International Journal of Biology and Chemistry

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VOLUME 15 NUMBER 2 2022 ISSN 2218-7979 eISSN 2409-370X

International Journal of

Biology

and Chemistry

Al-Farabi Kazakh National University

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al-Farabi Kazakh National University, al-Farabi ave., 71, 050040, Almaty, Kazakhstan website: http://ijbch.kaznu.kz/

Any inquiry for subscriptions should be sent to:

Prof. Mukhambetkali Burkitbayev, al-Farabi Kazakh National University al-Farabi ave., 71, 050040, Almaty, Kazakhstan

e-mail: [email protected], Mukhambetkali [email protected]

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EDITORIAL

The most significant achievements in the field of natural sciences are reached in joint collaboration, where important roles are taken by biology and chemistry. Therefore publi- cation of a Journal, displaying results of current studies in the field of biology and chemistry, facilitates highlighting theoretical and practical issues and distribution of scientific discoveries.

One of the basic goals of the Journal is to promote the extensive exchange of information between the scientists from all over the world. We welcome publishing original papers and materials of biological and chemical conferences, held in different countries (by prior agreement, after the process of their subsequent selection).

Creation of International Journal of Biology and Chemistry is of great importance, since scientists worldwide, including other continents, might publish their articles, which will help to widen the geography of future collaboration.

The Journal aims to publish the results of the experimental and theoretical studies in the field of biology, biotechnology, chemistry and chemical technology. Among the em- phasized subjects are: modern issues of technologies for organic synthesis; scientific basis of the production of biologically active preparations; modern issues of technologies for processing of raw materials; production of new materials and technologies; study on chemical and physical properties and structure of oil and coal; theoretical and practical issues in processing of hydrocarbons; modern achievements in the field of nanotechnology; results of studies in various branches of biology, chemistry and related technologies.

We hope to receive papers from the leading scientific centers, which are involved in the application of the scientific principles of biological and chemical sciences on practice and fundamental research, related to production of new materials, technologies well ecological issues.

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IRSTI 31.23.17:31.17.29 https://doi.org/10.26577/ijbch.2022.v15.i2.01

S.M. Adekenov

JSC “International research-production holding “Phytochemistry”, Karaganda, Kazakhstan e-mail: [email protected]

(Received October 12; received in revised form 2 November 2022; accepted 4 November 2022)

Mechanocomposites based on

1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide

Abstract. Current paper discusses the results of complex formation of 1(10)β-epoxy-5,7α(Н),6β(Н)-guai- 3(4),11(13)-dien-6,12-olide with polyvinylpyrrolidone, disodium salt of glycyrrhizin acid and magnesium carbonate. Inclusion complexes with disodium salt of glycyrrhizic acid were obtained by mechanochemical treatment into micelles formed by the associated molecules of glycyrrhizic acid. Polyvinylpyrrolidone and magnesium carbonate form host-guest type complexes with 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)- diene-6,12-olide. Mechanocomposites were obtained in laboratory ball mill MSHL-1 (Itomak, Russia) with 1 to 6 hours of processing time. Studies on water solubility of obtained mechanocomposites demonstrate that water solubility of 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide in a mechanocomplex with disodium salt of glycyrrhizic acid after 2-hour mechanochemical treatment increased by 4.61 times, in the mechanocomplex with polyvinylpyrrolidone by 4.42 times, and with magnesium carbonate by 1.66 times. The surface morphology of the obtained mechanocomposites was studied by scanning electron microscopy (magnification x500). After mechanochemical treatment, the original shape of the particles of the initial components has changed and it is impossible to isolate individual components, except for the formed agglomerates. The resulting substances are polydisperse powders with particles (5-20 µm in size) and their aggregates. The results obtained indicate that the increase in the water solubility of the substance based on 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide has been achieved by the formation of supramolecular complexes after mechanochemical treatment with polyvinylpyrrolidone, disodium salt of glycyrrhizic acid and magnesium carbonate.

Keywords: 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide, mechanocomposites, complexing agents, glycyrrhizic acid disodium salt, polyvinylpyrrolidone, magnesium carbonate, water solubility, photomicrographs.

Introduction

Natural compounds with immunomodulatory activity are widely used in treatment of various diseases, including autoimmune and inflammatory diseases in addition to cancer. Immunomodulators are agents that have the ability to enhance the host defense re- sponse and can be used prophylactically in combi- nation with other therapeutic agents. The anticancer activity of these immunomodulators is due to their anti-inflammatory, antioxidant action, as well as the induction of apoptosis, antiangiogenesis, and antime- tastasis [1–2].

An analysis of the available literature data [1- 4] indicates that the sesquiterpene lactone 1(10) β-epoxy-5,7α,6β(Н)-guai-3(4),11(13)-diene-6,12- olide (1), which has in its structure, in addition to the γ-lactone ring, an epoxy function and an olefinic double bond in the carbon backbone, can be consid-

ered as a potential immunomodulator. It is a colorless crystalline substance of the composition C₁₅H₁₈O₃ with Tm 101-104°, [α]_D +45° (with 0.3 chloroform).

The main disadvantage of 1(10)β-epoxy- 5,7α,6β(Н)-guai-3(4),11(13)-diene-6,12-olide, as well as other natural sesquiterpene lactones, is its poor solubility in water, which has a negative effect on its bioavailability and reduces the specific pharmacological activity in the body. Therefore, it is considered practically important to modify the natural guaianolide molecule by converting it into water- soluble complexes.

One of the modern ways to increase the solubility of a medicinal substance is a mechanochemical method of processing, which includes physicochemical transformations of solid components and their mixtures under conditions of intense shock-attrition effects [5-7]. The essence of the technology is to ob- tain solid dispersions of medicinal substances with

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5 S.M. Adekenov

excipients of various chemical nature. At the same time, there is an increase in the solubility of the starting substances and, accordingly, the effective- ness of their pharmacological action. Depending on the physicochemical properties of the substrate, solid dispersions are obtained by dispersing medicinal substances in molecular form or in an amorphous state with the formation of water-soluble inclusion complexes with excipients of the “guest-host” type [8].

Current paper discusses the results of complex formation of 1(10)β-epoxy-5,7α(Н),6β(Н)-guai- 3(4),11(13)-dien-6,12-olide with polyvinylpyrrolidone, disodium salt of glycyrrhizin acid and magnesium carbonate. Inclusion complexes with disodium salt of glycyrrhizic acid were obtained by mechanochemical treatment into micelles formed by the associated molecules of glycyrrhizic acid.

Materials and methods

Preparation of solid dispersions. The preparation of solid dispersions was carried out in a MSHL-1 ball mill (Itomak, Russia) with a drum with a fluoroplastic lining. Processing mode: total loading of the components of the processed mixture from 18 to 22 g,

drum volume – 300 ml, grinding media – steel balls (diameter 22 mm, loading 675 g). The processing time ranged from 1 to 6 hours. Optimal mass ratios of components: guaianolide/Na₂GA – 1:10, guaianolide/MgCO₃ – 1:5 and guaianolide/PVP – 1:10.

Complexing agents. The following were used as complexing agents:

- Disodium salt of glycyrrhizic acid (2) (Na₂GA) – a derivative of plant saponin, (CFS, 98%) manufactured by Shaanxi Sciphar Biotechnology Co. Ltd.

(Xi’an, China). The gross formula is C₄₂H₆₀O₁₆Na₂. It is a gray powder with a mustard tint. Doesn’t melt.

Sublimates at a temperature of ~400 °C. Easily soluble in water.

- Polyvinylpyrrolidone (3) (PVP) – a synthetic polymer manufactured by Huangshan Bonsun Phar- maceuricals Co., Ltd. (Huangshan, China). General formula (C₆H₉NO)_n. It is a white, yellowish-white powder, odorless. Has a sweetish taste. T_m = 150 °С.

Well dissolved in water, ethanol and methanol.

- Substance of basic magnesium carbonate (MgCO₃) produced by Biochem Chemopharma (France) of pharmacopoeial purity (Manufacturer’s pharmacopoeial monograph 42-3989-08). It is a white powder, odorless and tasteless, practically insoluble in water.

O O O

H

2 1 3

4 5

6 7

8 9 10

11 12

13 14

15

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Results and discussion

The preparation of solid dispersions of 1(10) β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene- 6,12-olide was carried out in a MSHL-1 ball mill with a drum with a fluoroplastic lining. Process- ing mode: acceleration of grinding media – 1g, total loading of components of the processed mixture from 18 to 22 g, drum volume – 300 ml, grinding media – steel balls (diameter 22mm,

loading 675g). The processing time ranged from 1 to 6 hrs.

As can be seen from Figure 1, the best result of the dissolution of 46.6 ± 0.13 seconds is a two- hour mechanochemical treatment of 1(10)β-epoxy- 5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide with disodium salt of glycyrrhizic acid at a ratio of 1:10. 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)- diene-6,12-olide and its mechanocomposite with magnesium carbonate are practically insoluble in water.

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Figure 1 – Dependence of the dissolution of mechanocomposites

1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide with polyvinylpyrrolidone and disodium salt glycyrrhizic acid from the time of mechanochemical processing

Figure 2 – Effect of mechanochemical treatment time on the complexation of 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide

The results presented in Figure 2 show that dur- ing a 2-hour mechanochemical treatment, the water solubility of 1(10)β-epoxy-5,7α(Н),6β(Н)-guai- 3(4),11(13)-diene- 6,12-olide in the mechanocomplex with the disodium salt of glycyrrhizic acid increased by 4.61 times, and in the mechanocomplex with polyvinylpyrrolidone by 4.42 times, and with magnesium carbonate by 1.66 times.

The results obtained indicate that the increase in the water solubility of the substance based on 1(10) β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene- 6,12-olide has been achieved by the formation of supramolecular complexes by the method of mechanochemical processing. The following were used

as complexing agents: a water-soluble derivative of plant saponin – disodium salt of glycyrrhizic acid and synthetic polymer polyvinylpyrrolidone.

The micrographs shown in Figure 3 character- ize the surface morphology of the obtained samples.

1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)- diene-6,12-olide consists of crystalline particles and their agglomerates. The disodium salt of glycyrrhizic acid consists of spherical hollow particles with a smooth surface. After mechanochemical treatment, the original shape of the particles of the initial components has changed and it is impossible to isolate individual components, except for the formed agglomerates.

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7 S.M. Adekenov

A B C

D E

Figure 3 – Electron micrographs of the initial components and the complexes obtained on their basis.

Note: А – 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide;

B – 1(10)β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide + polyvinylpyrrolidone (m/t time=2 h);

C – 1(10)β-epoxy-5,7α(H),6β(H)-guai-3(4),11(13)-diene-6,12-olide + disodium salt of glycyrrhizic acid (time m/t=2 h;);

D – Polyvinylpyrrolidone; E – Disodium salt of glycyrrhizic acid (x500). Magnification in all cases: x500

As can be seen in Figure 3, the resulting substances are polydisperse powders with particles 5–20 µm in size and their aggregates. In this case, in the above complexes, hydrogen bonds are formed in the intermolecular space. As a result of mechanical treatment of mixtures of powders after the initial grinding, the process of aggregation of microparticles takes place. Microcomposites are formed, consisting of submicron particles and having a very developed contact between the phases. In this case, the inclusion of the substrate molecule into micelles, which are formed due to the associated molecules of glycyrrhizic acid. And polyvinylpyrrolidone and 1(10) β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene- 6,12-olide form guest-host complexes.

Mechanical treatment of guaianolide with basic magnesium carbonate, polyvinylpyrrolidone and disodium salt of glycyrrhizic acid transforms the crystalline substance into an amorphous state. The process is accompanied by the formation of coordina- tion bonds between the molecules of 1(10)β-epoxy- 5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide and complexing agents by donor acceptor mechanism. The process of obtaining complexes occurs in the solid phase, which makes it possible to exclude the use of organic solvents. The results obtained cor- relate with existing information discussed below.

Carrying out mechanical activation in mills is the most common operation in mechanochemistry.

The advantages of this method are one-step, rela- tive simplicity, the ability to increase bioavailability without changing the molecular structure. The process takes place in the solid phase, which allows the exclusion of toxic solvents. High-intensity mechanical treatment can lead to the breaking of strong covalent bonds, while low-intensity mechanochemical treatment allows the molecules of biologically active substances to “penetrate” into the space in- side the macromolecule or self-associates of the ex- cipient, forming a supramolecular complex due to hydrogen bonds and van der Waals forces. The absence of covalent interaction between the molecules of biologically active substances and complexing agents indicates that the structure of the original substance does not change. To improve the solubility of simvastatin lactone and increase its oral bioavailability, its complexes with arabinogalactan and disodium salt of glycyrrhizic acid were obtained by mechanochemical activation. Pharmacokinetic tests in vivo on laboratory animals show a significant increase in the bioavailability of simvastatin after its administration in the form of a complex with the disodium salt of glycyrrhizic acid or with arabinogalactan [9].

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By mechanochemical treatment of triterpene betulin diacetate with arabinogalactan (ratio 1:9), mechanocomposites were obtained. It was estab- lished by gel permeation chromatography that mechanochemical treatment leads to a change in the molecular weight distribution [10]. Mechani- cal activation of a mixture of betulin diacetate with aerosil leads to homogenization of the mixture as a result of dispersion of the components and the formation of mechanically activated composites. Us- ing physicochemical methods, electron microscopy, Infrared spectroscopy and X-ray phase analysis, it was proved that mechanical activation leads to the formation of composites of betulin diacetate with aerosil and amorphization of crystalline diacyls. The water solubility of mechanically activated betulin diacetate composites increased from 0.8 to 6.1 g/mL [11]. Pharmaceutical solid dispersions of curcumin polyphenol with macromolecular polysaccharide arabinogalactan were obtained by mechanical processing. The complexes obtained by mechanochemical treatment demonstrated an increased solubility of curcumin up to 10.5 times compared to pure curcumin [12]. Composites of praziquantel with disodium salt of glycyrrhizic acid were obtained by mechanochemical processing at mass ratios of components 1:5, 1:10, and 1:20. In this case, the greater the mass ratio, the greater the increase in the solubility of praziquantel in water [13].

Conclusion

The results of the study show that both disodium salt of glycyrrhizic acid and polyvinylpyrroldone can be used as complexing agents in the mechanochemical treatment of sesquiterpene lactone 1(10)β-epoxy- 5,7α(Н),6β(Н)-guai-3(4),11(13)-diene-6,12-olide.

The obtained mechanocomplexes of 1(10)β-epoxy- 5,7α(Н),6β(Н)-guai-3(4),11(13)-dien-6,12-olide with polyvinylpyrrolidone and disodium salt of glycyrrhizic acid have an increased water solubility. This does not change the molecular structure of 1(10) β-epoxy-5,7α(Н),6β(Н)-guai-3(4),11(13)-diene- 6,12-olide, which is an important factor to preserve the pharmacological activity of the sesquiterpene lactone molecule.

Acknowledgment

The work was carried out within the frame- work of the project No. АР14870517 “Develop- ment of a water-soluble form of cyclopentadie- none guayanolide and its production technology”,

funded by Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan.

References

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Med. Chem. – Anti-Cancer Agents, № 5, pp. 239- 249. doi.org/10.2174/1568011053765976

5. Sali N., Csepregi R., Kőszegi T., Kunsá- gi-Máté S., Szente L., Poór M. (2018) Complex formation of flavonoids fisetin and geralbol with β-cyclodextrins. Journal of Luminescense, № 194.

pp. 82-90. doi.org/10.1016/j.jlumin.2017.10.017 6. Zhang K., Zhang M., Liu Z., Zhang Y., Gu L., Hu G., Chen X., Jia J. (2016) Development of quercetin-phospholipid complex to improve the bioavailability and protection effects against carbon tetrachloride-induced hepatotoxicity in SD rats. Fi- toterapia, № 113, pp. 102 – 109. doi.org/10.1016/j.

fitote.2016.07.008

7. Apanasenko I. E., Selyutina O.Yu, Polyakov N.E., Suntsova L.P, Meteleva E.S., Dushkin A.V., Vachali P., Bernstein P.S. (2015) Solubilization and stabilization of macular carotenoids by water soluble oligosaccharides and polysaccharides. Archives of Biochemistry and Biophysics, V. 572, pp. 58-65. doi.

org/10.1016/j.abb.2014.12.010

8. Dushkin A.V., Suntsova L.P., Khalikov S.S.

(2013) Mechanochemical technology for increasing the solubility of medicinal substances [Mekhano- himicheskaya tekhnologiya dlya povysheniya rast- vorimosti lekarstvennyh veshchestv]. Fundamental research, №. 1. pp. 448-457.

9. Kong R., Zhu X., Meteleva E. S., Chisty- achenko Yu. S., Suntsova L.P., Polyakov N.E., Kh- vostov M.V., Baev D.S, Tolstikova T.G., Yu J., Du- shkin A.V., Su W. (2017) Enhanced solubility and

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bioavailability of simvastatin by mechanochemically obtained complexes. International Journal of Phar- maceutics, № 534, pp. 108-118. doi.org/10.1016/j.

ijpharm.2017.10.011

10. Kuznetsova S.A., Shakhtshneider T.P., Mikhailenko M.A., Malyar Yu.N., Zamai A.S., Boldyrev V.V. (2013) Betulin diacetate mechanocomposite and its antitumor activity [Mekhanokom- pozit diacetata betulina i ego protivoopuholevaya aktivnost’]. Journal of Siberian Federal University.

Chemistry, №. 2, pp. 192-202.

11. Molyar Yu.N., Kuznetsova S.A., Shakht- shneider T.P., Mikhailenko M.A. Obtaining Com- posites of Betulin Diacetate and Dipropionate with Aerosil [Poluchenie kompozitov diacetata i dipro- pionata betulina s aerosilom]. Journal of Siberian Federal University. Chemistry, №2, pp. 277-286.

12. Zhang Q., Suntsova L., Chistyachenko Y.S., Evseenko V., Khvostov M.V., Polyakov N.E., Dush- kin A.V., Su W. (2019) Preparation, physicochemical and pharmacological study of curcumin solid dispersion with an arabinogalactan complexation agent. International Journal of Biological Macro- molecules, V. 128, pp. 158-166. doi.org/10.1016/j.

ijbiomac.2019.01.079

13. Lyakhov N.Z., Dushkin A.V., Meteleva E.S., Chistyachenko Yu.S., Polyakov N.E., Av- gustinovich D.F., Vishnevskaya G.B., Tsyganov M.A., Mordvinov V.A., Sorokina I.V., Tolstikova T.G., Orlovskaya I.A., Toporkova L.B., Khvostov M.V. Composition based on praziquantel for the treatment of opisthorchiasis [Kompoziciya na os- nove prazikvantela dlya lecheniya opistorhoza] Pat.

2681649 RF.

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IRSTI 34.27.19; 34.29.25; 34.45.21 https://doi.org/10.26577/ijbch.2022.v15.i2.02

A.E. Aitynova^1* , N.A. Ibragimova² , T.M. Shalakhmetova¹ , T.E. Gapurkhaeva² , A.V. Krasnoshtanov² , S.T. Kenesheva²

1Al-Farabi Kazakh National University, Almaty, Kazakhstan

2JSC “Scientific Center of Anti-infectious Drugs”

*e-mail: [email protected]

(Received 21 June 2022; received in revised form 12 July 2022; accepted 15 July 2022)

Antimicrobial effect of extract from root of Arctium tomentosum Mill.

(woolly burdock) against several reference strains

Abstract. Microorganisms are one of the main reasons for infectious diseases, however conditionally pathogenic ones are members of the normal human microbiome. Certain circumstances make them become pathogenic to organism. Treatment of microbial diseases majorly is carried out by antibiotics, using of which may lead to antibiotic resistance. Moreover, it causes deficiency of gut microflora, subsequent dysbiosis and weakening of immunity. Thus, replacement of antibiotics by naturally derived medicinal drugs will be an optimal way of fighting pathogenic microorganisms. Since plants are rich in various biologically active compounds, extracts from them will exert antimicrobial, anti-inflammatory, antioxidant properties and etc. In this investigation extract from Arctium tomentosum Mill. obtained by supercritical carbon dioxide extraction was studied for antimicrobial effect. Four reference microbial strains, including gram positive (Staphylococcus aureus), gram negative (Staphylococcus epidermidis, Escherichia coli) and fungi (Candida albicans) were treated. Ampicillin, Chloramphenicol and Nystatin were used for positive control. Procedure of antimicrobial assay was carried out by the method of two-fold serial dilutions in corresponding nutrient medium. As a result minimum bactericide and fungicide concentrations of the extract from A. tomentosum Mill. were determined. Against S. aureus it was 10.4 mg/ml and 41.7 mg/ml against S. epidermidis, while against E. coli it was 20.8 mg/ml. Minimum fungicidal concentration was 5.2 mg/ml. Also ranges of concentration of the studied extract that stop reproduction and development of selected microorganisms were determined. Thus, concentration range of bacteriostatic activity for S. aureus was 2.6–5.2, while for S. epidermidis was 2.6-20.8 mg/ml. Bacteriostatic activity of the studied extract against E.coli was not observed, however concentration range of fungistatic activity was 1.3–2.6 mg/ml.

Obtained results show that extract from Arctium tomentosum Mill. has antimicrobial activity, so it can serve as a base for formation of phytopreparate for the treatment of diseases with microbial origin.

Keywords: extract from Arctium tomentosum Mill., supercritical CO₂-extraction, bacteriostatic activity, fungistatic activity, gram positive, gram negative microorganisms, fungi.

Introduction

Microorganisms that are conditionally pathogenic are members of the normal human microbiome.

Among them are Candida albicans, Staphylococcus epidermidis and Escherichia coli residing as harm- less and lifelong commensals [1-3]. Certain circumstances make them become pathogenic and causing diseases, for example superficial infections of the skin caused by C. albicans[4], nosocomial infections caused by S. epidermidis[5] and severe food borne diseases caused by E.coli[6]. Treatment of diseases with microbial origin majorly is carried out by using of antibiotics. However it leads to not less im-

portant condition of antibiotic resistance, becoming more distributed in human population around the world [7]. Moreover treatment by antibiotics causes deficiency of gut microflora, subsequent dysbiosis and increasing the risk of intestinal inflammation [8].

Concerning these issues, replacement of antibiotics by naturally derived medicinal drugs will be an optimal way of fighting against pathogenic microorganisms. Shifting towards biologically active compounds obtained from herbal extracts may not only reduce side effects from antibiotics but also create a potent source of phytopreparates possessing antibacterial and antifungal properties.Thus the presence of secondary metabolites and various biologically

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11 A.E. Aitynova et al.

active compounds in plants of the genus Arctium (Asteraceae family) will help us to produce extracts with antimicrobial, as well as other benefit properties including anti-inflammatory and antioxidant [9-10].

Moreover, if taking into account that plants of this genus are commonly weeds, using them as a potent source for production of biologically active extract will not trigger any damage on ecosystem stability and bioavailability of these plants. The most distributed species from genus Arctium is Arctium lappa L.

(also known as “greater burdock”), which from earli- er time is used in traditional medicine cause it serves anti-inflammatory properties [11]. Also, its healing activity for treatment of gastrointestinal diseases was approved [12,13]. Presence of proteins, phenols and polyphenolic compounds in root, leaves and seeds of plants from the genus Arctium also make them a good source of valuable substances [14]. For example extract from root of Arctium lappa L. is known for detoxifying properties and ability to clear toxins from bloodstream [15]. Thereby, since the liver is the main participant of detoxifying process, treatment by this extract will conduct hepatoprotective influence [16].Also an oral drug from the Arctium lappa L.

fruit extract exerts antitumor effect on pancreas due to the presence of arctigenin [17].Extract from this plant’s fruit also contains inulin, compound known for its ability to lower blood sugar, so it can be used as a prophylactic treatment of early stages of diabetes mellitus [18].As well as for endogenous therapy Arc- tium species have been used in traditional medicine for various skin conditions including eczema psoria- sis, rashes, boils and etc.) [19].

Another member of the genus Arctium is Arctium tomentosum Mill., commonly known as “woolly burdock” cause of cobwebby hairs that densely cover its flower head. Along with greater burdock it was inter- changeably in traditional medicine cause of similar- ity in their biological activity [20]. In the monograph of the European Medicines Agency Arctium tomen- tosum Mill. is mentioned as a species with equivalent plant material to Arctium lappa L. [21]

According to investigation of chemical constitu- tion of A. lappa different parts, extract of its fruits rich in phenolic compounds, such as lignans; leaves except for lignans also contain flavonoids; roots are rich in phenolic acids, polysaccharides and unsatu- rated fatty acids [22-24].Exactly presence of these phytochemicals plays role in manifestation of anti- inflammatory [25,26] and antioxidant properties [27,28]. Concerning A. tomentosum, presence of arctiin was approved in roots and seeds at concentrations of 0.68% 10.3% respectively [29]. Namely

this compound along with arctigenin has been ef- fectively studied in vitro and in vivo for anti-inflammatory properties [30]. Recent studies have shown that extracts from inflorescences of A. tomentosum contain campesterol, squalene, sterols and lupeols, while its leaves are rich in tocopherols and sterols [31]. However the way of extraction phytochemicals from different parts of the plants are quite significant for their maximum yield. According recent studies supercritical carbon dioxide extraction is much more promising technique for obtaining of majority of biologically active compounds from plant [32]. The present work designed to investigate the antimicrobial effects of extract obtained by this method from root of Arctium tomentosum Mill.

against four microbial strains, namely C. albicans, S. aureus, S. epidermidis and E. coli.

Plant material and extraction. Samples of Arc- tium tomentosum Mill. root were collected from Ak- sai Gorge located in the western central part of the Trans-Ili Alatau mountains, which are part of the mountain system of the Northern Tian-Shan. Extract from root of Arctium tomentosum Mill. was obtained by supercritical CO₂-extraction with using of liquid carbon dioxide as reagent (GOST 8050-85).

Test microorganisms. Four reference strains of microorganisms including gram positive (S. aureus), gram negative (S. epidermidis, E. coli) and fungi (C.

albicans) were investigated. All microorganisms were obtained from American Type Culture Collec- tion (ATCC), USA.

Test antimicrobials. Following antibiotics were used as positive control: Ampicillin (10 μg) against S. aureus and S. epidermidis, Chloramphenicol (30 μg) for E. coli. and Nystatin (100 μg) for C. albi- cans. Solvent for studied extract – dimethyl sulfoxide (DMSO) was used as negative control.

Preparation of microorganisms’ suspension. The stock inoculum for each strain was prepared by the direct colony method. Bacterial turbidity equivalent to 0.5 McFarland standards was obtained, which cor- responds to: ~1.5×10⁸ CFU/ml for bacteria; ~1-5×10⁶ CFU/ml for C. albicans.

Antimicrobial activity assay. The procedure of testing antimicrobial activity was carried out by the method of two-fold serial dilutions in a liquid nutrient medium – Muller-Hinton Broth (Himedia, India) for bacteria and Sabouraud Dextrose Broth (Himedia, India) for yeast in sterile 96-well polystyrene culture plates (BIOLOGIX, China) [33,34].

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Inoculation and its conditions. Inoculation of bacteria was carried out by adding 10 μl of working inoculum into each well of culture plate, that contain 100 μl of a mixture (extract from root of A. tomento- sum and Muller-Hinton Broth) and antibiotic. Thus, final concentration of cells per well was ~2-8×10⁵ CFU/ml. Inoculation of fungi was carried out by adding 100 μl of working inoculum into each well of culture plate, that contain 100 μl of a mixture (extract from root of A. tomentosum and Sabouraud Dextrose Broth) and antimycotic. Thus, final concentration of yeast cells per well was ~0.5-2.5 ×10³ CFU/ml.

Bacteria were incubated for 18-24 hours and yeast for 46-50 hours in thermostat (Binder, Germany) at a temperature of 37°C.

Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC). For the determination of the MBC and MFC blocks of broth plates with absence of growth were inoculated to fresh nutrient broth. The broths then were incubated according to growth requirement of each organism.

Bactericidal activity was approved by the absence of turbidity in the fresh nutrient broth. The minimum bactericidal/fungicidal concentration was considered as the lowest concentration of the studied extract in the well, which completely stopped the growth of bacteria/fungi on the plates.

The present work was designed to investigate the antimicrobial effects of CO2-extract from root of Arctium tomentosum Mill. against four microbial strains C. albicans, S. aureus, S. epidermidis and E.

coli. As a result minimum bactericide (MBC) and fungicide concentrations (MFC) of the extract from root of A. tomentosum Mill. were determined. The smallest concentrations of the extract that fully inhib- ited growth of tested microorganisms on the plates after sowing on the corresponding medium broth were considered as MBC and MFC (presented on Figures 1-4 and Tables 1-2).

A B C

Figure 1 – Antimicrobial activity of studied groups against Staphylococcus aureus ATCC 6538-P strain.

Note: Control of solvent (DMSO), B – Ampicillin, C – extract from root of Arctium tomentosum Mill.

From the Figure 1 we can observe that extract from root of A. tomentosum shows minimum bactericide concentration against S. aureus from 5^th dilution, namely 10.4 mg/ml, while Ampicillin does it from 6^th dilution namely 0.125 mg/ml.

From the Figure 2 we can observe that extract from root of A. tomentosum shows minimum bactericide concentration against S. epidermidis from 3^rd dilution, namely 41.7 mg/ml, while Ampicillin does it from 10^th dilution namely 0.0039 mg/ml.

From the Figure 3 we can observe that extract from root of A. tomentosum shows minimum bactericide concentration against Escherichia coli from 4^rd dilution, namely 20.8 mg/ml, while Chloramphenicol does it from 2^nd dilution namely 5 mg/ml.

From the Figure 4 we can observe that extract from root of A. tomentosum shows minimum bactericide concentration against C. albicans from 6^th dilution, namely 5.2 mg/ml, while Nystatin does it from 7^th dilution namely 0.0078 mg/ml.

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A B C

Figure 2 – Antimicrobial activity of studied groups against Staphylococcus epidermidis ATCC 6538-P strain.

Note: Control of solvent (DMSO), B – Ampicillin, C – extract from root of Arctium tomentosum Mill.

A B C

Figure 3 – Antimicrobial activity of studied groups against Escherichia coli ATCC 6538-P strain.

Note: A – Control of solvent (DMSO), B – Chloramphenicol, C – extract from root of Arctium tomentosum Mill.

A B C

Figure 4 – Antimicrobial activity of studied groups against Candida albicans ATCC 6538-P strain.

Note: A – Control of solvent (DMSO), B – Nystatin, C – extract from root of Arctium tomentosum Mill.

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Table 1 – Minimum bactericidal (MBC) and fungicidal concentration (MFC) of the studied groups Test-strains

Values of MBC and MFC, mg/ml Control of solvent (DMSO) Positive control (antibiotic/

antimycotic) Extract from root of Arctium tomentosum Mill.

S. aureus

No bactericidal activity

0.125 10.4

S. epidermidis 0.004 41.7

E. coli 5.000 20.8

C. albicans 0.008 5.2

From the Table 1 we can see values of minimum bactericidal and fungicidal concentration of studied extract and control groups against selected reference strains. Minimum bactericidal concentration of the extract from root of A. tomentosum against S. aureus is 10.4 mg/ml and 41.7 mg/ml against S. epidermi- dis. The same condition was observed against E. coli at the concentration of 20.8 mg/ml. Regarding fungicidal activity extract from root of A. tomentosum shows it at the minimum fungicidal concentration of 5.2 mg/ml.

Concentrations of the studied extract that mani- fest disruption of microorganism’s reproduction and development, namely bacteriostatic activity also was investigated (Table 2).

Table 2 – Bacteriostatic and fungistatic activity of the studied groups

Test-strains Range of concentrations

S. aureus 2.6 – 5.2 mg/ml

S. epidermidis 2.6 – 20.8 mg/ml E. coli No bacteriostatic activity

C. albicans 1.3 – 2.6 mg/ml

From the Table 2 we can see obtained concentration ranges of bacteriostatic and fungistatic activity of the studied extract against used reference strains.

Bacteriostatic activity of the extract from root of A.

tomentosum was observed at the concentration range of 2.6 – 5.2 mg/ml against S. aureus and 2.6 – 20.8 mg/ml against S. epidermidis. No bacteriostatic effect of the extract was detected against E. coli, however fungicide activity against C. albicans was at the concentration range of 1.3 – 2.6 mg/ml.

Results of conducted investigation represent that the extract from root of Arctium tomentosum Mill.

has antimicrobial activity against reference strains of bacteria and fungi that were used. Several stud-

ies concerning antimicrobial properties of chemical constituents extracted from various plants were conducted recently. Our research is in accordance with conducted studies of Arctium species. As it is known from previous investigations, extract from woolly burdock contains arctiin, which is responsible for antibacterial activity [35]. Since arctiin has low bioavailability in human organism, it becomes activated when transformed to secondary metabolite – arctigenin with participation of microflora from human and animal gastrointestinal tract. Moreover, arctigenin is effective not only against pathogenic bacteria, but also against parasites and viruses, and serves as an effective immunomodulator [36]. Also extract of woolly burdock contains terpenes – campesterol and lupeol that are bioactive against microorganisms.

Mechanism of their activity is based on complexation of cell layering proteins (adhesins, substrates and etc) with subsequent inactivation of their intermembrane space disulphide bond, which leads to breakage of bacterial cell shell [37]. Roots of woolly burdock are also rich in sterols and flavonoids, which are able to inhibit synthesis of bacterial cell wall causing their death [38]. Concerning fatty acids, extract of woolly burdock contains docosapentaenoic, eicosapentaenoic and hexadecanoic acids that are able to integrate into cell membrane of microbe and induct its lysis with final phagocytosis. This totally may serve a function of endogenous antibiotic [39]. Other mechanisms of non-saturated fatty acids activity include inhibition of respiration, impact on amino acids transportation and impairment of oxidative phos- phorylation of pathogenic microorganisms [40].

Conclusion

The conducted research shows the presence of antimicrobial properties of the extract from root of Arctium tomentosum Mill. Antibacterial activity of the studied extract against reference strains of Staph- ylococcus aureus ATCC 6538-P, Staphylococcus

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epidermidis ATCC 6538-P, Escherichia coli ATCC 6538-P was observed at the concentrations of 10.4 mg/ml, 41.7 mg/ml, and 20.8 mg/ml respectively.

Antifungal activity was observed at the concentration of 5.2 mg/ml against reference strain of Candida albicans ATCC 6538-P. Moreover presence of bacteriostatic activity at the concentration range of 2.6 – 5.2 mg/ml against S. aureus and 2.6 – 20.8 mg/ml against S. epidermidis was approved, as well as fungicide activity against C. albicans at the concentration range of 1.3 – 2.6 mg/ml. Obtained results show that extract from root of Arctium tomentosum Mill.

has antimicrobial activity due to the presence of biologically active compounds in the plants of the genus Arctium. Thus we can state that the studied extract may be recommended for production of phytopreparate, which will be helpful in the treatment of diseases with microbial origin for fighting bacteria and fungi, as well as for disruption of their growth and development.

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IRSTI 34.27.29; 34.27.19; 34.27.17 https://doi.org/10.26577/ijbch.2022.v15.i2.03

S.S. Bakiyev , I.T. Smekenov , N. B. Baltakhozha , A. Kauysbekov ,A.K. Bissenbaev*

*Al-Farabi Kazakh National University, Almaty, Kazakhsta

*e-mail: [email protected]

(Received 9 August 2022; received in revised form 29 August 2022; accepted 2 September 2022)

Isolation, identification and physiological growth characteristics of Pseudomonas parafulva from diseased Acipenser baerii

Abstract. The article presents the results of isolation of the bacterial pathogen Pseudomonas parafulva from a diseased Siberian sturgeon (Acipenser baerii) grown in a recirculating aquaculture system. The studied diseased individuals of the Siberian sturgeon (Acipenser baerii) were characterized by reduced activity, did not consume compound feed, on the body of some individuals there were hemorrhages at the bases of the fins, as well as deep penetrating ulcers. Internal organs (liver, spleen and kidneys) and washes from open ulcers were used to isolate the bacterium. As a result of studies of biological materials, the dominant bacterium was used for further identification. The bacterium is characterized as a gram- negative, motile oxidase-positive rod, in the Voges-Proskauer and methyl red test negative, in the O/F test it is characterized as oxidative. As a result of molecular genetic analysis of the 16S rRNA gene sequence of the isolated bacterium, 99% identity with other strains of P. parafulva was determined. Subsequently, the isolated strain was named AB004, and the 16S rRNA sequence was registered in the National Center for Biotechnology Information (NCBI) database under registration number OK634400. According to the results of the physiological characteristics of AB004, the optimal indicators of cultivation in the Luria- Bertani (LB) medium are: NaCl concentration – 1%, pH value – 7.0, temperature – 37°C.

Keywords: recirculating aquaculture system, Acipenser baerii, pseudomonosis, Pseudomonas parafulva, 16S rRNA gene.

Introduction

The annual reduction of sturgeons in natural conditions contributed to the development of industrial aquaculture of these valuable species. The main reasons for the significant reduction are: deterioration of the general ecological situation of natural habitats, including changes in the water regime, hydrochemi- cal composition, and also poaching [1, 2]. It is for the purpose of restoring the natural numbers of sturgeon populations that cultivation and artificial reproduction are carried out in facilities with a recirculating aquaculture system, where all optimal growing conditions are created [3]. One of the valuable objects of sturgeon breeding is the Siberian sturgeon (Acipenser baerii). In the world sturgeon aquaculture, the Sibe- rian sturgeon (Acipenser baerii) occupies a leading position, as it is characterized by a fast growth rate and significantly early puberty (5–6 and 6–8 years for male and female respectively) among sturgeon species [4, 5].

But despite the creation of optimal growing conditions in industrial aquaculture, there is a risk of disease in sturgeons caused by bacterial pathogens of the genus Pseudomonas. The following representatives of the Pseudomonas genus are the main bacteria that are of a massive nature infecting fish in aquaculture: P. aeruginosa, P. fluorescens, P. putida [6–8].

Bacteria of the genus Pseudomonas are the causative agents of pseudomonosis disease affecting sturgeons (Acipenseridae), salmonids (Salmonidae) and cypri- nids (Cyprinidae) [9, 10]. Hemorrhagic septicemia and ulcers, as well as clouding of the eyes, are observed in fish as a result of damage by bacteria of the genus Pseudomonas [11]. Mortality of fish as a result of diseases caused by bacteria of the genus Pseudo- monas can reach 100% [12].

P. parafulva is characterized as a Gram-negative, motile, non-spore forming bacterium. Able to live in a wide temperature range from 4 to 37°C, a taxonomically close species of Pseudomonas putida [13].

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19 S.S. Bakiyev et al.

The study used diseased sturgeons (Acipenser baerii) reared in conditions of recirculating aquaculture systems (RAS), Uralsk, Kazakhstan. Diseased fish were transported in the microbiological laboratory of Zhangir Khan West Kazakhstan Agrarian Technical University. The fish were examined for the presence of external parasites. On the body of diseased fish were found ulcers with deep penetrating muscle necrosis, hemorrhages on the fish body and in the pelvic fins, branchial ischemia, and inflammation of the anus.

To isolate bacterial pathogens, biological materials of internal organs and washes from ulcers on the fish body were selected. To isolate bacteria, Luria- Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar) was used at pH 7.0 [14]. Cultures of bacteria were grown at a temperature of 30ôС within 24 hours. Dominant colonies of bacteria were selected for further research. A single colony of bacteria was inoculated into LB medium and grown for 18 hours at 37ôC. The isolated bacterial culture was preserved on LB agar at 4ôC and in LB at – 75ôC with the addition of sterile glycerol 50%

(v/v).

Analysis of the physiological and biochemical properties of AB004 was carried out with the following tests shown in Table 1. Identification media (Condalab, Spain; TM Media, India) were used for the analysis. Results were observed after incubation according to the manufacturer’s instructions.

To isolate total DNA, an overnight bacterial culture cultivated at 37^oC in LB medium. 400 μl of an overnight culture of bacteria was taken into 0.5 ml Eppendorf tubes, centrifuged at 6000 prm for 5 minutes. The resulting cell pellet was resuspended in 200 μl of autoclaved distilled water, then boiled at 100^oC in a thermo bath for 10 minutes. The cell culture after

boiling was centrifuged at 20000 g for 10 minutes at 5^oC. The resulting supernatant was used as the total DNA of the studied bacterial culture. The obtained DNA was processed using nanodrop (Thermo Sci- entific). The obtained bacterial DNA was stored in a freezer at – 20^оС [15]. To amplify the 16S rRNA gene, we used universal bacterial primers 27F:

5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R:

5’-GGCTACCTTGTTACGACTT-3’ [16]. The studied amplicons were sequenced by Biofidal (Vaulx- en-Velin, France; http://www.biofidal-lab.com).

BLAST sequence searches via the NCBI website and phylogenetic tree was constructed by the neighbor- joining method in the MEGA XI software according to Han et al. (2017) [17].

To study antibiotic resistance, commercial discs (Condalab, Spain) were used, which included 19 antibiotics with a concentration from 1 to 300 μg (Table 2). To determine antibiotic resistance, Mueller-Hin- ton agar was used and incubated with AB004 at 35°C for 24 hours [18].

To determine the growth characteristics of AB004, the following factors were selected: pH (3.0, 5.0, 7.0, 9.0), temperature (27, 32, 37, 42^oC) and concentration of NaCl (0-5%) [19]. Strain AB004 was grown in LB medium until the optical density of the culture was reached at OD₆₀₀ equal to 1.0. This suspension (0.5 ml) was used for inoculation and growth measurement. Incubation was carried out on a shak- er at 150 rpm, cell growth was determined by the change in the optical density (OD₆₀₀) of the culture every hour for 10 hours. All experiments were carried out in triplicate.

AB004 is a gram-negative, motile bacterium capable of growing in LB medium with NaCl concentration of 0-5%, pH 7.0-9.0 at 27-42^oC (Table 1).

Table 1 – Biochemical characteristics of AB004

# Characteristics Reaction # Characteristics Reaction

1 Gram stain - 18 D-xylose +

2 Morphology rod 19 Lactose -

3 Motility + Growth under conditions:

4 Oxidase + 20 0% NaCl +

5 Methyl red - 21 1% NaCl +

6 Voges-Proskauer test - 22 2% NaCl +

7 O/F test О 23 3% NaCl +

8 Hydrolysis of gelatin - 24 4% NaCl +

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# Characteristics Reaction # Characteristics Reaction

9 Hydrolysis of esculin - 25 5% NaCl +

10 H₂S formation - 26 4^oC +

11 Indole formation + 27 27^oC +

12 Lysine decarboxylase - 28 32^oC +

13 Ornithine decarboxylase - 29 37^oC +

14 Arginine dihydrolase + 30 42^oC +

15 ONPG - 31 pH 3.0 -

Acid formation from: 32 pH 5.0 -

16 Sucrose - 33 pH 7.0 +

17 Trehalose - 34 pH 9.0 +

Note: “+” – positive; “-” – negative; “O” – oxidative

Table continuation

The strain showed a positive reaction to oxidase, indole formation, arginine dihydrolase, and also forms acid from D-xylose. A negative reaction was observed in tests for lysine and ornithine decarboxylase, H₂S formation, ONPG, sucrose, trehalose, lactose. AB004 is not capable of hydrolyzing gelatin and esculin. According to the results of the O/F test, the bacterium was identified as oxidative. In terms

of the main biochemical characteristics, the isolated strain AB004 is relatively similar to the previously studied P. putida MTCC 7525 isolated from soil samples in India [20]. Thus, it was determined that P. parafulva is not only taxonomically but also bio- chemically similar to the bacterium P. putida [13].

The results of the AB004 antibiotic resistance analysis are presented in Table 2.

Table 2 – Sensitivity of AB004 to different antibiotics

Group Antibiotic Disk Content

(μg) AB004

Sensitivity Zone diameter (mm) Penicillins

Oxacillin 1 R 0

Penicillin G 10 R 0

Ampicillin 10 R 0

Amoxicillin 10 R 0

Quinolones Enrofloxacin 5 R 15.5±0.3

Norfloxacin 10 S 25±0.7

Cephalosporins Cefazolin 30 R 0

Aminoglycosides Gentamicin 10 S 16.3±0.4

Streptomycin 10 I 13.2±0.2

Nitrofurans Nitrofurantoin 300 R 0

Tetracyclines Tetracycline 30 I 15.5±0.3

Oxytetracycline 30 R 14.5±0.3

Macrolides Erythromycin 15 R 7.6±0.4

Lincomycins Lincomycin 10 R 0

Rifamycins Rifampicin 5 R 10.7±0.4

Coumarins Novobiocin 30 R 0

Amphenicols Chloramphenicol 10 R 8±0.7

Florfenicol 30 R 11.3±1.6

Folic acid synthesis inhibitors Trimethoprim + sulfamethoxazole 25 R 0

Note: R – resistant, I – intermediate, S – sensitive

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21 S.S. Bakiyev et al.

AB004 was found to be resistant to oxacillin, penicillin G, ampicillin, amoxicillin, enrofloxacin, cefazolin, nitrofurantoin, oxytetracycline, erythromycin, lincomycin, rifampicin, novobiocin, chloramphenicol, florfenicol, trimethoprim + sulfamethoxazole. Antibi- otics were found to be sensitive for AB004: norfloxacin and gentamicin. Medium sensitivity was observed for streptomycin and tetracycline.

As a result of antibiotic resistance studies, it was determined that the isolated bacterium Pseudomonas parafulva AB004 is characterized as a multi-resistant strain that has shown resistance to 15 out of 19 antibiotics studied. The strain showed moderate resistance to streptomycin and tetracycline. Gentamicin and norfloxacin were found to be sensitive antibiotics for the isolate of Pseudomonas parafulva. So, it is gener- ally known that the antibiotics gentamicin and norfloxacin have a strong bactericidal effect on a wide range of gram-negative bacterial pathogens, which include Pseudomonas spp., Enterobacter spp. and Shigella spp. [21, 22]. Thus, as a result of the study of antibiotic resistance, it is possible to use norfloxacin and gentamicin to inactivate the isolated strain of Pseudomonas parafulva.

The 16S rRNA sequence was 1457 bp in length with GenBank accession number OK634400 (Fig- ure 1). In the analysis, 99.38-99.52% identity was observed with strains Pseudomonas parafulva

(KX345930.1), Pseudomonas parafulva strain PRS09-11288 (CP019952.1), Pseudomonas paraful- va JCM 11244 (LC507438.1). Based on the results obtained, phylogenetic trees were built to determine the relationship with other representatives of Pseudo- monas spp. (Figure 2).

As a result of the study of the sequenced region of the 16S rRNA gene, it was determined that the isolated bacterium is identified as a representative of the genus Pseudomonas, the species Pseudomonas parafulva.

Figure 1 – Agarose gel electrophoresis of PCR product of the 16S rRNA gene of isolate P. parafulva

Figure 2 – Unrooted neighbor-joining phylogenetic tree based on 16S rRNA gene