Composition for preventing, alleviating or treating cognitive dysfunction, containing oxya chinensis sinuosa extract as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating or treating cognitive dysfunction, containing an Oxya chinensis sinuosa extract as an active ingredient. More specifically, when the Oxya chinensis sinuosa extract of the present invention was administered to a scopolamine-induced cognitive dysfunction animal model, cognitive functions of learning ability and memory were enhanced, and the expression of choline acetyltransferase (ChAT) and neuronal nuclear antigen (NeuN) proteins was increased. Therefore, the Oxya chinensis sinuosa extract of the present invention can be effectively used as a material for health functional foods, medicines, feed additives for companion animals, and the like for preventing, alleviating or treating cognitive dysfunction.

Description

Composition for preventing, improving or treating cognitive dysfunction containing rice locust extract as an active ingredient

[0001] The present invention relates to a composition for preventing, improving or treating cognitive dysfunction containing a rice locust extract as an active ingredient.

[0002]

[0003] This research was supported by the National Research Foundation of Korea (Mid-career Researcher Support Program) funded by the government (Ministry of Science and ICT) (NRF-2021R1A2C1005980).

[0004] Cognitive ability encompasses memory, spatial and temporal perception, judgment, language, and computational abilities, enabling individuals to carry out daily life without assistance. However, any form of brain damage, whether rapid neuronal cell death caused by stroke or trauma, or slow neuronal cell death resulting from degenerative brain diseases, immediately leads to irreversible dysfunction of neural circuits. Ultimately, cognitive ability declines, leading to conditions such as forgetfulness, memory impairment, dementia, and Alzheimer's disease, making it impossible to maintain a normal level of daily life. The number of patients with cognitive impairment, which not only causes personal suffering but also leads to a decline in the productive population, is expected to increase to over 74.7 million by 2030, making the development of health functional foods and pharmaceuticals for these disorders an urgent issue.

[0005] Meanwhile, companion animals, unlike the traditional concept of pets, are living creatures that share emotional bonds and coexist with humans. In modern society, companion animals are increasingly recognized as family members, leading to increased interest in their nutrition and health, and the development of diverse related products. Cognitive impairment (cognitive dysfunction) in companion animals is classified as a disease similar to dementia in humans. Given the inability of companion animals to communicate, key symptoms that may indicate cognitive impairment include decreased memory and intimacy with family members, changes in sleep patterns, deviations in elimination habits, changes in eating habits, and aversion to unfamiliar environments. With the average lifespan of companion dogs and cats estimated at approximately 15 years, cognitive impairment typically begins to develop in companion animals around the age of 11. As in humans, cognitive impairment in companion animals is believed to be closely related to brain function. Therefore, there is a growing need to develop pharmaceuticals or functional feeds that address cognitive impairment and improve brain function in companion animals.

[0006] Meanwhile, the rice grasshopper (Oxya chinensis sinuosa) belongs to the Orthoptera order and the Locust family. It is 30–38 cm long, has a yellow-green body, and a yellow-brown head and thorax. It inhabits grasslands near rice paddies and cultivated fields. It was recognized as a major pest of rice, and its numbers declined due to the use of pesticides. However, its numbers have recently been on the rise again.

[0007] Prior art related to cognitive dysfunction includes Korean Patent Publication No. 2023-0140344, which discloses a "composition for improving cognitive function and preventing or treating degenerative brain diseases, comprising a wasp venom derived from the black hornet as an active ingredient," and Korean Patent Publication No. 2022-0166062, which discloses a "composition for preventing, improving, or treating degenerative brain diseases, comprising a mixture of licorice, sorghum, and silkworm pupa as an active ingredient." However, the "composition for preventing, improving, or treating cognitive dysfunction, comprising a rice locust extract as an active ingredient," according to the present invention, has not yet been disclosed.

[0008] The present invention was derived from the above-mentioned needs, and the present invention provides a composition for preventing, improving, or treating cognitive dysfunction containing a rice grasshopper extract as an active ingredient, and more specifically, the present invention was completed by confirming that when the rice grasshopper extract of the present invention was administered to an animal model of cognitive dysfunction induced by scopolamine, cognitive functions such as learning ability and memory were improved, and the expression of ChAT (choline acetyltransferase) and NeuN (neuronal nuclear antigen) proteins was increased.

[0009] In order to solve the above problem, the present invention provides a health functional food composition for improving cognitive function or memory containing a rice locust extract as an effective ingredient.

[0010] In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction containing a rice locust extract as an active ingredient.

[0011] In addition, the present invention provides a veterinary composition for preventing or treating cognitive dysfunction containing a rice locust extract as an active ingredient.

[0012] In addition, the present invention provides a pet feed additive for improving cognitive function or memory containing a rice locust extract as an effective ingredient.

[0013] In addition, the present invention provides a pet food containing the above feed additive.

[0014] The present invention relates to a composition for preventing, improving or treating cognitive dysfunction containing a rice locust extract as an active ingredient. Specifically, when the rice locust extract of the present invention is administered to an animal model of cognitive dysfunction induced by scopolamine, cognitive functions such as learning ability and memory are improved, and the expression of ChAT (choline acetyltransferase) and NeuN (neuronal nuclear antigen) proteins is increased.

[0015] Figure 1 is a schematic diagram of an experiment to confirm the efficacy of improving cognitive function by administering the rice locust extract of the present invention to a mouse model of cognitive dysfunction induced by scopolamine.

[0016] Figure 2 shows the results of confirming the change in body weight according to the administration of the rice locust extract of the present invention in a mouse model of cognitive dysfunction induced by scopolamine. CTL is a control group treated with physiological saline, Sco is a group administered scopolamine (1 mg / kg) alone, Sco+Don is a positive control group administered scopolamine and donepezil (3 mg / kg), Sco+Ocs is a group administered scopolamine and rice locust extract (10 g / kg), and Ocs is a group administered rice locust extract alone.

[0017] Figure 3 shows the results of a water maze test confirming the memory improvement effect of administering the rice locust extract of the present invention to a mouse model of cognitive dysfunction induced by scopolamine. A is the result measuring the number of times the target point was passed, B is the result measuring the time spent at the target point, and C is the result measuring the time taken to find the target point.

[0018] Figure 4 shows the results of confirming the memory improvement effect of administering the rice locust extract of the present invention in a mouse model of cognitive dysfunction induced by scopolamine through a Y-maze test.

[0019] Figure 5 shows the results of confirming the memory improvement effect of administering the rice locust extract of the present invention in a mouse model of cognitive dysfunction induced by scopolamine through a light / dark test.

[0020] Figure 6 shows changes in the expression of ChAT (choline acetyltransferase) and NeuN (neuronal nuclear antigen) proteins following administration of the rice locust extract of the present invention in a scopolamine-induced cognitive dysfunction mouse model. (A) is a band photograph and (B) is a graph quantifying the same. α-tubulin is a loading control.

[0021] In order to achieve the purpose of the present invention, the present invention provides a health functional food composition for improving cognitive function or memory containing a rice locust extract as an effective ingredient.

[0022] The above rice grasshopper extract can be prepared by a method including, but not limited to, the following steps:

[0023] (1) A step of extracting rice grasshoppers by adding an extraction solvent;

[0024] (2) a step of filtering the extract of step (1); and

[0025] (3) A step of preparing an extract by drying the filtered extract of step (2).

[0026] In the above step (1), the extraction solvent is preferably selected from water, C1 to C4 lower alcohol, or a mixture thereof, more preferably water, but is not limited thereto.

[0027] In the above manufacturing method, the extraction method may be any conventional method known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent is preferably added in an amount of 1 to 20 times the weight of the rice grasshopper, more preferably 5 to 15 times, and the extraction time is preferably 1 to 30 hours, and even more preferably 5 to 20 hours, but is not limited thereto. In the above method, the drying in step (3) is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

[0028] The health functional food composition for improving cognitive function or memory of the present invention is preferably manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

[0029] When the health functional food composition of the present invention is used as a food additive, the rice grasshopper extract can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use (prevention or improvement). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0030] There are no specific restrictions on the types of the above foods. Examples of foods to which the above rice locust extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0031] When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumarin or stevia extract, or a synthetic sweetener such as saccharin or aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in mixtures. The proportion of these additives is not particularly important, but the composition of the present invention is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0032] In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction containing a rice locust extract as an active ingredient.

[0033] The above cognitive impairment may be any one selected from, but is not limited to, dementia, Alzheimer's disease, ischemic stroke, traumatic brain injury, amnesia, Parkinson's disease, Pick's disease, Creutzfeldt-Jakob disease, and mild cognitive impairment.

[0034] The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-mentioned effective ingredient, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. For parenteral administration, it is preferable to select a method such as topical application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection.

[0035] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective amount may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0036] In addition, the present invention provides a veterinary composition for preventing or treating cognitive dysfunction containing a rice locust extract as an active ingredient.

[0037] The veterinary composition comprising the rice locust extract of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition comprising the rice locust extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0038] The veterinary composition comprising the rice locust extract according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using a method well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of a powder, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, etc.

[0039] The veterinary composition according to the present invention may vary depending on the age, sex, and weight of the animal, but may be administered once or several times a day, and the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, weight, age, etc. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0040] In addition, the present invention provides a pet feed additive for improving cognitive function or memory containing a rice locust extract as an effective ingredient.

[0041] In the present invention, the term "companion animal" means a dog, cat, rabbit, ferret, guinea pig, hamster, etc. raised for companion purposes, and the scope of the animal includes without limitation any animal that lives with humans.

[0042] The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term 'feed' in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for or intended for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0043] In addition, the present invention provides a pet food containing the above feed additive.

[0044] The pet food of the present invention may further include additives commonly included in pet food in the art, and specifically, may further include a vitamin mixture including vitamin C and vitamin D, a mineral mixture including iron and copper, a gelling agent such as agar, taurine, etc. The content of the additives may be determined by the amount generally combined in the art, and is not limited thereto. However, the pet food of the present invention is characterized in that it does not include preservatives, coloring agents, sweeteners, or seasonings.

[0045]

[0046] Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0047]

[0048] Manufacturing Example 1. Manufacturing of rice locust (Oxya chinensis sinuosa) extract

[0049] Rice locust powder was purchased from Goesan Farmer and used in the experiment. 10 g of rice locust powder was homogenized with 30 ml of sterilized water. The mixture was then stirred at 180 rpm for 12 hours for extraction. After centrifugation at 1,500 rpm for 15 minutes, the supernatant was filtered through a 0.2 μm pore-size cellulose filter, frozen in liquid nitrogen, and then freeze-dried to produce a powder.

[0050]

[0051] Manufacturing Example 2. Measurement of extraction yield and re-dissolution

[0052] After measuring the weight of the freeze-dried rice locust extract of Manufacturing Example 1, the extraction yield (%) was calculated by substituting it into the calculation formula.

[0053] Calculation formula 1: Extraction yield (%) = (Weight after drying) / (Weight of powder used before extraction) × 100

[0054] As a result, the extraction yield of the rice locust extract according to Manufacturing Example 1 was confirmed to be 15.33%. The powder obtained after freeze-drying was calculated in sterilized water and re-dissolved.

[0055]

[0056] Example 1. Establishment of animal experiment design

[0057] Six-week-old male ICR mice weighing 20–25 g purchased from Damul Science (Daejeon, South Korea) were used as experimental animals. For the cognitive dysfunction induction experiment, the mice were divided into five groups (n=8): (1) control group (CTL; ​​saline), (2) Sco (Scopolamine 1 mg / kg, Sigma-Aldrich, #S0929, anticholinergic drug), (3) Sco+Don (Scopolamine 1 mg / kg + Donepezil 3 mg / kg; acetylcholinesterase inhibitor), (4) Sco+Ocs (Scopolamine 1 mg / kg + rice grasshopper powder extract 10 g / kg), (5) Ocs (rice grasshopper powder extract 10 g / kg).

[0058] As shown in Fig. 1, scopolamine and donepezil were injected intraperitoneally using a syringe, and the rice locust extract (Ocs) was administered orally. The drugs were administered a total of 16 times daily over a 19-day experimental period, and on the 19th day after the final administration, the mice were sacrificed and brain tissues were extracted for various histological and biochemical analyses. The isolated brain tissues were either lyophilized or lysed to measure antioxidant activity using Western blotting and ELISA analysis.

[0059]

[0060] Example 2. Confirmation of the efficacy of rice locust extract in improving memory and cognitive function in a mouse model of scopolamine-induced cognitive disorder.

[0061] Cognitive dysfunction was induced in mice by intraperitoneal injection of scopolamine for 10 days. As described above, donepezil and rice locust extract (Ocs), as positive controls, were administered intraperitoneally and orally, respectively, 30 minutes prior to scopolamine treatment. After drug administration, the mice were weighed, and behavioral analyses were performed on a time-based basis to assess their spatial memory, including the Morris water maze test, Y-maze test, and light / dark test.

[0062]

[0063] 1) Weight change

[0064] As disclosed in Fig. 2, the body weight of the scopolamine-only administration group (Sco) did not increase during the experimental period compared to the normal group (CTL), whereas the body weight of the scopolamine and rice locust extract of the present invention administration group (Sco+Ocs) showed an increase.

[0065]

[0066] 2) Morris water maze test

[0067] To assess the spatial memory of mice, the Morris water maze test was performed. Specifically, mice rely on exploratory skills to find a submerged escape platform. This test used a circular pool (122 cm) with a hidden platform. Mice were trained continuously for four days before drug administration to ensure they found a short, direct path from the starting position to the hidden platform. After drug administration, the maze test was performed, and spatial acquisition was measured and data analyzed using SMART 3.0 software (Ver. 3.0, Harvard Apparatus, Holliston, MA 01746, USA).

[0068] As a result, as shown in Fig. 3A, in the case of the scopolamine-only administration group (Sco) compared to the control group (CTL), the number of times the target point was passed was significantly reduced to 5, and in contrast, in the scopolamine and rice locust extract of the present invention administration group (Sco+Ocs), the number of times the target point was passed was increased to 15.

[0069] In addition, in the results of measuring the time spent at the target point, the time spent in the scopolamine-only administration group (Sco) was significantly reduced compared to the control group (CTL), whereas the time spent in the scopolamine and rice locust extract administration group of the present invention (Sco+Ocs) was increased (Fig. 3B).

[0070] Finally, the time taken for the mouse to find the target point again was measured. As shown in Fig. 3C, the scopolamine-only administration group (Sco) took about 40 seconds compared to the control group (CTL), whereas the scopolamine and the rice locust extract of the present invention administration group (Sco+Ocs) took about 20 seconds, confirming that the time taken to find the target point was reduced.

[0071]

[0072] 3) Y-maze test

[0073] The present invention conducted a Y-maze analysis to evaluate spatial and reference memory by rice locust extract. The Y-maze experiment was conducted by first informing each experimental group of the location of food, then changing the location of the food, and measuring the time required to find the food.

[0074] Each experimental group of mice was made aware of the location of the food, the location of the food was changed, and the time taken was measured. As a result, as shown in Fig. 4, in the case of the group administered scopolamine alone (Sco), the dwell time was confirmed to be approximately 10 seconds because the group did not recognize the location where the food had previously been (target zone). However, in the group administered scopolamine and the rice grasshopper extract of the present invention (Sco+Ocs), the dwell time was confirmed to have increased to approximately 30 seconds.

[0075]

[0076] 4) Light / Dark test

[0077] A light / dark test was conducted on each experimental group of mice. The light / dark test was conducted by exposing each group of mice to a light and dark environment and measuring the time they spent in each environment.

[0078] As a result, as disclosed in Fig. 5, in the case of the scopolamine-only administration group (Sco), the mice stayed for 90 seconds and 200 seconds, respectively, under light / dark conditions, but in the scopolamine and rice locust extract administration group of the present invention (Sco+Ocs), the mice stayed for 150 seconds and 120 seconds, respectively.

[0079]

[0080] Example 3. Confirmation of the efficacy of rice locust extract on brain neurons in a mouse model of scopolamine-induced cognitive disorder.

[0081] To determine the effects of the rice locust extract of the present invention on neurons in mice with cognitive dysfunction, the brains of each group were extracted on the 19th day after 16 drug administrations over 19 days, homogenized using a tissue lysis buffer, and centrifuged at 4°C and 12,000 rpm for 20 minutes to precipitate the brain tissue. The supernatant was recovered and used as a protein sample for Western blot analysis.

[0082] As a result, as disclosed in Fig. 6, there was almost no difference in the expression of ChAT (choline acetyltransferase) protein in the scopolamine-only administration group (Sco) compared to the control group (CTL), but in the case of the scopolamine and rice grasshopper extract of the present invention administration group (Sco+Ocs), the expression of ChAT protein increased.

[0083] In addition, in the case of NeuN (Neuronal nuclear antigen) protein expression, it was confirmed that the rice grasshopper extract of the present invention (Sco+Ocs) increased to a level similar to that of the control group (CTL) compared to the scopolamine-only administration group (Sco).

Claims

1. A health functional food composition for improving cognitive function or memory containing a rice grasshopper extract as an effective ingredient.

2. In paragraph 1, the extraction solvent of the rice grasshopper extract is water, C 1 ~C 4 A health functional food composition for improving cognitive function or memory, characterized by comprising low-grade alcohol or a mixture thereof.

3. A health functional food composition for improving cognitive function or memory, characterized in that the composition in the first paragraph is in one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

4. A pharmaceutical composition for preventing or treating cognitive dysfunction containing a rice grasshopper extract as an active ingredient.

5. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that in paragraph 4, the cognitive dysfunction is any one selected from dementia, Alzheimer's disease, ischemic stroke, traumatic brain injury, amnesia, Parkinson's disease, Pick's disease, Creutzfeldt-Jakob disease, and mild cognitive dysfunction.

6. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that in paragraph 4, in addition to the effective ingredient, it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

7. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that the composition in claim 4 is manufactured in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

8. A veterinary composition for preventing or treating cognitive dysfunction containing a rice grasshopper extract as an active ingredient.

9. A pet feed additive for improving cognitive function or memory containing rice grasshopper extract as an effective ingredient.

10. Pet food containing feed additives of Article 9.

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