RU2745401C1 - Method for inducing the secretion of biologically active compounds in sea snail rapana venosa val. - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: method involves stimulating the release of biologically active compounds, while shellfish are selected from their natural habitat at sea water temperatures from 17.2 C to 8.2 ºC. For stimulation, a combined stimulating solution consisting of 25% magnesium sulfate (MgSO₄) and 0.04% serotonin solution (C₁₄H₁₉N₅O₂·H₂SO₄) is used, which is injected into the proboscides or leg muscle of hungry sea snails with a syringe. For shellfish with a shell height of 46,3059,27 mm, the volume of the combined stimulating solution is 0.5 ml, for shellfish with a shell height of 6,077 mm - 1 ml, for shellfish with a shell height of 114.14 mm-1.5 ml.

EFFECT: invention provides possibility of obtaining biologically active compounds.

4 cl, 6 dwg, 1ex

Description

The invention relates to the field of biotechnology and is intended to obtain a natural product for pharmacological purposes.

It is known that all mollusks belonging to the superfamily Muricoidea have a very high content of esters strictly localized in the middle region of the dye-secreting hypobranchial gland [Andrews, 1991]. Choline esters are represented by murexin, dihydromurexin and sensioylcholine. Thus, in R. bezaor (Linn.), A closely related species of R. venosa, the content of murexin and sensioylcholine was 400 and 100 mg g ^{-1 of} fresh soft tissues, respectively [Roseghini et al., 1996]. The authors found that all compounds had a strong neuromuscular blocking effect, causing paralysis of the muscles of invertebrates, including paralysis of the articular muscles of bivalve molluscs.

The predatory gastropod mollusk Rapana venosa feeds on bivalve mollusks, but mussels are the main food item (Chukhchin, 1984). When attacked, adults paralyze the victim, releasing a biotoxin of a complex biochemical composition (Cesari and Mizzan, 1993). The biotoxin is produced in the hypobranchial gland, which is divided into three sections: two lateral ones that secrete mucus, and the middle one, colored milky white, secreting a dye purple (Chukhchin, 1970; Ponte, Modica, 2017). It is known that violet tyrian is formed from the secretion of the hypobranchial glands of gastropods from the precursor salt of the choline ester of tirindoxyl sulfate after a series of enzymatic, oxidative, and photochemical reactions [Benkendorff et al., 2001]. These bioactive secondary metabolites belong to the chemical class of indole alkaloids, which are natural products for the production of drugs [Grabley, Sattler, 2003]. Found that derivatives of indirubin effectively inhibit the proliferation of cancer cells [Leclerc et al., 2001; Meijer et al., 2003; Kim et al., 2007], and also induce apoptosis in human breast and prostate cancer cells [Brown, Wouters, 1999; Nam et al., 2005; Kim et al., 2007]. It is widely accepted that cell death after DNA damage by anticancer agents is mainly the result of apoptosis [Brown, Wouters, 1999].

It is known that to obtain substances contained in the hypobranchial gland of gastropods (Nucella lapillus, Murex brandaris, M. trunculus, Purpura capillus, P. patula, Dicathais orbita), it is excised, while the enzyme (purpurase) is released, which removes the sulfate group the formation of tirindoxil. Tyrindoxyl sulfate is isolated from the glands by ethanol extraction [Cooksey, 2001].

A known method of stimulating the release of biologically active compounds in the gastropod mollusk Dicathais orbita Gmelin, 1781 is an endemic of southern Australia and the coastal waters of New Zealand. [Warwick James Noble. Aspects of life history and ecology of Dicathais orbita Gmelin, 1781 related to potential aquaculture for bioactive compound recovery // A thesis submitted to the Flinders University, in candidature for the degree Doctorate of Philosophy. School of Biological Sciences Flinders University. 2014. 156 p.https: //flex.flinders.edu.au/file/20844afe-ccb6-4112-bb2a-6d3784d3ed73/l/Thesis-Noble-2014.pdf]. To induce the secretion of biologically active compounds in the molluscs D. orbita of three size classes (23-33 mm, 33.5-53 mm and> 53.5 mm), four chemicals were tested, dissolved in seawater: 5% ethanol; benzocaine, 200 mg · l ^-1 ; sodium pentabarbital, concentration 1 mg · l ^-1 and 0.5 M magnesium chloride. Fresh seawater was used as a control. The clams were immersed in a stimulant solution for one hour, with each clam exposed to only one substance. It was found that only solutions of ethanol and benzocaine stimulated the release of biologically active compounds. According to the author, this is due to the effect of impaired control of the nervous system for relaxation of the leg muscle and stress response in D. orbita, which leads to the expulsion of mucus containing bioactive precursors of purple tyrian. After the animals were exposed to these substances, their nutrition was monitored for another 5 days. However, according to the author, the possibility of long-term sublethal consequences cannot be ruled out.

The objective of the invention is to develop a method for intravital multiple stimulation of the release of biologically active compounds (BAC) in the rapaid R. venosa.

The technical result of solving the problem is that a technology has been developed for the in vivo production of biologically active compounds in the predatory gastropod molluscum rapana R. venosa - a raw material for the production of medicines. This technology was developed for the first time and allows obtaining a significant amount of UAS. In addition, an additional technical result is the possibility of in vivo sex determination of mollusks, which is also important for scientific purposes when studying the population structure of mollusks.

The claimed technical result is achieved due to the fact that in the Method for induction of the secretion of biologically active compounds in Rapana venosa Val. 8.2 ° C. For stimulation, a combined stimulating solution is used, consisting of 25% magnesium sulfate (MgSO ₄ ) and 0.04% serotonin solution (C ₁₄ H ₁₉ N ₅ O ₂ H ₂ SO ₄ ), which is injected into the trunk of hungry rapa with a syringe or muscle of the leg. For mollusks with a shell height of 46.30 ÷ 59.27 mm, the volume of the combined stimulating solution is 0.5 ml; for mollusks with a shell height of 60 ÷ 77 mm - 1 ml; for molluscs with a shell height of 114.14 mm - 1.5 ml. The combined stimulating solution is prepared by dissolving 4 mg of serotonin in 10 ml of distilled water and 10 ml of 25% magnesium sulfate, the volumes of which are combined in a 1: 1 ratio. In addition, to stimulate the release of biologically active compounds, mollusks with a shell height of more than 60 mm and a total weight of more than 30 g are selected. In addition, after the introduction of the combined stimulating solution, an in vivo sex determination of the rapana is carried out.

The claimed method meets the criteria: novelty and inventive step, tk. For the first time, conditions were proposed for the induction of the secretion of biologically active compounds in Rapana venosa, which do not require complex and expensive equipment or the use of expensive chemical reagents. The conducted patent research, as well as the study of available scientific publications related to the subject of the invention, did not find solutions that have features similar to the claimed technical solution, which confirms the conclusions about compliance with the patentability criteria.

The invention is illustrated by illustrations, where:

FIG. 1 - A - cage with rapa; B - rapanas in the laboratory before stimulating the release of biologically active compounds;

FIG. 2 - Stimulating solution injection sites: 1 - trunk, 2 - leg muscle;

FIG. 3 - The process of release of biologically active compounds with rapa beans after stimulation: A - freshly isolated yellow hypobranchial gland secretion; B - the secret of the hypobranchial gland after oxidative and photochemical reactions - pink and purple;

FIG. 4 - Isolation of biologically active compounds with rapans after stimulation (Table);

FIG. 5 - Dependence of the proportion (in%) of the rapana specimens that have released biologically active compounds on the temperature of the water in the sea;

FIG. 6 - Male rapana. The arrow indicates the penis - penis.

The claimed method is the result of scientific research. The material for the research was the rapa shells R. venosa with a shell height of 46.30 to 114.1 mm and an age of 2+ to 4+ years. The mollusks were collected in November 2019 in the Sevastopolskaya Bay at a depth of 9 to 15 m under the "mussel farm (outer roadstead of the Sevastopol Bay). Subsequently, the mollusks were kept in a plastic cage (top sheathed with a case to prevent their crawling), (Fig. 1A) and suspended in the sea at a depth of 2.5 m (salinity 18%) .Feed for rapan were live mussels with sizes from 60 to 79 mm (average length -70 mm), grown on a mussel farm.

Before the start of stimulation, the total weight of the mollusks was determined (OHAUS balance, accurate to 0.01 g). The mass of soft tissues of mussels consumed by rapa beans was calculated using the formula (Pirkova et al., 2019):

During 10/28/19 - 03/10/2020, with an interval of 2-3 weeks under laboratory conditions, the release of biologically active compounds in rapa was stimulated using a combined solution: magnesium sulfate (MgSO ₄ - 12.5%) with a solution of serotonin (C ₁₄ H ₁₉ N ₅ O ₂ · H ₂ SO ₄ - 0.02%). The stimulating solution was prepared in the following way: 4 mg of serotonin was dissolved in 10 ml of distilled water and 10 ml of 25% magnesium sulfate was added. The composition of the stimulating solution was selected by the authors on the basis of information on the synergistic effect of serotonin and magnesium sulfate on the physiological functions of mollusks associated with nutrition. It has been proven that serotonin is a neuromodulator that controls nutrition in invertebrates (Dayan and Huys, 2009). Thus, an increase in the concentration of serotonin in the hemolymph of a mollusk after its introduction into the leg muscle or through the trunk into the digestive system is a signal to stimulate the synthesis of ALS. And magnesium sulfate (magnesia), causing relaxation of smooth muscles and vasodilation (https://apteka.103.by/magnija-sulfat-instruktsiya/#3 - 06/02/2020), promotes its unimpeded release. Thus, the combined solution of serotonin and magnesium sulfate is an effective stimulant for the release of biologically active compounds in rapana, without negatively affecting the viability of mollusks,

Using a syringe, the solution was injected into the trunk or into the leg muscle of the rapana (Fig. 2). Then the mollusks were laid out on a tray with the mouth up (Fig. 1B). After the appearance of a cloudy yellow liquid in the mouth, the rapans were placed on weighing bottles or measuring glasses downward with a siphonal channel (Fig. 3). For the period from 28.10.2019 to 10.03.2020, with an interval of 2-3 weeks, 10 stimulations of ALS secretion were performed in rapan (Table 1).

The optimal volume of the injected stimulating solution was experimentally determined depending on the size of the mollusks. For mollusks with a shell height of 46.30 to 59.27 mm, it was 0.5 ml; for rapan with sizes from 60 to 77 mm - 1 ml; with a shell height of 114.14 mm - 1.5 ml (No. 20). After the introduction of the stimulating solution into the leg muscle, the release of ALS began in 2-3 minutes; when it was introduced into the trunk, the onset of excretion in different individuals was noted in the interval from 3 to 11 min.

The dependences of the average volume released by BAS on the shell height (1) and on the total weight (2) of the molluscs have been established:

where: V is the average volume of BAS, ml; e is the base of the natural logarithm (2.71828), H is the height of the rapana shell, mm.

where: W is the total weight of the rapana, g.

It was revealed that the larger the size or total weight of the rapana, the greater the volume of biologically active compounds it can release after stimulation. Thus, the maximum volume of BAS (8.5 ml) was isolated by a shellfish with a shell height of 114.1 mm and a total weight of 81.31 g; and the minimum volume (0.4 ml) was isolated with rapana with size and weight characteristics: H = 50.32 mm and W = 20.71 g

The tendency of the dependence of the proportion of individuals that isolated ALS on the water temperature in the sea was investigated: the maximum proportion of individuals that released ALS was observed at a water temperature of 16 ° С, the minimum - at 8.2 ° С (Fig. 5).

За период наблюдения среднее количество положительных откликов на стимуляцию выделения биотоксина у самок и самцов рапаны достоверно не отличалось (соответственно 4,1 и 4,2).Rapana is a dioecious mollusk. The sex of the rapana was previously determined by destroying the shell of the rapana (Chukhchin, 1984; Savini et all., 2004; Saglam et all., 2009; Saglam et all., 2015; Bondarev, 2016). The inventors have found that after the administration of the stimulating solution, when the body of the mollusk is relaxed and the leg cap is easily pushed back, it is possible to determine the sex of the rapana in vivo, as shown in FIG. 6. In the studied sample, the sex ratio is equal and amounted to:

During the observation period, the average number of positive responses to the stimulation of biotoxin secretion in female and male rapanas did not differ significantly (4.1 and 4.2, respectively).

The signal for the release of biotoxin (or biologically active compounds) by rapa is the physiological need for food and the availability of food. According to V.D. Chukhchina (1984), the intensity of feeding depends on the size of the rapana and the temperature of the water. The break between meals can be about 4 days. According to the author's data, at water temperatures below 10 ° C, mollusks stopped feeding (Chukhchin, 1984).

During the experiment, the sea water temperature changed in the autumn-winter period from 17.2 to 8.2 ° C. The amount of mussels consumed by rapanas did not depend on the water temperature within the specified values. So, at a temperature of 9.8 ° C, the number of mussels consumed in two weeks by 20 rapans was 10 specimens. (with an average value of the diet - 0.22 g · specimen ^-1 · day ^-1 ), and at a temperature of 16.0; 8.2 and 10.4 ° С for the same observation period - 1 mussel (or 0.02 g · specimen ^-1 · day ^-1 ). It is known that at a water temperature of 10.0 ° C, the linear growth of the rapana stops (Chukhchin, 1984). By the difference between adjacent total weight values, you can determine the shellfish that have recently eaten. Such individuals, as a rule, did not excrete ALS after stimulation. Therefore, ALS induction is recommended to be performed in hungry rapa.

After the introduction of the stimulating solution, the rapans secrete biologically active compounds in the form of a cloudy yellowish substance (see Fig. 3A). Depending on the intensity of light and the duration of exposure to air, the yellow color of the BAS turns to pink or red, and then to purple (see Fig. 3). The formation of purple was accompanied by the release of a volatile substance with a pungent odor, as pointed out by V.D. Chukhchin (1970) - mercaptan.

During the entire study period, the survival rate of molluscs was 100%.

An example of the implementation of the method.

Rapana R. venosa were collected in November 2019 in the Sevastopolskaya Bay in the amount of 20 specimens. at a depth of 9 to 15 m under the mussel farm (outer roadstead of the Sevastopol Bay). The sizes of shells - mollusks ranged from 46.30 to 114.1 mm, and the age - from 2+ to 4+ years.

During 10/28/19 - 03/10/2020, with an interval of 2-3 weeks in laboratory conditions, the release of biologically active compounds in rapa was stimulated (a total of 10 stimulations were carried out). To induce the secretion of biologically active compounds (BAC), a combined solution was used: magnesium sulfate (MgSO ₄ - 12.5%) with a serotonin solution (C ₁₄ H ₁₉ N ₅ O ₂ · H ₂ SO ₄ - 0.02%). The stimulating solution was prepared immediately before the stimulation in the following way: 4 mg of serotonin was dissolved in 10 ml of distilled water and 10 ml of 25% magnesium sulfate was added. Using a syringe, the solution was injected into the trunk or into the leg muscle of the rapana. The volume of the injected solution depended on the size of the mollusks: with a shell height from 46.30 to 59.27 mm, it was 0.5 ml; with a shell height of 114.14 mm - 1.5 ml (No. 20); for rapan with sizes from 60 to 77 mm - 1 ml. After the introduction of the stimulating solution into the leg muscle, the release of ALS began in 2-3 minutes; when it was introduced into the trunk, the onset of excretion in different individuals was noted in the interval from 3 to 11 minutes in the form of a cloudy yellow liquid filling the mouth of the mollusk. After that, the rapan was placed on weighing cups or measuring glasses downward with a siphonal channel to collect BAS. Hungry mollusks responded positively to stimulation.

The volume of BAS depended on the size (total live weight) of the rapana: the maximum volume - 8.5 ml was isolated once with the rapana with a shell height of 114.1 mm and a total weight of 81.31 g; and the minimum - 0.4 ml was isolated with rapa with a shell height of 50.32 mm and a total weight of 20.71 g. As a result of 10 stimulations of induction of biologically active compounds from 20 specimens. rapan received 137.45 ml of BAS.

Literature sources taken into account:

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Claims (4)

1. A method for inducing the secretion of biologically active compounds in Rapana venosa Val., Including stimulating the release of biologically active compounds and collecting them, characterized in that the mollusks are taken from their natural habitat at a sea water temperature in the range from 9.8 to 17 ° C, and for stimulation, a combined stimulating solution is used, consisting of 25% magnesium sulfate (MgSO ₄ ) and 0.04% serotonin solution (C ₁₄ H ₁₉ N ₅ O ₂ H ₂ SO ₄ ), which is injected into the trunk of the hungry rapa using a syringe or a leg muscle, and for mollusks with a shell height of 46.30 ÷ 59.27 mm, the volume of the combined stimulating solution is 0.5 ml, for mollusks with a shell height of 60 ÷ 77 mm - 1 ml, for mollusks with a shell height of 114.14 mm - 1.5 ml. 2. The method according to claim 1, characterized in that the combined stimulating solution is prepared by dissolving 4 mg of serotonin in 10 ml of distilled water and 10 ml of 25% magnesium sulfate, the volumes of which are combined in a 1: 1 ratio. 3. The method according to claim 1, characterized in that to stimulate the release of biologically active compounds, mollusks with a shell height of more than 60 mm and a total weight of more than 30 g are selected. 4. The method according to claim 1, characterized in that after the introduction of the combined stimulating solution, an in vivo sex determination of the rapana is carried out.

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