WO2016139519A1

WO2016139519A1 - Immunostimulating composition for preventing or treating fish diseases

Info

Publication number: WO2016139519A1
Application number: PCT/IB2015/056707
Authority: WO
Inventors: Wojciech GÓRECKI; Wojciech FIAŁKOWSKI; Łukasz PIJANOWSKI; Monika KOMOROWSKA
Original assignee: Uniwersytet Jagielloński
Priority date: 2015-03-05
Filing date: 2015-09-03
Publication date: 2016-09-09
Also published as: PL411449A1; PL226061B1

Abstract

A pharmaceutical composition of immunostimulating effect intended for fish, for the most for administration by immersion, was revealed.

Description

IMMUNOSTIMULATING COMPOSITION FOR PREVENTING OR TREATING FISH DISEASES

The invention concerns the immuno stimulating formulation intended for fish, in particular for administration by immersion. The core advantage of the invention is that the formulation is administered by immersion with no simultaneous effect on water chemistry. Composition of the immunostimulant is entirely non-toxic (within the range of suggested doses) and may be administered both to ornamental fish and fish intended for human consumption without a withdrawal period.

Initial research performed within the last decade confirmed the capacity of herbal extracts' use in health prevention and treatment of diseases in multiple aqueous animal species. At present, herbs and herbal products continue to play an increasingly important role in aquaculture. In aquaristics, antibiotics have been regularly administered On preventive basis', which results in an increasing antibiotic resistance (Musa N et al. 2008). Immuno stimulants may, in certain cases, replace antibiotics, e.g. in catfishes (Siluridae) (Bich Hang BT et al. 2014). At present, there is no alternative for therapeutic products based on cancerogenic malachite green, methylene blue or formalin, gradually withdrawn from the zoological market. The products containing the a/m substances, despite their harmful effect on the environment, have been frequently used without investigation of the reason behind the problems observed in aqueous animals. In this context, opportunity of limiting the chemotherapeutics use by introducing the immuno stimulating formulations is of particular significance.

Impact of different herbal mixtures on the fish physiological processes and their antibacterial and antiviral effect in aqueous organisms have been researched by scientific team throughout the world (Diigenci SK et al. 2003; Muniruzzaman M & Chowdhury MBR, 2008). According to scientific reports, extracts from multiple plant species may demonstrate immuno stimulating properties (Govind et al. 2012). Active substances contained in such extracts may be administered to fish in different developmental stages, including in alevin (Bricknell I &. Dalmo RA, 2005). However, relevant development of organs responsible for immunological processes is a must. In the case of extracts administered with feed, effectiveness of plant extracts effect on the fish immunological system depends in majority from feeding effectiveness. In this case, immunostimulating additives have no effect on animals taking in no feed. Multiple plant extracts, including coneflower extract (Echinacea) obtained for human and livestock immunostimulants production, has a specific bitter and tart taste, being the effect of, among others, saponins, flavonoids and phenolic compounds may reduce feed acceptance (Popek at al. 2010). The advantage of formulation administered to water is the opportunity of stimulating the immunological mechanisms regardless of quantity and frequency of fish feeding.

In recent years, there has been a significant progress in the area of herb cultivation, acquisition and production of plant extracts. At present, the focus is on standardization of raw material intended for drug, immunocosmetics, dietary supplements or feed additives production (Kolodziej et al. 2010). Quality, stability and availability of the obtained plent extract are of particular importance. High class plant extracts available on the market enable production of fish formulation featuring precise concentration of active substances. The claimed solution, as opposed to the scientific reports quoted above, is based on a standardized product of determined stability.

At present, there are several commercial products of similar effect available on the market, however their administration method and composition differs from the applied invention. The Mosura company produces a plant formulation in a form of PRO Tonic, enhancing immunity of shrimp and fish. This dietary supplement (feed) is intended for the most for shrimp (however, according to the manufacturer's instruction, it may be also used for fish breeding. The formulation improves immunological system by nutrients and immuno stimulants and contains unspecified concentration of active substances. The manufacturer provided no data proving the effectiveness and effect of the product.

The Ecological Laboratories INC company, known under the MICROBE-LIFT brand, produces a herbal formulation enhancing fish immunity. This medicinal product is effective in bacterial and fungal inflammations, abscesses, ecchymoses, skin slime, mouth opening abscesses and fin rot. It enhances immunological system without the risk of strain resistance development as in the case of antibiotics. This facilitates elimination of pathogens from the infected site, which - deprived of a host - decease quickly. However this product is intended exclusively for aquaristic use and may not be administered in treating fish intended for human consumption. The product contains unspecified concentration of active substances. The manufacturer provided no data proving the effectiveness and effect of the product.

The patent application KR101107034 (B l) describes a feed additive for fish composed of plant materials. According to the published description in the application, this extract demonstrates antibacterial, antifungal and antiparasitic effect, however its composition and administration method is completely different from the applied invention.

The patent application CN102091242 (A) describes the mixture of traditional Chinese herbs in applicable proportions, used for treatment of various diseases in fish. However its composition is completely different from the applied invention. The mixture is based on the natural, non- standardized plant extracts, thus the effectiveness of the product may vary depending on batches used for component production.

The patent application CN101507744 (A) describes herbal composition along with preparation method. The product is intended for aquaristic use, however its composition and administration method is completely different from the applied invention. The formulation is based on natural non-standardized plant extracts.

The patent application RU2011128157 (A) describes the production method of a biologically active feed additive intended for livestock, birds and fish. It comprises probiotics and medicinal herbs (among other purple coneflower (Echinacea purpurea)). However, its complete composition, due to the type of used substances and their concentrations, is different from the applied invention. There is also a difference in administration method and effect described in the patent application for the product. No concentration of active substances contained in the herbal extracts used in the product is provided.

Natural immunostimulats constitute a comprehensive group of formulations obtained from bacteria, fungi and certain plants. Due to diversified directions of their effect, there is no one proper name encompassing all their properties. The literature describes them as natural immuno stimulants, immunomodulators, immunoaugmentors or biological response modifiers (BRM). Immunostimulating effect of purple coneflower (Echinacea purpurea) is commonly known in humans and animals, including fish. Its effect on the immunological system in oral administration was described in Nile tilapia (Oreochromis niloticus) (Aly SM & Mohammed MF, 2009; Aly SM et al. 2008), carp (Cyprinus carpio) (RenYongLin, 2008) and guppy (Poecilia reticulata) (Guz L et al. 2011). The a/m quoted scientific research demonstrated that the active substances of this plant, such as flavonoids, have immunostimulating properties and enhance immunity to bacterial infections.

Enriching water or feed in vitamins in aquaristics is made by special multi-vitamin supplements. These formulations have a form of fluids poured directly into the aquarium and/or sprinkled on the feed directly before administration to fish. There are several vitamin supplements for fish currently available on the market and offered by the largest manufacturers of aquaristic products. These are of universal nature, which means that they may be administered to vast majority of ornamental fish. Their composition comprises usually any and all necessary vitamins in proportions selected by a given manufacturer (there are minor differences between the products), e.g. DENNERLE V-Maxx vitamin formulation for fish in drops. These products are not manufactured and administered for enhancing immunity.

The aim of the invention is providing a product based on natural extracts and vitamins and demonstrating highly effective immunostimulating effect, intended for all fish species and applicable for multi-species shocking in aquaculture and aquaristics. Providing a formulation administered to water and applicable for immersion and having the standardized composition is of particular importance.

The subject-matter of the invention is a pharmaceutical composition intended for use in fish prevention or treatment, for the most as immunostimulating formulation, consisting in three parts: A, B and C, advantageously in weight proportions A:B:C as 1000:2:2, provided that part A constitutes a standardized purple coneflower extract in a solvent selected from the group covering: water or water solution of propylene glycol or glycerin, in particular of 70% concentration, provided that the drug extract ratio (DER) is 1 : 10, part B is thiamine, in particular in a form of thiamine hydrochloride or mononitrate, whereas part C is ascorbic acid or its salt, for the most in a form of ascorbic acid of 90-99% purity or ascorbic acid phosphate.

Advantageously, the composition according to the invention is intended for administration by immersion.

Advantageously, the composition according to the invention is intended for administration in a semi-static system, with weekly change of 50% of water into fresh one before the formulation is applied.

Advantageously, the composition according to the invention is intended for administration by immersion in 2-6 doses, every seven days.

Advantageously, the composition according to the invention is intended for administration by immersion thinned to 1 : 1000.

The formulation according to the invention, in the developed concentration, demonstrates immunostimulating effect observed in administration by immersion, which is the entirely new method of administration of such formulation.

The composition according to the invention comprises of purple coneflower (Echinacea purpurea) extract, thiamine (vitamin Bi) and ascorbic acid (vitamin C). The product is non-toxic in concentrations exceeding lOg/dm for aqueous animals and fully biodegradable in short time perspective. Due to composition, the formulation may be administered to fish intended for human consumption without a withdrawal period and ornamental fish. What's important, as opposed to multiple chemotherapeutics, this product meets the safety requirements for humans and animals. In the researched quantities, the composition demonstrates no cancerogenic or mutagenic effect and may be administered to multi-species fish stocking.

The developed formulation is technologically easy to produce and use. The contained substances are easily available in trade and maintain minimum monthly stability required for formulations 12-18. The composition is unstable in water and quickly biodegradable, which is its significant advantage. The product causes no direct changes to water chemistry. Upon its application, no long-term (below 7 days) changes in concentration of phenolic compounds being one of the key active substances of the purple coneflower extract and antioxidizing activity were observed.

The composition according to the invention may be widely applied in fishery and aquaristics. Its administration results in enhanced breeding and farming quality by improving immunological parameters of fish. In most cases, the formulation may support reducing losses during animal transport and quarantine, thanks to which it may contribute to better profitability of fish breeding, in particular fry. The formulation may be administered to fish intended for human consumption without a withdrawal period. In fishery, application of drugs may result in periodical withdrawal of treated fish batch from sales. Application of the invention provides the opportunity of faster recovery of fish, which may influence the convalescence period reduction.

The developed formulation is also intended for animal quarantine. What's important, as opposed to multiple chemotherapeutics, this product meets the safety requirements for humans and animals. In the researched quantities, the composition demonstrates no cancerogenic or mutagenic effect and may be administered to multi- species fish stocking. No special precautions are necessary. The formulation damages no fishing gear and remains no difficult to remove or permanent stains on breeding containers, which is frequent for chemotherapeutics based on such colorants as: malachite green and methylene blue. Leaving no permanent deposits on aquariums is of significance for aquaristics, in which esthetics play an important role.

The developed formulation is technologically easy to produce and use and maintains a minimum monthly stability required for formulations 12-18. In the case of placing the product on the zoological and aquaculture market, maintaining long stability is a key, since it may take several months before the product reaches the breeder, during which it will be transported between warehouses and stores. The product requires no special storage conditions, which is necessary for certain medicinal products and probiotics. As already mentioned, the formulation is unstable in water and biodegradable, which is a great advantage for the product intended for aquaculture. It causes no changes to water chemistry parameters.

The developed product may be comprehensively used in fishery and aquaristics. There is an expanding niche on the zoological market related to availability of products intended for fish care and convalescence of demonstrated effectiveness. According to estimations, app. 90% of ornamental fish available in zoological stores is imported from Asia. These animals, upon long journey, are usually stressed and in poor physical condition. The effects include losses in the acclimatization and quarantine period. Health problems related to bacterial and fungal infections are also observed during the quarantine. To prevent them effectively, enhancing fish immunity in advance is necessary. The composition according to the invention facilitates regeneration of immunity lost due to stress or change of conditions and mitigate the bacterial and fungal infection effects.

Upon sorting and transport, due to which fish lose their immunity, application of immuno stimulants is recommended. The problem frequently faced by fish breeders is temporary discontinuation of feed intake by animals exposed to stress. Therefore upon transport or sorting, administration of levamisole, beta-glucans or plant extracts proves to be ineffective. Reduced immunity in fish is also usually stated upon the winter period, when fish do not feed, which is strengthened by negatively influencing environmental factors (e.g. low water temperature). The effective fish protection method in this case is application of formulation administered by immersion in autumn, during transport. Active substances will be absorbed via gills and skin.

Administration of extract containing vitamins by immersion (to water) is a new solution in aquaculture. This application method of immunostimulating formulation will be applied in artificial breeding in closed circuits, hatching and the entire aquaristics.

Example 1. Formulation of composition according to the invention

In the exemplary embodiment, the immunostimulating composition has a form of a single stable liquid constituting a usable solution. The composition comprises of three parts: A, B and C, in which part A is a standardized purple coneflower extract, interchangeably on water basis (solvent: extraction water) or glycol basis (solvent: propylene glycol and extraction water in 70% to 30% ratio), or glycerin basis (solvent: glycerin and extraction water in in 70% to 30% ratio), part B is vitamin Bi (thiamine in the form of thiamine hydrochloride or mononitrate of high purities), part C is vitamin C (ascorbic acid 90-99% or ascorbic acid phosphate intended for feeds or food). In part A, the drug extract ratio (DER) is 1: 10.

The composition is prepared in A:B:C ratios as 1000:2:2. Parts B and C are dissolved in part

A in ambient temperature. If purple coneflower water extract is used, the composition shall require preservatives in a form of potassium sorbate according to commonly adopted principles of food or feed products preservation.

Example 2. Application of the composition according to the invention

Fish are placed in a breeding or quarantine tank of stabilized physical properties and chemical properties of water and operating, stable filtration and/or aeration system. Fish should be exposed to formulation in the semi-static system i.e. with weekly change of 50% of water into fresh water before application. To obtain a immunostimulating effect, the procedure is repeated 2 - 6 times, every seven days. From the prepared formulation, 1 ml per each water liter in the tank is measured out. Then the prepared solution is poured into the water with fish and shuts down (if installed) the filtration system for 24 hours from administration of the formulation. Aeration system should remain operating.

Example 3. Effects of application of the invention on fish and water environment

The aim of research was demonstrating the presence between the experimental groups, of significant, distributed in time differences in concentration of phenolic compounds in water and antioxidizing activity.

The 45-day research were performed in the period between June and half-July 2014. Throughout this period, every 7 days, the purple coneflower and vitamin extract was administered to water. Each time before the extract and vitamin application, 50% of water in the tanks was changes. In most cases, before and upon the extract administration, chemical composition of water was analyzed with a view to presence of phenolic compounds and antioxidizing properties. To verify the extract and vitamin effect on fish, the osmotic shock tests were performed. These tests were performed three times in two-week intervals.

The research was performed on 8 groups of zebrafish (Danio rerio). Each group was subject to three experimental repetitions and comprised of 20 subjects. The first group (control group) received neither extract nor vitamins, whereas the second group (B+C) received only vitamin Bi 2 mg/dm 3 and vitamin C 2 mg/ dm 3. The subsequent four study groups received purple coneflower extract in quantity of 10 mg/1, 100 mg/1, 1000 mg/1 and 10000 mg/ dm . The remaining two groups received purple coneflower extract in quantity of

1000 mg/ dm 3 and 10000 mg/ dm 3 and vitamin Bi 2 mg/dm 3 and vitamin C 2 mg/ dm 3. The experiment was repeated 3 times. The limit of quantification of phenolic compounds for the applied method was 0.005 [mg-dm^"

3 ], limit of quantification for the analyzed compounds reached 0.002 [mg-dm -^"3 ]. The analyzed antioxidizing activity was expressed in percentage scale (%).

Within the performed experiment, the water samples to be analyzed were taken in 1., 5., 14., 20., 35. and 42. day of experiment. On day 14, 35 and 42, water samples to be analyzed were taken before and after administration of the extract, whereas in the remaining days only once - before administration of the extract. Phenolic compounds content in water [mg-dm^" ] is presented on Fig. 1, whereas Fig. 2 presents value of water antioxidizing activity.

Phenolic compounds and antioxidizing activity are the indicators to be used in the immunological and immunogenetic studies. Antioxidizing and phenolic compounds (polyphenols) eliminate oxygen free radicals and are responsible for different processes taking place in the organism. Monitoring of these parameters enables tracking water chemistry and polyphenols decomposition rate. Fast decomposition rate of these compounds in water, with simultaneous maintenance of effect on fish may be of importance from the perspective of ecology and environment protection. Formulations based on plant extracts have been currently the ones of the most desired products on the market. Own research results demonstrate that addition purple coneflower extract to water in quantity up to 1000 mg of extract - dm -^"3 water, and in quantity of up to 1000 mg extract - dm -^"3 water with vitamins Bi and C or vitamins Bi and C alone, does not provide statistically significant changes of phenolic compound concentration and antioxidizing activity in water. Changes to concentrations of the studies compounds were stated in the subjects with added 10000 mg/dm 3 and 10000 mg/dm 3 of purple coneflower with additional vitamins Bi and C, however upon several days their quantities started to decrease. Adding purple coneflower in groups of

10000 mg/dm 3 and 10000 mg/dm 3 with additional vitamin Bi and C resulted in a significant increase of phenolic compound concentration and antioxidizing activity. These changes were observed between the samples taken at the same day before administering of the formulation and after it, as well as between the samples after administering of the formulation and on the 7. day upon administration. In the latter one, decrease in phenolic compound concentration and antioxidizing activity was clearly visible. The obtained results prove that the composition according to the invention is unstable in water and requires fast biodegradation, which is its obvious advantage. The second important observation is lack of noticeable, significant changes to concentration of phenolic substances and antioxidizing activity in groups of 1000 mg/dm 3 and 1000 mg/dm 3 with additional vitamins Bi and C. Despite noticing the effect in the form of increased fish immunity upon purple coneflower and vitamins administration, no changes to water chemistry were observed. These results may recommend application of purple coneflower and vitamins application in the ecological immuno stimulating formulations for fish even in very high concentrations up to 10000 mg/dm . However, further research works, due to economic conditions and demonstrated effectiveness already at 1000 mg/dm should be carried out with particular focus on concentrations within the range of 1000- 10000 mg/dm³.

Example 4. Immunostimulating effect of composition according to the invention.

Fish in natural environment are permanently exposed to negative environmental factors, including among others water thermal conditions, oxidation, salinity and reaction outside the optimum range for a given species. In the environment, changes to water parameters are subject to natural daily and seasonal changes. These fluctuations are usually of gradual nature. The other case is when due to activities of the breeder, sudden and shock-inducing changes to water chemistry and temperature occur. These events take place especially during transport, quarantine or result from therapy. Recently, facing the limited number of medicinal products available on the market, the aquarists and fishermen use increasingly often kitchen salt as the substance of comprehensive biocidal, including antiparasitic effect. Fish sensitivity to effect of sodium chloride dissolved in water is specific for each individual species. A commonly discussed case is zebrafish - a popular laboratory and aquarium fish demonstrating high sensitivity to chemical substances, in particular kitchen salt constituting at the same time highly effective medicinal product. Considering the above, to determine the effectiveness of immunostimulating formulation increasing the immunity to negative environmental factors, the salting out tests with the use of sodium chloride were performed. Under the performed studies, three osmotic shock tests with the use of NaCl as stress- inducing factor were carried out, providing a lethal effect distributed in time. The experiments were performed with the use of fry aged 90 days of average mass of 0.2 g.

The primary objective of the study was demonstrating the presence between the study groups of significant and distributed in time differences in survival rate of fish subject to lethal factor. The secondary objective was determining fish immunity growth rate in time.

The 45-day studies were performed between June and half-July 2014. Throughout this period, every 7 days, the purple coneflower and vitamin B i and C extract was administered to water. Each time before the extract and vitamin application, 50% of water in the tanks was changes. In most cases, before and upon the extract administration, chemical composition of water was analyzed with a view to presence of phenolic compounds and antioxidizing properties. To verify the extract and vitamin effect on fish, the osmotic shock tests were performed. These tests were performed three times in two-week intervals- Each study group comprised of 10 subjects. The research was performed on 8 groups of zebrafish (Danio rerio). The first group (control group) received no extract nor vitamins, whereas the second group (B+C) received only vitamin Bi 2 mg/dm³ and vitamin C 2 mg/ dm³. The subsequent four study groups received purple coneflower extract in quantity of 10 mg/1, 100 mg/1, 1000 mg/1 and 10000 mg/ dm³. The remaining two groups received purple coneflower extract in quantity of 1000 mg dm³ and 10000 mg/ dm³ and vitamin Bi 2 mg/dm³ and vitamin C 2 mg/ dm³. The test results for the individual groups are presented in percentage value.

Figures 3 - 14 present the results of three osmotic shock test carried out on zebrafish. hi each test, three time points were selected, which most precisely illustrate the impact of purple coneflower and vitamins on fish immunity to environmental factor. Statistically significant differences in fish survival between the groups, expressed as percentage value, were highlighted in the tables in yellow and red.

In the first test (Fig. 3) carried out on 4-month old fry, significant differences occurred in 24., 27. and 30. minute. Upon 24. minute, the group survival was observed only for 1000 mg/1, 10000 mg/1, 1000 mg/1 B+C and 10000 mg/1 B+C, whereas in the control group and 10 mg/1 group mortality rate reached 30% and in the group with only vitamins administered, complete stocking mortality was recorded (Fig. 4). In 27. minute (Fig. 5) and 30. minute (Fig. 6) significant differences occurred between the following groups: control group, 10 mg/1 and B+C and groups of 1000 mg/1, 1000 mg/1 B+C and 10000 mg/1 B+C.

In the second test (Fig. 7), statistically significant differences were observed in 54. minute (Fig. 8) between the groups of 10 mg/1, 100 mg/1 and B+C, and the groups of 1000 mg/1 B+C and 10000 mg/1 B+C, 57. minute (Fig. 9) between the control group, 10 mg/1, 100 mg/1 and B+C, and the groups of 1000 mg/1 B+C and in 60. minute (Fig. 10) between the control group, 10 mg/1, 100 mg/1 and B+C, and the groups of 10000 mg/1 B+C.

In the third test (Fig. 1 1 ), statistically significant differences were observed in48. minute (Fig. 12) between the control group, 10000 mg/1 B+C, and the groups of 1000 mg/1, 1000 mg/1 B+C and 10000 mg/1 B+C, whereas in 51. minute (Fig. 13) and 54. minute (Fig. 14) between the control group, 10 mg/1, 100 mg/1, 10000 mg/1 B+C and B+C, and the groups 1000 mg/1, 1000 mg/1 B+C and 10000 mg/1 B+C.

The performed studies lead into the following conclusions: I. In the groups, in which the extract at 10000 mg/1 was added, fish mortality over the 45-day study was higher by app. 30%.

II. There were three time points (9 minutes) in each of three tests, in which highly significant differences in fish survival between the groups were observed.

III. In the salting out tests, higher fish survival in the groups with added purple coneflower extract at 1000 and 10000 mg/1 was stated.

IV. Only in the groups of 1000 mg/1 and 10000 mg/1, with vitamins added simultaneously with extracts, a positive impact on fish survival was demonstrated - vitamins alone provided no such effect.

V. When administering purple coneflower in concentration of 10000 mg/1 (group of 10000 mg/1 B+C), fish gained higher immunity already after two applications of the extract - test 1.

VI. Upon 4^th and 6^th administration of extract, better results were obtained for concentration of 1000 mg/1 (group of lOOOmg/1 B+C).

VII. For further studies on the formulation, the purple coneflower concentrations within the range of 1000 - 10000 mg/1 should be selected.

Claims

1. A pharmaceutical composition intended for use in fish prevention or treatment, for the most as immuno stimulating formulation, consisting in three parts: A, B and C, advantageously in weight proportions A:B:C as 1000:2:2, provided that part A constitutes a standardized purple coneflower extract in a solvent selected from the group covering: water or water solution of propylene glycol or glycerin, in particular of 70% concentration, provided that the drug extract ratio (DER) is 1: 10, part B is thiamine, in particular in a form of thiamine hydrochloride or mononitrate, whereas part C is ascorbic acid or its salt, for the most in a form of ascorbic acid of 90-99% purity or ascorbic acid phosphate.

2. Composition according to claim 1, characterized in that it is intended for administration by immersion.

3. Composition according to claim 1, characterized in that it is intended for administration in semi-static system, with weekly change of 50% of water into fresh water before application of the formulation.

4. Composition according to claim 1, characterized in that it is intended for administration by immersion in 2 - 6 repetitions, every seven days.

5. Composition according to claim 1, characterized in that it is intended for administration by immersion thinned to 1: 1000.