Bulletin of Experimental Biology and Medicine, Vol. 156, No. 6, April, 2014 IMMUNOLOGY AND MICROBIOLOGY

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Comparative Analysis of Antibacterial Properties and Chemical Composition of Glycyrrhiza glabra L. from Astrakhan Region (Russia) and Calabria Region (Italy) O. V. Astaf’eva and L. T. Sukhenko

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 156, No. 12, pp. 799-802, December, 2013 Original article submitted December 13, 2012 We compared antibacterial activity of various extracts of two licorice subspecies against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. Diethyl carbonate extracts of Glycyrrhiza glabra root from Astrakhan region (Russia) exhibited maximum activity against the test microbial strains; activity of Astrakhan licorice was superior among 50% ethanol extracts from Astrakhan (Russia) and Calabria (Italy). Antibacterial activity is directly proportional to the content of glycyrrhizin and 18β-glycyrrhetinic acid in the extracts. According to preliminary data, the content of these chemical components in Glycyrrhiza glabra root from Astrakhan region is higher than in licorice growing in Italy, which is presumably related to climate and geographic characteristics of Astrakhan region. Key Words: licorice; glycyrrhizin; 18β-glycyrrhetinic acid; antibacterial activity Licorice Glycyrrhiza glabra L. is widely used as a medicinal plant. The Glycyrrhiza glabra species includes three variants: Persian or Turkish licorice (Glycyrrhiza glabra var violacea), Russian licorice (Glycyrrhiza glabra var gladunlifera), and Spanish or Italian licorice (Glycyrrhiza glabra var typical). The major component of licorice root is glycyrrhizin, a triterpene saponin presented by a mixture of glycyrrhetinic acid potassium, calcium, and magnesium salts. Glycyrrhizin molecule consists of a hydrophilic (two molecules of glucuronic acid; Fig. 1, a) and hydrophobic (glycyrrhetinic acid; Fig. 1, b) parts [4] Glycyrrhizin and glycyrrhetinic acid exist in the form of 18α- and 18β-stereoisomers [5]. Glycyrrhizin exhibits anti-inflammatory, antimicrobial, hepatoprotective, antitumor, antiulcer, and immunomodulatory activity [2,7]. Antiviral (against HIV-1) and antibacterial activities of 18β-glycyrrhetinic Astrakhan State University, Russia. Address for correspondence: [email protected]. O. V. Astaf’eva

acid were also revealed; glycyrrhizin also exhibits pronounced antiviral activity because it disturbs synthesis of virus envelope [1-3]. The content of the major active components in the plant determining its biological activity depends on various factors (climate, geography, gathering time, drying method, etc.). Here we studied the dependence of antibacterial activity on the extractant and consequently on the content of the major bioactive components.

MATERIALS AND METHODS Extraction fractions of different Glycyrrhiza glabra samples were analyzed on an Agilent 1200 liquid chromatograph (Agilent Technologies) connected with an API 4000 mass spectrometer (Applied Biosystem/ MDS SCIEX) and equipped with an Agilent ZORBAX 3.5 μ Extend-C18 80Å 4.6×150 mm column (Agilent Technologies). The mobile phase consisted of methanol and 0.05% formic acid. Detection was

0007-4888/14/15660829 © 2014 Springer Science+Business Media New York

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Bulletin of Experimental Biology and Medicine, Vol. 156, No. 6, April, 2014 IMMUNOLOGY AND MICROBIOLOGY

Fig. 1. Chemical formulas of glycyrrhizin (a) and 18-glycyrrhetinic acid (b).

performed at wavelengths of 254, 280, 275, 330, and 350 nm. During chromatography, the composition of the mixture was changed from 0 to 100%. Flow rate was 0.5 ml/min, sample volume 10 μl. First, we studied the chromatographic behavior of the standard samples of glycyrrhizin and 18β-glycyrrhetinic acid (Sigma-Aldrich) and then analyzed various samples of raw material collected in Astrakhan (Russia) and Calabria (Italy) regions. Chemical components of licorice were extracted by different methods. The roots (20 g) of all three variants were extracted (1:5) with 50% ethanol. The mixture was allowed to stay for 7 days in a darkglass bottle at constant agitation. The extracts were filtered, sterilized, and evaporated in a dry-air sterilizer at 85oC. Glycyrrhiza glabra roots collected in Astrakhan and Calabria were also treated with other extractants, such as 96% ethanol, ethyl acetate, and diethyl carbonate. Crushed raw material (30 g) was placed in a flask with a ground joint, 150 ml extractant was added (100% ethanol, ethyl acetate, or diethyl carbonate), attached to a reflux condenser, and heated on a water bath for 24 h. The extracts were then filtered.

Antibacterial activity was tested on Staphylococcus aureus VKPM V-1899, Escherichia coli SK VKPM V-1911, and Bacillus subtilis VKPM V-1919 from All-Russian Collection of Industrial Microorganisms, State Research Institute of Genetics and Selection of Industrial Organisms. Antibacterial activity was evaluated by the agar diffusion method as described elsewhere [8]. Pure solvents (ethanol, ethyl acetate, and diethyl carbonate) were used as the control. Extracts were prepared and antibacterial activity was evaluated in Laboratories of Biotechnologies, Astrakhan State University. Analysis of chemical composition was performed by O. V. Astaf’eva at Ca’Foscari University of Venice (Italy) within the framework of International Postgraduate Research on chemistry in cooperation with Astrakhan State University. Glycyrrhiza glabra L. was kindly provided by Prof. Gabriele Capodaglio (Ca’Foscari University of Venice).

RESULTS The highest glycyrrhizin concentration was found in 50% ethanol extract of licorice gathered in Astrakhan

TABLE 1. Glycyrrhizin and 18-Glycyrrhetinic Acid Content in Extracts of Glycyrrhiza glabra Root from Astrakhan Region (Russia) and Calabria Region (Italy) Substance concentration, mg/g dry weight

Fraction in dry material, wt %

Fraction glycyrrhizin

18-glycyrrhetinic acid (range)

glycyrrhizin

18-glycyrrhetinic acid

50% extract of G. glabra (Astrakhan region)

516

1×10–4-4×10–4

51.6



Diethyl carbonate extract of G. glabra (Astrakhan region)

108

10.81

10.8

1.1

Diethyl carbonate extract of G. glabra (Calabria region)

0.42

5×10–4-1×10–3

0.04



O. V. Astaf’eva and L. T. Sukhenko

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TABLE 2. Antibacterial Activity (Growth Zone Diameter, mm) of Glycyrrhiza glabra Root Extracts (M±m) Glycyrrhiza glabra Root Extract

E. coli

St. aureus

B. subtilis

G. glabra (Astrakhan region)

14.1±3.6

11.5±2.3

10.5±6.8

50% ethanol

G. glabra (Calabria region)

18.7±3.6

4.65±1.30

8.7±5.9

1

/2 G. glabra (Astrakhan region)

11.9±0.5

4.0±0.6

13.7±5.2

1

/2 G. glabra (Calabria region)

5.9±0.7

0

4.2±0.4

Diethyl carbonate G. glabra (Astrakhan region)

28.2±1.6*

25.3±2.8*

24.80±4.02*

1

11.9±2.5*

10.0±0.8*

14.8±5.9

9.1±0.9*

9.5±1.2*

11.1±0.8*

10.2±3.1*

5.8±1.3

7.8±2.3

11.5±0.7*

9.5±2.3

9.5±2.3

9.5±0.7*

5.6±0.7*

9.0±1.7

14.1±0.8

13.6±4.2*

13.8±1.7

0

8.2±1.5

11.7±2.4*

G. glabra (Astrakhan region)

16.5±2.1

8.5±0.7

10.2±1.8

1

11.0±1.6

5.3±1.8

9.5±1.7

G. glabra (Calabria region)

14.1±0.8*

11.3±5.2*

9.1±2.7

1

10.2±2.9*

0

7.8±1.2

Ethanol

0

0

0

Ethyl acetate

0

0

0

Diethyl carbonate

0

0

0

/2 G. glabra (Astrakhan region)

G. glabra (Calabria region) 1

/2 G. glabra (Calabria region)

Ethyl acetate G. glabra (Astrakhan region) 1

/2 G. glabra (Astrakhan region)

G. glabra (Calabria region) 1

/2 G. glabra (Calabria region)

96% ethanol

/2 G. glabra (Astrakhan region) /2 G. glabra (Calabria region)

Note. 0: no antimicrobial effect. *p0.05 in comparison with the control.

region. This result can indicate either higher efficiency of extraction with extractant mixture in comparison with ethanol [6] or higher glycyrrhizin content in this raw material. The concentration of glycyrrhizin in the extract of licorice from Calabria region was considerably lower than in the extract licorice growing in the Astrakhan region (Table 1). Antibacterial activity against non-pathogenic E. coli, St. aureus, and B. subtilis strains was analyzed. Among 50% ethanol extracts of Russian and Italian licorice variants, extract of Calabrian licorice exhibited higher activity against E. coli. However, 1:2 dilution of this extract reduced its activity by 3 times, while extract of licorice root from the Astrakhan region retained its activity. Extracts of licorice root from the Astrakhan region exhibited more potent antibacterial effect against St. aureus and B. subtilis. After dilution of these extract with water, only extracts of

licorice grown in the Astrakhan region retained their antibacterial activity (Table 2). Comparison of diethyl carbonate, ethanol, and ethyl acetate extracts of licorice root from the Astrakhan region and Calabria region showed that diethyl carbonate extracts exhibited the highest antibacterial activity against all three types of non-pathogenic cultures; it should be noted that Russian licorice root extract was 2.5-fold more effective in this respect. This can be explained by higher content of glycyrrhizin and 18β-glycyrrhetinic acid in the Glycyrrhiza glabra root from the Astrakhan region. On the contrary, ethyl acetate extract of licorice root from the Calabria region exhibited 1.5-fold higher antimicrobial activity. Ethanol extracts of both licorice variants exhibited similar antibacterial activity against E. coli and St. aureus. Thus, diethyl carbonate extracts of Glycyrrhiza glabra root from Astrakhan region (Russia) exhibited

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Bulletin of Experimental Biology and Medicine, Vol. 156, No. 6, April, 2014 IMMUNOLOGY AND MICROBIOLOGY

maximum activity against the test microbial strains; activity of Astrakhan licorice was superior among 50% ethanol extracts from Astrakhan (Russia) and Calabria (Italy). It can be concluded that extracts of the two Glycyrrhiza glabra variants exhibited antibacterial activity, but to a different extent. Antibacterial activity directly depended on the content of glycyrrhizin and 18β-glycyrrhetinic acid in the extracts. The content of these chemical components in Glycyrrhiza glabra root from Astrakhan region is higher than in licorice from Italy, which is presumably related to climate and geographic characteristics of Astrakhan region [8].

REFERENCES 1. O. A. Plyasunova, I. P. Egorycheva, N. V. Fedyuk, et al., Vopr. Virusol., 37, No. 5, 235-237 (1992). 2. N. M. Asl and H. Hosseinzadeh, Phytother. Res., 22, No. 6, 709-724 (2008). 3. L. Badam, J. Commun. Dis., 29, No. 2, 91-99 (1997). 4. R. K. Harwanch, K. C. Patra, S. K. Pareta, et al., Pharmacologyonline, 2, 1032-1038 (2011). 5. V. S. Jatav, S. K. Singh, P. Khatri, and A. K. Sharma, Int. J. Pharm. Front. Res., 1, No. 1, 170-185 (2011). 6. T. Nomura, T. Fukai, and T. Akiyama, Pure Appl. Chem., 74, No. 7, 1199-1206 (2002). 7. S. Saxena, Nat. Prod. Radiance., 4, No. 5, 358-367 (2005). 8. L. Sukhenko, Efektivni nastroje modernich Ved-2011. Praha (2011), pp. 53-56.

Comparative analysis of antibacterial properties and chemical composition of Glycyrrhiza glabra L. from Astrakhan region (Russia) and Calabria region (Italy).

We compared antibacterial activity of various extracts of two licorice subspecies against Staphylococcus aureus, Escherichia coli, and Bacillus subtil...
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