (Figure 5).
c) Furthermore, in the beam test, the db/db-control diet group showed a decline in balance function compared to the db/m-control diet group, whereas the epalrestat diet and fig extract diet groups showed significant improvements in balance function compared to the db/db-control diet group (Figure 6).

Example 3: Effects of figs on patients with diabetic neuropathy (1) Study outline In order to investigate the effectiveness of figs on diabetic neuropathy, subjects with diabetic neuropathy were asked to take dried mango (control group) or dried fig (fig group) every day for two months. The evaluation items in this study were nerve function and sensory function related to the diagnosis of diabetic neuropathy, subjective symptoms, and the evaluation by a doctor who regularly treats diabetic neuropathy.

(2) Subjects In order to examine the effectiveness of figs against diabetic neuropathy, patients with diabetic neuropathy were included. The criteria for diabetic neuropathy were set with reference to the "Diabetic Neuropathy Association Diagnostic Criteria 2002 Revised Edition" described in the Diabetes Treatment Guidelines 2019. Specifically, the history of diabetes was asked, and if the patient had been diagnosed with diabetes and had an HbA1c value of 6.0 or higher, it was deemed "diabetes is present" and was a required item. In addition, the condition items were "neuropathy" if two or more of items 1-3 of the diagnostic criteria shown below were met, or if item 4 was met. Regarding item 2, vibration sense, it was deemed "applicable" if a decrease was observed in either the medial malleolus or the first toe. In addition, the nerve conduction test was performed on both feet, and if the conduction velocity or amplitude showed abnormal values on both sides, it was deemed "neuropathy is present" (the criteria for abnormal values are described for each measurement item).
In addition, the following 10 exclusion criteria were set and confirmed at the time of informed consent.

<Diagnostic criteria for diabetic neuropathy>

<Exclusion criteria>

(3) Participant recruitment and subject selection flow The participant recruitment and subject selection flow is shown in Figure 7. Through recruitment, 80 people aged 30-65 years old with diabetes and suspected diabetic neuropathy who did not fall under the exclusion criteria were recruited. A screening test was conducted on the 80 candidates, and 40 participants who met the diagnostic criteria for diabetic neuropathy were selected. Just before the final measurement, three people in the control group and one person in the fig group withdrew for personal reasons, so the number of participants who completed the final measurement was 17 in the control group and 19 in the fig group.

(4) Method The study was conducted in an open-label manner. However, participants were not informed of which test product was expected to have activity. Moreover, the examiners performed the measurements without knowing which test product each participant had taken. The subjects were divided into two groups to avoid bias in the following items among those measured during the screening test (nerve conduction velocity, amplitude, Achilles tendon reflex, vibration sense, HbA1c, fasting blood glucose level, age, and male/female ratio). The study period was two months, and participants were instructed to take about 50 g of dried mango (Tomizawa Shoten, Philippines) or dried fig (Tomizawa Shoten, Turkey, Smyrna variety) every day during the study period. A questionnaire and various measurements were conducted at the start of the study (initial measurement), one month later (intermediate measurement), and two months later (final measurement). The following items were measured in the screening test and this study (initial measurement, intermediate measurement, and final measurement), respectively.

<Screening test>
Nerve conduction test, Achilles tendon reflex, vibration sense, blood analysis, interview <Main test (initial measurement, intermediate measurement, final measurement)>
Nerve conduction test, Achilles tendon reflex, vibration sense, blood analysis, interview, sensory threshold test, plantar sensation test, gait, questionnaire (Achilles tendon reflex, vibration sense, and interview were not performed in the initial measurement, and the screening test values were used as the initial measurement results)

(a) Questionnaire survey At the start of the study, participants were asked whether they had any subjective symptoms of numbness using the questionnaire below, and at the end of the study, participants were asked about any changes in symptoms compared to when the study began. The number of people with numbness in each group was as follows: 13 in the control group, 13 in the fig group.

<Questionnaire regarding subjective symptoms of numbness>

(b) Evaluation of neurological and sensory functions (nerve conduction test)
Measurements were taken with the subject lying face down using a nerve conduction measuring device (Omron, HDN-1000). The area where the sensor would come into contact was wiped with a special alcohol sheet (prep pad). Special gel was applied to the electrode parts of the device, and the electrodes were placed at the center of the ankle and Achilles tendon, with the sensor part placed on the midline of the gastrocnemius muscle. With the subject relaxed, a weak pulsed current was passed through the body, and nerve conduction velocity and amplitude were measured. This was performed on both the left and right feet.

"Achilles tendon reflex"
The following measurements were taken by the nurses:
The test participants were asked to kneel backwards on a chair, and the Achilles tendon was tapped 10 times or more using a hammer for testing the Achilles tendon reflex to confirm whether the foot plantar flexed. If the reflex was absent, the reflex was further measured using the augmentation method. This was performed on both the left and right feet. The scores were 1: normal (plantar flexion), 2: weakened (absent with the normal method, plantar flexion with the augmentation method), and 3: absent (absent with the normal method, absent with the augmentation method).
The augmentation method is a method of measuring tendon reflexes in a state where tendon reflexes are more likely to occur by having the participant perform an unrelated movement (clasping their right and left hands together and pulling on each other), as tendon reflexes are less likely to occur when the participant is nervous or concentrating their attention on the test area.

Vibration sense test
The following measurements were taken by the nurses:
The participants sat on a chair with their legs extended. A vibration sense test tuning fork (C128) was vibrated and placed on the medial ankle and the tip of the first toe. The participants were asked to report the time when they no longer felt the vibration, and this was taken as the measurement. The test was performed on both the left and right feet. The scores were 1: normal (sensed for 10 seconds or more) and 2: decreased (disappeared in less than 10 seconds).

"Perception threshold test"
Measurements were taken while lying on one's back using a Neurometer (CPT/C; Neurotron). The area where the sensor was to be placed was wiped with a dedicated alcohol sheet, and the sensor was attached to the measurement site (first toe). Starting with an imperceptible low-intensity electrical stimulus, the current intensity (perception threshold) was recorded at the point where the participant could perceive the stimulus while gradually increasing the current stimulus. Measurements were taken in the following order: 2000 Hz (Aβ fiber: vibration), 250 Hz (Aδ fiber: touch), and 5 Hz (C fiber: pain, warm and cold, autonomic nerves). The measurements were taken on both the left and right feet.

Plantar sensation test
The subjects placed their feet on a plantar sensation evaluation device (Asuka Electric Works, Ltd.) in a sitting position, and the minimum threshold at which they could sense the vibration of the contactor against the sole of the foot was measured. The minimum threshold was measured at a total of four locations, the ball of the big toe and the ball of the little toe of each foot.

(c) Other "Blood Analysis"
Blood samples were collected under fasting conditions by standard methods. That is, participants had dinner by 9 p.m. the previous day, but did not have breakfast. In addition, to prevent hypoglycemia, participants did not take any diabetes medications on the morning of the measurement session. There were no restrictions on other medications.

"Medical interview and doctor's assessment"
A doctor who routinely treats diabetic neuropathy interviewed the subjects about their history of diabetes, whether they had any subjective symptoms of sensory abnormalities in the lower limbs, and the appearance of both feet (below the ankles). Measurements were taken three times in total: at the screening test, during the midterm measurement, and at the end of the test.
Based on the interview and test values (nerve conduction test, Achilles tendon reflex, vibration sense, and perception threshold test), a comparison was made for each participant with the results at the start of the study. The results were judged on a six-point scale: "markedly improved,""improved,""slightlyimproved,""nochange,""slightlyworsened," and "worsened."

(5) Permissible Items During the study period, participants were permitted to continue using any oral medications that they had been taking on a daily basis.

(6) Statistics The obtained values are shown as mean ± standard error. Between-group comparisons were performed using chi-square tests for questionnaires, doctor's judgments, Achilles tendon reflexes, and vibration sense tests. Details are described for each item. For other items, statistical analysis was performed using a linear mixed model with "group" and "time" as fixed effects and "participant" as random effects. In this study, the purpose was to examine the presence or absence of an effect of figs, so "time" was set to two points, the initial measurement and the final measurement. In addition, when measurements were performed on both the left and right feet or at multiple locations, the site was also analyzed as a random effect. Details are described for each item. When there was an interaction, the change from the "initial measurement" at the "final measurement" was calculated, and between-group comparisons were performed using unpaired t tests.
For those who declined the final measurement, the results of the interim measurements for questionnaires, doctor's judgment, Achilles tendon reflex, and vibration sense test were treated as the final measurement results. For items analyzed using linear mixed models, statistics were performed with missing data.

(7) Results 1) Participant background The participant background of this study is shown in Table 4. The test product intake rate was 90% or higher for all participants. The medication status for diabetes mellitus is shown in Table 5.

2) Effect on Blood Glucose Levels Blood analysis was performed to examine the effect of ingestion of each test product on blood glucose levels (Figure 8).
Figure 8: Changes in HbA1c and blood glucose levels over time. Graphs show mean ± standard error. Statistical analysis was performed using a linear mixed model with fixed effects of "group" and "time" and random effect of "participant."

No interactions were observed when comparing the two groups. Furthermore, a paired t test was used to compare HbA1c and fasting blood glucose levels at the end of the study with the initial values for each group, but no significant differences were observed. From these results, no effect was observed on blood glucose levels from taking the test products.

3) Nerve function/sensory function "Achilles tendon reflex"
To examine the effect of fig on nerve function, the Achilles tendon reflex was measured (Table 6). At the end of the test, if the score improved from the initial stage in both legs, the test was deemed "effective," and the number and percentage of subjects who "were effective" are shown (Table 6).
A significant difference was found between the control group and the fig group in the number of people who responded positively, indicating that consuming figs improves neurological disorders.

Nerve conduction test
To investigate the effects of fig on nerve function in detail, nerve conduction studies were performed (Figure 9).
Figure 9: The trends in the average values of nerve conduction velocity (A) and amplitude (B) in both legs are shown. The trends in the average values of nerve conduction velocity (C) and amplitude (D) in the right leg are also shown. Note that an interaction was observed in the nerve conduction velocity of the right leg, so the change from the initial value at the end of the study is shown (E). The graph shows the mean ± standard error. (A, B) Statistical analysis was performed using a linear mixed model, with fixed effects of "group" and "time" and random effects of "participant" and "measurement site". (C, D) Statistical analysis was performed using a linear mixed model, with fixed effects of "group" and "time" and random effect of "participant". (E) Statistical analysis was performed using a t-test. * p<0.05 vs. control

No significant change was observed in the amplitude in both legs between the control and fig groups (Figure 9B). On the other hand, the nerve conduction velocity in both legs tended to improve in the fig group compared to the control group (Figure 9A). Therefore, for reference, the results of conduction velocity and amplitude in the right leg are shown (Figures 9C-E). No significant change was observed in the amplitude in the right leg between the two groups (Figure 9D), but an interaction between "group" and "time" was observed in the conduction velocity (Figure 9C). Therefore, the change in conduction velocity in the right leg from the initial value at the end of the study was calculated and compared between the groups (Figure 9B). As a result, a significant change was observed in the fig group compared to the control group, indicating that fig intake improves nerve damage.

Vibration sense test
A vibration sense test was conducted to examine the effect of fig on sensory function. At the final measurement, the number and percentage of subjects who answered "effective" were shown when the score improved from the initial stage in both feet (Table 7). There was no significant difference between the control group and the fig group in the number of subjects who answered "effective," but the intake of figs did lead to improvement.

Plantar sensation test
To investigate the effect of fig on sensory function in detail, a plantar sensory test was performed (Figure 10).
Figure 10: (A) Plantar sensory thresholds were measured at four locations, two on each side (thenar eminence and hypothenar eminence), and the progress of the average values was shown. (B) Because an interaction between "time" and "group" was observed, the amount of change from the initial value at the end of the study is shown. The graph shows the mean ± standard error. (A) Statistical analysis was performed using a linear mixed model, with "group" and "time" as fixed effects and "participant" and "measurement site" as random effects. (B) Statistical analysis was performed using a t-test. * p<0.05 vs. control

As a result, an interaction was observed between "group" and "time course" (Figure 10A). Furthermore, a significant difference was observed in the amount of change from the initial value at the end of the study in the fig group compared to the control group (Figure 10B), demonstrating that fig intake improves sensory function loss caused by diabetic neuropathy.

"Perception threshold test"
In order to examine the effects of fig on nerve function and sensory function in detail, a sensory threshold test was performed. The sensory threshold test is a test in which currents of different frequencies are passed through the skin to quantify the sensory threshold, and it is said that it can evaluate the function of Aδ fibers and C fibers related to pain sensation that cannot be captured by nerve conduction tests. Specifically, the sensory thresholds of Aβ fibers at 2000 Hz, Aδ fibers at 250 Hz, and C fibers at 5 Hz can be quantified, and the normal values are 2000 Hz: 155-337 μA, 250 Hz: 58-144 μA, and 5 Hz: 27-87 μA, respectively. The average values at each frequency of the participants in this study were 2000 Hz: 286 ± 20 μA, 250 Hz: 113 ± 9 μA, and 5 Hz: 80 ± 5 μA, which are close to the upper limit of normal values, and it was confirmed that the sensory threshold increases due to diabetic neuropathy. Therefore, the sensory threshold of fig was actually examined (Figure 11).
Figure 11: (A) Perceptual sensory thresholds were measured for the left and right at each frequency, and the changes over time in the average values were shown. (B) Because an interaction between "time" and "group" was observed at each frequency, the amount of change from the initial value at the end of the study was shown. The graph shows the mean ± standard error. (A) Statistical analysis was performed using a linear mixed model, with "group" and "time" as fixed effects and "participant" and "measurement site" as random effects. (B) Statistical analysis was performed using a t-test. ## p<0.01 vs. "initial" by Bonferroni test, ** p<0.01 vs. control

As a result, an interaction between "group" and "time" was observed at all frequencies, and furthermore, only in the fig group, the perception threshold was significantly lower at the end compared to the initial value (Figure 11A). Furthermore, the amount of change from the initial value at the end also tended to be lower in the fig group compared to the control group at each frequency, with a significant difference observed at 250 Hz (Figure 11B). These results demonstrate that fig intake improves the decline in nerve and sensory function caused by diabetic neuropathy.

4) Overall assessment and subjective symptoms (Doctor's assessment)
A doctor who treats diabetic neuropathy on a daily basis interviewed participants (at screening, mid-point, and end), and each participant was compared with the start of the study based on the interview and test values (nerve conduction test, Achilles tendon reflex, vibration sense, and perception threshold test). Results were judged on a six-point scale: "markedly improved,""improved,""slightlyimproved,""unchanged,""slightlyworsened," and "worsened." A score of "slightly improved" or better was considered "effective," and a chi-square test was performed.
In the fig intake group, 80% of the participants were judged to be "slightly improved" or better, a significant difference was observed compared to the control group (Table 8), making it clear that fig intake improves diabetic neuropathy.

"Subjective symptoms"
At the start of the study, 26 participants who reported having subjective symptoms of numbness were asked about any changes in symptoms at the end of the study. Evaluation was done on a six-point scale: "markedly improved,""improved,""slightlyimproved,""unchanged,""slightlyworsened," and "worsened." Evaluations of "slightly improved" or higher were considered "effective," and a chi-square test was performed.
As a result, in the fig intake group, 85% of participants who had numbness at the start of the study were rated as "slightly improved" or better, showing a significant difference compared to the control group (Table 9). These results demonstrate that fig intake improves the subjective symptoms of numbness caused by diabetic neuropathy.

Claims (3)

An agent for preventing or improving diabetic neuropathy, comprising an ethanol extract of fig fruit as an active ingredient , wherein the neuropathy is selected from a decrease in nerve conduction velocity, a decrease in sensory function, a decrease in balance function, and myelin disorder. A food for preventing or improving diabetic neuropathy, comprising an ethanol extract of fig fruit as an active ingredient , wherein the neuropathy is selected from a decrease in nerve conduction velocity, a decrease in sensory function, a decrease in balance function, and myelin disorder . The agent according to claim 1 or the food according to claim 2, which suppresses neuropathy without lowering blood sugar levels.