ES2249153B1 - MASLINIC ACID AS AN ADDITIVE FOR ANIMAL PRODUCTION. - Google Patents

Abstract

Maslinic acid as an additive for animal production. Use of maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural products or concentrates thereof that contain it in the preparation, alone or in combination with other agents, of applicable products to animal production characterized in that the product is an inhibitor of the activity of serine proteases, an enhancer of enzymes producing NADPH, of the growth of the liver, smooth muscle and their respective protein proteins, of protein turnover, of body growth and of the ordering and compaction and development of glycogen granules and the rough endoplasmic reticulum of liver cells.

Description

Maslinic acid as an additive for production animal.

Object of the invention

Animal production has a series of essential health and economic conditions for its economic viability Regardless of other parameters, the expense corresponding to food intake, time of use of facilities, with all that this entails, and loss of morbidity benefit in the specific animal species, as well as the yield of animal mass obtained, are factors decisive for the success of the approach economic-business In addition to these parameters, it is necessary to take into account the achievement of the lowest possible consumption of natural resources and cause the least environmental impact in what to the productive activity it refers. In this context, the use of maslinic acid (2-alpha, 3-betadihydroxiolean-12-en-28-oic), a natural protease inhibitor, obtained from waste solids of the industrial milling of the olive, supposes a substantial improvement in different histological parameters and enzymes that result, among other things, in improvements from the point economic, sanitary and environmental view, such as time of breeding, amount of intake, animal morbidity, excretion of nitrogen, consumption of natural resources (water) and time of use of the facilities of this activity and its corresponding expenses.

State of the art

Maslinic acid [Dictionary of Natural Products on CD-ROM, Chapman & Hall, ISSN 0966-2146 ver. 5: 1 (1996); Maslinic Acid CAS [4373-41-5]] is a triterpene little distributed in the vegetable kingdom [Phytochemical and EthnobotanicalDB, http://www.ars-grin.gov/duke/plants.html], there are no commercial sources of it . Biological activities of this type of Pentacyclic triterpenic acids [Selective Inhibition Of Cyclic Amp-Dependent Protein Kinase By Amphiphilic Triterpenoids And Related Compounds, Wang, BH, Polya, GM, Phytochemistry , 41 (1): 55-63, (1996)] have been studied to a considerable extent, particularly that of oleanolic acid [Induction Of Differentiation In The Cultured F9 Teratocarcinoma Stem Cells By Triterpene Acids Lee, HY, Chung, HY, Kim KH, Lee JJ, Kim KW, Journal of Cancer Research & Clinical Oncology . 120 (9): 513-518, (1994); Effect Of Oleanolic Acid On Hepatic Toxicant-Activating And Detoxifying Systems In Mice, Liu, J., Liu, YP, Parkinson, A., Klaassen, CD, Journal of Pharmacology & Experimental Therapeutics . 275 (2), 768-774, (1995); Antagonistic
Effect of Oleanolic Acid on Anaphylactic Shock, Zhang, LR, Ma, TX, Acta Pharmacologica Sinica . 16 (6), 527-530, (1995); Anti-Angiogenic Activity of Triterpene Acids Shon, KH, Lee HY, Chung, HY, Young, HS, Yi, SY, Kim, KW, Cancer Letters , 94 (2), 213-218, (1995); Pharmacology of Oleanolic Acid and Ursolic Acid, Liu, J., Journal Of Ethnopharmacology , 49 (2), 57-68, (1995)].

one

However, works are beginning to be abundant for direct derivatives of this acid, which are demonstrating interesting activities [Antitumor activity of amooranin from Amoora rohituka stem bark, Rabi, T., Curr. Sci., 70 (1), 80-1 (1996); Preparation of triterpene derivatives and medicinal compositions for treatment of nephritis, Ohgi, T., Yoshifusa, H., Kandori, K., PCT Int. Appl. WO 9600236 A1 (1996); Study of terpenoid exhibiting DNA topoisomerase II inhibition, (CA 124: 196916) Nozaki, HY et al, Tennen Yuki Kagobutsu Toronkai Koen Yoshishu, 37, 349-54 (1995)]. One of these derivatives is precisely maslinic acid, which has recently been published that has a potent in vitro inhibitory activity of the AIDS virus protease (HIV-1) [Anti-HIV Triterpene Acids from Geum japonicum, Xu, HX; Zeng, F .; Wan, M .; Sim, Keng-Yeow J. Nat. Prod ., 59 (7), 643-645 (1996)]. Other derivatives of oleanolic acid, such as equinocytic acid (16-hydroxyoleanolic acid) have demonstrated inhibitory effects against HIV replication in H-9 cells with EC50 values of 2.3 mM [Anti-AIDS agents, 21. Triterpenoid saponins as anti-HIV principies from fruits of Gleditsia japonica and Gymnocladus chinensis, and a structure-activity correlation, Konoshima, Takao; Yasuda, Ichiro; Kashiwada, Yoshiki; Cosentino, L. Mark; Lee, Kuo-Hsiung, J. Nat. Prod ., 58 (9), 1372-7, (1995)]. Many other direct derivatives have been shown to be antagonists of leukotriene D4 [Leukotriene D4 antagonists in Tripterygium wilfordii, Morota, Takashi; Saitoh, Kazuko; Maruno, Masao; Yang, Chun-Xin; Qin, Wan-Zhang; Yang, Bing-Hui, Nat. Med., 49 (4), 468-71 (1995)] and most hopeful is that a pharmacophoric search for HIV-1 protease inhibitors, conducted at the National Cancer Institute (Betsheda , USA) has designated a derivative of maslinic acid as a promising basis for future development in this activity [Discovery of Novel, Non-Peptide HIV-1 Protease Inhibitors by Pharmacophore Searching, Wang, Shaomeng; Milne, GWA; Yan, Xinjian; Posey, Isadora; Nicklaus, Marc C .; GrahamX Lisa; Rice, William G., J. Med. Chem ., 39 (10), 2047-54 (1996)] Both oleanolic acid and maslinic acid are abundantly found in the olive skin wax [The Lipids of Olea -Europaea 4. Pentacyclic Triterpene Acids in Olives, Bianchi, G., Pozzi, N., Vlahov, G., Phytochemistry , 37 (1), 205-207, (1994)], and a patent developed by our research team and whose ownership is held by the University of Granada [Procedure for industrial use of 3β-hydroxiolean-12-en-28-oic (oleanolic) and 2α, 3β-dihydroxiolean-12-en-28-oic acids (maslinic) contained in the by-products of olive milling. Spanish patent P9601652. International Patent W098 / 0433], allows to obtain these two acids industrially, separately and in high degree of purity, from solid by-products of the industrial milling of the olive, by any of the procedures now employed (presses, continuous in three phases and in the so-called two-phase), which constitutes an affordable and inexhaustible source of them. The established separation process is extremely simple and efficient, allowing us to isolate these products from the complex original mixtures.

As a result of the biological tests carried out, so far, two patents have been registered by the University of Granada to obtain medicines as protease inhibitors for the treatment of diseases caused by protozoa of the genus Cryptosporidium [Use of maslinic acid as inhibitor of serine proteases for the treatment of diseases caused by parasites of the genus Cryptosporidium (P9701029)]. Tests conducted on MDCK cell line show a 92.3% infection inhibition rate at 37 mg / mL. In the case of AIDS-causing viruses, tests have resulted in the patent Use of Maslinic Acid as a protease inhibitor for the treatment of disease caused by acquired immunodeficiency viruses (P9702528).

Moreover, the proper use of certain protease inhibitors may constitute a excellent tool in directed enzymatic modification, by an example of the balance in protein turnover during cell growth causing controlled changes of same.

Description of the invention

For the development of the present invention, we have made a series of experiences aimed at determining the usefulness of maslinic acid, isolated natural product industrially by-products of the milling of the olive, both in the optimization of cell growth by the modification of the balance existing in the processes of continuous protein turnover as in the decrease of morbidity of the animal population, by significantly reducing the infection processes generated during crop stress and its corresponding manipulation.

Our most standardized works and the most significant results have been carried out in fish culture in general, and in rainbow trout ( Oncorhynchus mykiss ) in particular, due to its commercial importance in inland aquaculture.

Embodiment of the invention

The procedures used and the results Most significant found are summarized below:

Preparation of diets : the diet used in this work was prepared from powdered feed provided Ecotex 30 (trout-specific extruded food) (DIBAQ DIPROTEG). The composition of said feed is as follows: Fish meal; Fish oil; Wheat and by-products; Soybean flour; Vitamin mineral premix; Antioxidant and antifungal; Vitamins and minerals, whose final nutritional composition is Proteins 44%, Ashes 7%, Fat 30%, Fiber 1% and moisture 7%, yielding an energy content per kg of diet of: Gross energy: 260.08 kcal; Digestive energy: 218.56 kcal; Metabolizable energy: 186.8 kcal; Digestible protein: 90%.

The dose of inhibitor (maslinic acid) varied by each experimental group (5 groups in total): Control group (0 mg inhibitor / kg diet); Group 1: 1 mg inhibitor / kg diet; Group 2: 5 mg inhibitor / kg diet; Group 3: 25 mg inhibitor / kg diet; Group 4: 250 mg inhibitor / kg diet.

The process of mixing maslinic acid with the powdered diet was carried out with the utmost thoroughness, because It is extremely important that the end result be a diet perfectly homogeneous. The next step is the transformation of the powdered diet into grains with a diameter suitable according to the size of the fish over time. Be they prepared two different sized diets, one with a diameter of 1.5 mm and another with a diameter of 3 mm. The diets ready for Consumption is stored safe from light and moisture, and at a temperature of 4 ° C.

Experimental animals : the species used in this work was rainbow trout ( Oncorhynchus mykiss ), obtained from a local fish farm. The starting weight of trout in each of the experimental situations was approximately 20 grams. The admitted weight range ranged from 18 to 20 grams. Taking into account that during the acclimatization process to the test vats a small mortality rate is frequent in the population, mainly due to stress, 5% more animals were selected than will be necessary for the performance of the scheduled experiments.

During the acclimatization time to the new environment, the trout were uniformly distributed in independent 360-liter tanks, with a continuous flow of water (1.5 L / kg / min) previously deodorized and dechlorinated, at a constant temperature of 15 \ pm 1ºC, thanks to an automatic heating-cooling system. The water is oxygenated to saturation levels (8-9% O2) through diffusers installed to each of the vats and connected to a blower of 1 HP of power. To prevent bacterial diseases, chloramine-T, a topical antibiotic that protects against bacterial infections, was initially used. The dose used was 5 grams of chloramine-T dissolved in two liters of water, pouring it into the vats without water flow, and letting it act for 10 minutes. After that time, the water flow is reopened, initially abundant to remove excess accumulated chlorine and then adjusting it to the optimal conditions mentioned above. The disinfection was repeated regularly (approximately every 3 weeks), the vats being cleaned daily. Throughout the acclimatization process the trout were fed " ad libitum " with the control diet previously prepared. In this way the fish were getting used to the texture, size and taste of it.

Preparation of the batches. - After 10 days from their arrival the trout were in perfect condition (they showed no signs of disease and ate abundantly). Mortality during acclimatization was low (around 2%). The experimental batches were then prepared, for which 5 360-liter polyethylene tanks were prepared (one for each group: control; 1 mg / kg; 5 mg / kg; 25 mg / kg and 250 mg / kg), under the same conditions of water flow, degree of oxygen saturation, temperature, etc. than those used in acclimatization. The fish are weighed individually after being anesthetized with ethylene glycol monophenyl ether and immediately redistributed in the vats (120 animals / tank) with an initial average weight of 20.00 ± 0.16 grams. This moment constituted Time 0 of the experimentation period.

Animal feeding .- The diet began to be supplied the day after weighing. Each of the vats was already provided with their specific diet. From the first day until the end of the experiment the fish were going to receive a diet dose corresponding to 1.5% of the total weight of the tank, distributed in two doses: 9.00 am and 04.00 pm. The choice of dose and schedule was established according to the experience of other researchers, who ensure that they are the optimal feeding conditions to obtain the best growth rates [Fauconneau et al , 1984; Cowey, 1981)].

Growth control and dose readjustment : The first point to develop the complete fish growth curve was when the batches were made. That same day, 18 animals were sacrificed to determine the initial weight of the liver and white muscle (first point in the development of the growth curve of these two tissues) and for the sampling of the initial time, necessary for the determination of the growth related parameters (protein concentration, RNA, DNA and enzymatic measures). The fish are weighed every week until the end of the experiment to obtain the necessary points for a good growth curve of the whole fish. Samples for the development of tissue growth curves (liver and white muscle) are taken every 4 weeks.

Determination of specific growth rates body (G R), liver (K GH) and white muscle (K_ {GM}) and protein replacement

The specific speed of body growth (GR), which represents the percentage of weight gain per day, was determined using the following equation:

G_ {R} (% day) = \ frac {Ln \ W_ {n} - Ln \ W_ {0}} {t_ {n} -t_ {0}}

being:

W_ {0} = weight of the animal at time 0

W_ {n} = weight of the animal over time "n"

t_ {0} = initial time

t_ {n} = time "n"

n = number of days since start.

The determination of the specific rate of growth of the liver and white muscle (K GH and K GM, respectively) was carried out with an equation equivalent to the previous one, but using the daily percentage of tissue protein growth. The protein concentration was determined by the method of Lowry [Lowry et al , 1951]. The% increase in protein / day values coincide with the% increase in weight / day values of the liver and muscle [Peragón et al , 1981].

K_ {G} (% day) = \ frac {Ln \ P_ {n} - Ln \ P_ {0}} {t_ {n} -t_ {0}}

being:

P_n = protein content (mg / ml) of tissue in time "n".

P0 = protein content (mg / ml) of tissue at the beginning.

For the determination of the fractional rates of synthesis (K S) and degradation (K D) in the liver and white muscle of rainbow trout, we rely on the technique described by Garlick et al., Applied to study of fish by Harschemeyer, and modified by our research group [Garlick et al , 1980; Harschemeyer et al , 1983; Pocrnjick et al , 1983; Houlihan et al , 1986, 1988; Peragón et al , 1992].

For the study of the different parameters of protein replacement times corresponding to the 58, 153 and 226 days of starting the experiment to control the possible differences in protein biosynthesis that took place between the different experimental groups. For this study, They used 9 animals at each time point. The solution that is injected to the fish contained a mixture of L- [2,6 3 H] Phe (37 MBq / m; 100 mCi / ml and specific radioactivity 1640 dpm / nmol) and Phe 150 mM. The dose administered was 0.5 ml / 100 g weight body, and represents between 5 and 12.5% of blood volume total, assuming that the volume of blood of a fish represents between 10 and 40% of your body volume [Jones and Randall, 1978]. Said solution was injected into the caudal vein after anesthetize the animal with ethylene glycol monophenyl ether. Immediately after the injection the animals were returned to the Aerated water for recovery. Two of the 9 fish used in each point is sacrificed 2 minutes after the injection, and the rest at 45 minutes. In their respective times, the fish are, in addition, heavy, proceeding to the extraction of the liver and white muscle, whose weights are also noted.

Transformation of the labeled phenylalanine in β-phenylethylamine

Phenylalanine hydroxylase, an enzyme present in the sample, and active in the experimental conditions, catalyzes the transformation of phenylalanine (Phe) into tyrosine (Tyr). For ensure that the radioactivity we are measuring is the corresponding to the marked Phe and not to Tyr that could originate from it, both amino acids are transformed into their corresponding biogenic amines, β-phenylethylamine (β-PEA) and tyramine. A simple and effective way to achieve this is to use tyrosine decarboxylase (Tyr-DCase), capable of decarboxylate both tyrosine and phenylalanine at pH 6.3 and 50 ° C temperature 17. Of the two biogenic amines formed, only β-PEA can be extracted with chloroform: n-heptane, discarding tyramine. PH 6.3 is essential for Phe to separate from Cl - and anions decarboxylation can be carried out with the maximum performance and effectiveness

The determination of the specific radioactivity of the β-PEA from the two fractions it is done by using the scintillation counter and a program suitable. While determining the total concentration of β phenylethylamine (β-PEA) is carried to out according to the method of Suzuki [Suzuki and Yagi, 1976].

The fractional speed of synthesis of Proteins (K S) are calculated from the radioactivity specific for free Phe (dpm / nmol) and radioactivity specific mean of protein-bound Phe (dpm / nmol). The fractional rate of protein degradation (K D) was calculated by difference between the synthesis rate K_ {S} (% / day) KG growth rate (% / day) according to the following expression: K_ {D} = K_ {S} - K_ {G}.

To determine the retention efficiency of proteins (ERP), that is, the percentage of synthesized protein which is deposited in the tissue, the equation was used: ERP = (K_ {G} - K_ {S}) x 100.

The determination of RNA content in liver and white muscle of rainbow trout is based on the technique used by Munro and Fleck and modified by Peragón et al . [Munro and Fleck (1966); Peragón et al , 1992]. The determination of the concentration of DNA in liver and white muscle is based on the method of Labarca and Paigen [Labarca and Paigen, 1979].

Tissue treatment and enzymatic activities

The liver and white muscle frozen at -80 ° C are used, corresponding to each of the experimental situations and at different times of the experiment. The homogenates are prepared from a pool of 6 livers or 6 pieces of white muscle, using a suitable homogenization buffer (TH) in the following proportions: Liver : 1/10 (w / v) and Muscle : 1/5 (p / v) for all enzymes. The homogenates are centrifuged at 105,000xg for 1 hour at 4 ° C and the resulting supernatant is distributed in aliquots, which will be kept at -80 ° C until the same time that the enzymatic activity is to be measured.

NADPH producing enzymes

Determination of the activity of the malic enzyme (MS) in liver and white muscle of rainbow trout ( Oncorhinchus mykiss ). Based on the method of Ochoa et al with some modifications [Ochoa et al 1955].

Determination of the activity of glucose-6P dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the liver and white muscle of rainbow trout ( Oncorhinchus mykiss ). The reaction of G6PDH is of the simple and bidirectional REDOX type, and that of 6PGDH is a bidirectional oxidative decarboxylation. In both cases the reaction proceeds to the formation of reduction equivalents (NADPH). The basis is the spectrophotometric measurement at 340 nm of the appearance of NADPH after the action of both enzymes. When measuring two different enzymes we have to prepare two types of cuvettes:

- Cuvettes A : As a substrate they contain 1 ml of 6-phosphogluconate (6PG).

- Cuvettes B : Contains 0.1 ml of 6-phosphogluconate (6PG) and 0.1 ml of glucose 6-phosphate (G6P).

The cuvettes A will be used for the direct determination of the activity of glucose 6-P dehydrogenase. The activity of 6-phosphogluconate dehydrogenase is the result of the difference in the values obtained with cuvettes A and B. [Lupiáñez et al , 1987, Peragón et al , 1990].

Determination of isocitrate activity NADP + dependent dehydrogenase in the liver and white muscle of rainbow trout (Oncorhinchus mykiss). It is an enzyme NADP + dependent that catalyzes decarboxylation oxidative isocitrate to yield α-ketoglutarate (αKG). The appearance of NADPH at 340 nm. Technique based on the described method by Bernt and Bergmeyer (1983), with some modifications.

Growth related parameters Average values of the weights of the different groups at the beginning, half and end of the experiment

2

(n = 18; t = 226 days. Weights expressed in grams S.E.M. G_ {R} and L_ {S} in%)

Absolute daily intake

3

(n = 18; t = 226 days. Results expressed in mg or KJ / fish ± S.E.M.)

Relative daily intake [mq or J / g fish]

4

(n = 18; t = 226 days. Results expressed in mg or J / g fish ± S.E.M.)

Nutritional indices

5

\hskip0,3cm

(n=18; t=226 días. FE= g crecimiento/g ingesta. PER= g crecimiento/g proteína ingerida)

 \ hskip0,3cm 

(n = 18; t = 226 days. FE = g growth / g intake. PER = g growth / g protein ingested)

Claims (6)

1. Use of maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural or concentrated products thereof containing it, alone or in combination with other agents, of products applicable to animal production characterized by the fact that the product is an enhancer of white muscle growth, of its titular proteins and of body growth. 2. Use of maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural or concentrated products thereof that contain it in the preparation, alone or in combination with other agents, of products applicable to animal production, according to claim one, characterized in that the product is inhibitor of the activity of serine proteases. 3. Use of maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural or concentrated products thereof that contain it in the preparation, alone or in combination with other agents, of products applicable to animal production, according to first and second claim characterized in that at the molecular level the product is an enhancer of enzymes producing NADPH and protein turnover. 4. Use of maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural products or concentrates thereof that contain it in the preparation, alone or in combination with other agents, of Products applicable to animal production, according to previous claims, characterized by the use thereof in amounts comprised between 0.1 mg and 250 mg per kilogram per kg of feed. 5. Animal feed product characterized by containing maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural or concentrated products thereof containing it. 6. Animal feed product characterized by containing maslinic acid (2-alpha, 3-betadihydroxy-28-carboxy-lean-12-ene), its derivatives or natural or concentrated products thereof containing it in amounts between 0.1 mg and 250 mg per kilo per kg of food.