ORIGINAL SCIENTIFIC ARTICLE ORGINALNI NAUČNI RAD ORIGINAL SCIENTIFIC ARTICLE

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF MINOR L. EXTRACTS Tijana Cirovic1, Ana Barjaktarevic1, Snezana Cupara1, Violeta Mitic2, Jelena Nikolic2 and Vesna Stankov Jovanovic2 1University of Kragujevac, the Faculty of Medical Sciences, Department of Pharmacy, Kragujevac, Serbia 2University of Nis, the Faculty of Science and Mathematics, Department of Chemistry, Nis, Serbia

ANTIOKSIDATIVNA I ANTIMIKROBNA ANTIVNOST EKSTRAKATA BILJKE L Tijana Ćirovic1, Ana Barjaktarević1, Snežana Cupara1, Violeta Mitić2, Jelena Nikolić2 i Vesna Stankov Jovanović2 1Univerzitet u Kragujevcu, Fakultet medicinskih nauka, Katedra za farmaciju, Kragujevac, Srbija 2Univerzitet u Nišu, Prirodno-matematički fakultet, Katedra za hemiju, Niš, Srbija

Received/Primljen: 29.08.2019. Accepted/Prihvaćen: 10.09.2019.

ABSTRACT SAŽETAK

The aim of this study was to investigate in vitro antioxidant Heterorozna porodična hiperholesterolemija povezana je sa and antimicrobial potential of methanol and chloroform extracts velikim rizikom od ranog nastanka ishemijske bolesti i kardio- of Sanguisorba minor L. subsp. muricata Briq. herba. Total phe- vaskularne smrti. Gotovo da nema podataka o rasprostranjenosti nolic and flavonoid content of the investigated extracts were char- bolesti u ukrajinskoj populaciji. Cilj studije bio je proceniti acterized. Antioxidant activity was estimated by five different in učestalost porodične hiperholesterolemije kod pacijenata koji su vitro assays. Antioxidant potency composite index was calculated lečeni od „L.T. Malaja terapija, Nacionalni institut Nacionalne also. Antimicrobial activity was tested against nine bacterial and akademije medicinskih nauka Ukrajine “zbog rane ishemijske one fungus strains by the micro-well dilution assay. The methanol bolesti srca. Analizirani su podaci medicinske evidencije za 600 extract of S. minor contains more phenols and shows the stronger pacijenata lečenih u Institutu tokom 2015-2017. Rana ishemijska antioxidant and antibacterial activity in comparison to the chlo- bolest srca dijagnostikovana je kod 89 pacijenata. Verifikacija roform extract. However, the chloroform extract was superior to bolesti je izvršena ili na osnovu podataka koronarografije, ili na the methanol extract in content of flavonoids. Gram-positive bac- osnovu prethodnog infarkta miokarda sa K talasom. Da bi se teria were more sensitive than Gram-negative, to both extracts, identifikovali pacijenti sa porodičnom hiperholesterolemijom, Staphylococcus aureus being the most sensitive. Sanguisorba mi- korišćeni su mrežni kriterijumi holandske lipidne klinike. Sumn- nor extracts were not considerably active against Candida albi- jalo se na prisustvo porodične hiperholesterolemije kod više od cans. The previous research data about Sanguisorba minor are 14,8% pacijenata sa ranom ishemijskom bolešću srca. Među tim scarce, so this data represent the first report on antimicrobial ac- pacijentima, 2 (2,2%) je imala definitivnu dijagnozu; 27 (30,3%) tivity of S. minor. These results indicate that extracts of S. minor verovatno će imati dijagnozu, 26 (29,7%) - imalo je moguću di- subsp. muricata have evidence-based potential for more compre- jagnozu, a kod 34 (38,2%) pacijenata nije bilo verovatno da im se hensive studies. dijagnostikuje porodična hiperholesterolemija. Izraz "porodična hiperholesterolemija" nije spomenut u bolničkoj dijagnozi. Ovaj Keywords: Sanguisorba minor subsp. muricata, antioxidant ac- rad pokazuje da je uprkos učestaloj pojavi porodične hiperholes- tivity, antimicrobial activity terolemije primećeno da je doktor lekara na ovu bolest prilično nizak.

Ključne reči: Sanguisorba minor subsp. muricata, antioksida- tivna aktivnost, antimikrobna aktivnost

ABBREVIATIONS

ABTS: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); ANOVA: One-way analysis of variance; CLP: Cecal Ligation and Puncture; CUPRAC: Cupric Reducing Antioxidant Capacity; FRAP: Ferric-reducing antioxidant power; TBARS: Thiobarbituric Acid Reactive Species; TFC: Total Flavonoid Content; TPC: Total Phenolic Content; TRP: Total Reducing Power

Corresponding author: Ana Barjaktarevic University of Kragujevac, the Faculty of Medical Sciences UDK: 000.00-000.0/.0-000; 000.000/ Department of Pharmacy Ser J Exp Clin Res 2019; 20 (1): 3-13 69 Svetozara Markovica Street, 34000 Kragujevac, Serbia DOI: 10.2478/sjecr-2019-0044 E-mail: [email protected]

INTRODUCTION g for the chloroform extract. Dry extracts were obtained by the rotary vacuum evaporator and stored in the desiccator un- Sanguisorba minor (little burnet) is a perennial herba- til the experiment. ceous , belonging to the genus Sanguisorba, family . It is widespread throughout Europe, Africa and Total phenolic and flavonoid content Asia. It has been used as a traditional medicine for the treat- ment of conjunctivitis, fever and diarrhea in the form of in- Total Phenolic Content (TPC) of samples was determined fusions and tinctures (1, 2). Different subspecies of S. minor spectrophotometrically at 750 nm according to the Folin-Ci- have been found in Serbia, including subsp. muricata (3). ocalteu method. Gallic acid was used as a standard for the calibration curve and results were expressed as µg of gallic Sanguisorba genus expressed a wide range of acid equivalents per mg of dry weight (µg GAE per mg dw). pharmacological effects - such as hemostatic, antibacterial, All the measurements were done in triplicate (18, 19). anti-inflammatory, antioxidant, hypoglycemic, neuroprotec- tive or anticancer (3). The most investigated Sanguisorba Total Flavonoid Content of extracts (TFC) was estimated species has been Sanguisorba officinalis, while S. minor spe- by the aluminium chloride colorimetric method. The absorb- cies is less explored (4, 5). Among various active compounds ance was measured spectrophotometrically at 510 nm. All the isolated from the root and aerial parts of the S. minor, phe- measurements were done in triplicate. Rutin was used to per- nolic acids, triterpenoids, tannins and flavonoids have been form the standard curve, and the results were expressed as µg reported as its major components (6-8). The previous re- of rutin equivalent per mg dry weigh (µg RE/mg) (20). search also proved that S. minor contains high amount of pol- Antioxidant activity assays yphenols, β-carotene, vitamin E, and vitamin C which has been associated with the antioxidant potential of S. minor ABTS radical scavenging activity herba (9-11). The aqueous extract of S. minor subsp. muri- cata herba showed anti-inflammatory and anti-ulcerogenic Free radical scavenging activity of S. minor extracts was effects (12, 13), while the ethanol extract of S. minor herba determined by the ABTS radical cation decolorization assay. inhibits activity of acetylcholinesterase (2). To the best of our The absorbance of the mixture was measured at 734 nm. All knowledge, there have not been any investigations on S. mi- the measurements were carried out three times. Results were nor antimicrobial activity. Since S. officinalis has shown an- expressed as µg of Trolox equivalents (TE) per mg extract timicrobial properties in the previously published research, dry weight (µg TE per mg dw) (19). we have been encouraged to direct our research in this way (14-16). DPPH radical scavenging assay

THE AIM OF THE PAPER Free radical scavenging activity of the investigated ex- tracts was determined the application of the DPPH assay. The An autochthon growing plant from the region of Serbia, absorbance of samples was measured at 515 nm spectropho- S. minor subsp. muricata Briq. has not been characterized tometrically. All samples were made in triplicate. Trolox was neither chemically nor biologically. Therefore, the aim of this used as a positive control. Results were expressed as µg of study was to determine the amount of flavonoids and phenol Trolox equivalents (TE) per mg dry extract weight (µg TE compounds in methanol and chloroform extracts of S. minor per mg dw) (19). subsp. muricata herba as part of the chemical characteriza- Cupric Reducing Antioxidant Capacity (CUPRAC) tion. Biological activity of afore-mentioned extracts was assay evaluated by the antioxidant and antimicrobial testing of ex- tracts in vitro. The CUPRAC assay was performed according to the Di- mitrijevic et al. The absorbance of reaction mixture was MATERIALS AND METHODS measured at 450 nm spectrophotometrically. Results were expressed as µg of Trolox equivalents per mg of dry weight Plant material (µg TE per mg dw) (19).

Aerial parts of S. minor subsp. muricata were collected Ferric-Reducing Antioxidant Power (FRAP) assay from Kamenica village meadows, located in Sumadija region (Serbia) during a spring sunny day. Plant materials were au- The reducing power was determined by ferricyanide-fer- thenticated at the Institute for Biology and Ecology at the ric chloride method. The change of absorbance of the FRAP Faculty of Science (University of Kragujevac, Serbia), by reagent was monitored at 596 nm 595 nm. All the measure- standard botanical keys for the plant determination (3, 17). ments were done in triplicate. Results were expressed as a µg After drying in the shade at room temperature, aerial parts Fe/mg dw of Fe(II) equivalents per mg of dry weight (µg Fe (herba) were ground and extracted with reflux of methanol per mg dw) (19). and chloroform on boiling temperature for four hours. 35 g of the aerial parts of the plant and 300 mL of solvents were used. The yield of the methanol extract was 2.46 g and 0.61

Total Reducing Power (TRP) assay assay, positive control agents were doxycycline and nystatin in the concentration range of 0.01-20.0 µg/mL. Dimethyl sul- The method of the reducing power assay was based on foxide (DMSO, 100%) was taken as a negative control, in the the reduction of Fe(III) hexacyanate to Fe(II) hexacyanate. concentration range from 0.02 to 50.0 mg/mL. After adding The absorbance of the reaction mixture was measured at 700 20 µL of 0.5% triphenyl tetrazolium chloride solution, bacte- nm, spectrophotometrically. All samples were made in tripli- rial and fungal growth was detected. Results were presented cate. Results were expressed as µg of the ascorbic acid equiv- as MIC/MBC or MIC/MFC (in mg/mL). The minimal inhib- alents per mg of dry extract weight (µg AAE per mg dw) itory concentration (MIC) was defined as the lowest concen- (19). tration of samples that produced complete growth inhibition of the tested microorganisms while minimal bactericidal/fun- Antioxidant Potency Composite Index-ACI gicidal concentration (MBC/MFC) was defined as the lowest Antioxidant Potency Composite Index (ACI) was calcu- concentration of extracts that killed ≥99.9% of the final inoc- lated according to the following equation: ACI=(sample ulum. All experiments were independently repeated three score/best score)×100 (21). times.

Antimicrobial activity assay Statistical analysis

Methanol and chloroform extracts of the S. minor subsp. All the experiments were performed in triplicate and the muricata herba were used for the evaluation of antimicrobial experimental data were expressed as mean±standard devia- activity by the micro-well dilution assay in vitro (CLSI 2009, tion (SD). Antioxidant activity was analyzed by five different with some modifications). The initial concentration of ex- methods which make it difficult to compare results with each tracts was 100 mg/ml. Antibacterial assays were carried other. Because of that, statistical analysis was performed with against three Gram-positive bacteria (Bacillus cereus, Enter- the Antioxidant Potency Composite Index (ACI) values. Lin- ococcus faecalis, Staphylococcus aureus) and six Gram-neg- ear regression was used to identify the correlation between ative bacteria (Escherichia coli, Pseudomonas aeruginosa, antioxidant activity and total phenolic and flavonoid contents Enterobacter aerogenes, Proteus mirabilis, Klebsiella pneu- of S. minor herba extracts. moniae, Salmonella enteritidis). Antifungal activity of the extracts was tested against Candida albicans.

Suspensions of test strains were prepared from fresh over- RESULTS night cultures transferred into normal saline (0.9% NaCl) un- Antioxidant activity assays der aseptic conditions. Density of each suspension was stand- ardized to 0.5 McFarland. The final bacterial suspension was 6 The antoxidant activity, the Total Phenolic (TPC) and To- adjusted to 10 CFU/mL. Serial dilutions of extracts were tal Flavonoid Content (TFC) of the tested S. minor herba ex- prepared and tested in the concentration range from 0.04 to tracts are shown in Table 1. These results represent mean 100.0 mg/mL in a 96/well microtiter plate with the inoculated value±standard deviation of three replicate experiments. Mueller-Hinton brott (for bacterial suspension) or the Sabouraud Dextrose agar (for fungal suspension). For this

Table 1. Antioxidant activity of S. minor herba extracts

S. minor ABTS DPPH CUPRAC FRAP TRP TPC TFC еxtracts µg TE/mg µg TE/mg µg TE/mg µg Fe/mg µg AAE/mg µg GAE/mg µg RE/mg

Methanol 77.26 95.06 289.09 205.62 0.58 132.80 506.50 ±0.16 ±0.28 ±0.24 ±2.53 ±0.015 ±3.87 ±5.00

Chloroform 53.83 40.31 182.90 78.22 0.06 67.87 889.00 ±1.35 ±0.54 ±0.55 ±0.44 ±0.005 ±0.77 ±43.30

Antioxidant Potency Composite Index (ACI)

Antioxidant Potency Composite Index (ACI) values of the methanol and chloroform S. minor herba extracts are presented in Table 2.

Table 2. Antioxidant Potency Composite Index (ACI) for S. minor extracts by five different methods

S. minor еxtracts ABTS DPPH CUPRAC FRAP TRP Average Methanol 98.85 100 100 100 100 99.77 Chloroform 69.67 42.40 63.27 38.04 10.35 44.75

The correlation between total phenolic/flavonoid content and antioxidant potency composite index of S. minor extracts and antioxidant composite index of Sanguisorbae minor ex- (r2=0.9979 and r=0.9989). The total flavonoid content and tracts has been shown in Figures 1 and 2. There is a signifi- ACI of S. minor extracts are significantly negative correlated cant positive correlation between the total phenolic content (r2=0.9972 and r=0.9985).

Figure 1. Linear regression between the Total Phenolic Content (µg GAE/mg) and Antioxidant Potency Composite Index (ACI) of Sanguisorbae herba extracts

160

140 y = 1,1902x + 14,717 120 R² = 0,9979 100 80 60 index (ACI) 40 20

Antioxidant potency composite 0 0 20 40 60 80 100 120 Total phenolic content (µg GAE/mg)

Figure 2. Linear regression between the Total Flavonoid Content (µg RE/mg) and Antioxidant Potency Composite Index (ACI) of Sanguisorbae herba extracts

900 800 700 600 y = -6,8855x + 1185,7 500 R² = 0,9972 400

index (ACI) 300 200 100

Antioxidant potency composite 0 0 20 40 60 80 100 120 Total flavonoid content (µg RE/mg)

Antimicrobial activity

The results of the micro-well dilution assay on S.minor herba extracts have been summarized in the Table 3.

Table 3. Antimicrobial activity of S. minor extracts against bacterial and fungus strains (MIC/MBC in mg/mL)

S. minor extracts Microorganisms ATCC Methanol Chloroform Escherichia coli 25922 3.13/3.13 3.13/12.50 Pseudomonas aeruginosa 9027 1.56/3.13 3.13/6.25 Salmonella enteritidis 13076 3.13/3.13 3.13/12.50 Proteus mirabilis 12453 3.13/3.13 6.25/6.25 Klebsiella pneumoniae 10031 3.13/3.13 6.25/6.25 Enterobacter aerogenes 13048 1.56/3.13 3.13/25.0 Gram-positive bacteria Bacillus cereus 11778 0.39/0.39 1.56/3.13 Staphylococcus aureus 25923 0.10/0.39 0.78/12.50 Enterococcus faecalis 19433 0.39/0.39 0.39/6.25 Fungi Candida albicans 14053 6.25/12.50 6.25/12.50

DISCUSSION Investigations on S. minor subsp. muricata are scarce or absent, which limits us to discuss our results in the light of Two different extracts of S. minor subsp. muricata herba the references data available solely on S. minor species, of were investigated and compared to their antioxidant, antimi- which S. officinalis has been the most investigated. There crobial potential-methanol and chloroform extract. In order have not been any reports on the content of total phenolics to chemically characterize this scarcely researched plant, for S. minor herba methanol or chloroform extract. However, contents of phenolics and flavonoids were determined and an the TPC was determined in methanol extract of S. officinalis attempt to understand their possible influence on the antioxi- herba (116.96±5.89 mg GAE/100mg), which is in concord- dant activity of the extracts was made. ance with the result of TPC in our investigated S. minor herba methanol extract (23). Antioxidant activity of plants has been thoroughly re- searched by several different methods in order to perform the Flavonoids have been documented to be strong scaven- best possible evaluation of all compounds contributing to this gers of the most oxidizing molecules, singlet oxygen and/or activity. In most cases, the antioxidant activity has been pos- different free radicals (24). In our study, chloroform extract itively correlated to the content of total phenolics, due to their contained a higher level of flavonoids (889.00±43.30 µg potential to scavenge free radicals (22). In absence of the ref- RE/mg), than the methanol extract (506.50±5.00µg RE/mg). erence data for this species from Serbia for phenolics, deter- Direct comparison of total flavonoids from our study and pre- mination of total content of phenolic compounds in investi- vious reports on S. minor spp. is not reproducible due to the gated extracts seems to have been a prerequisite. In our study different equivalents used in the result expression. we compared content of these antioxidant-contributing com- pounds in the methanol and chloroform extract. The metha- The antioxidant potential of S. minor subsp. muricata nol extract, due to the stronger polarity of its solvent, pos- methanol and chloroform extracts was evaluated by five in sessed a higher content of phenolics (132.80±3.87 µg vitro methods: ABTS, DPPH, CUPRAC, FRAP and TRP as- GAE/mg dw) in comparison to the chloroform extract says. Methanol extract showed a significantly higher antiox- (67.87±0.77 µg GAE/mg dw). These results demonstrate that idant activity than the chloroform extract, determined by all total phenolic content depends on the polarity of the solvent assays (Table 1). That difference may be due to strong differ- used. Previous studies also reported methanol as an effective ence in the polarity of these two solvents (25). Antioxidant solvent for the extraction of phenolic compounds (22, 16). Potency Composite Index values also confirmed that the methanol extract of S. minor herba showed much better antioxidant activity than the chloroform extract in all em- The previous published results indicated similar activity ployed assays (Table 2). Due to the lack of similar data on S. of S. officinalis herba extracts against both, the Gram-nega- minor subsp. muricata that could serve as the reference point, tive and Gram-positive bacteria (16). However, Shan et al. we declare that our results are within the range of antioxidant that suggested in their research that S. officinalis herba ex- activity found in Sanguisorba genus plants, which has been tract was more active against the Gram-positive bacteria. In well documented (4). our investigation S. minor extracts were also more active against the Gram-positive bacteria. We, therefore, share their The highly significant positive correlations between the observation that a possible cause may be different interac- TPC and ACI of S. minor extracts obtained in this study (Fig- tions of Sangusiorba sp. extracts with outer layers of the ure 1) support the hypothesis that phenolic compounds con- Gram-negative and Gram-positive bacteria, since both plants tribute significantly to the total antioxidant capacity of plants belonging to Sanguisorba genus exhibited the same prefer- species (25). The positive correlation between antioxidant ence to act stronger against the Gram-positive bacteria (27). activity and total flavonoid content of S. minor aerial parts extracts was reported (15). However, we observed an inverse CONCLUSION correlation between the ACI and TFC in our extracts (Figure 2). The chloroform extract showed the higher TFC, but the S. minor subps. muricata was investigated in vitro for its lower antioxidant activity. Therefore, we assume that flavo- antioxidant and antimicrobial properties. Methanol extract of noids in our investigated S. minor extracts do not contribute S. minor subps. muricata herba has a high content of phenols significantly to the antioxidant activity. and shows considerable antioxidant activity, measured by all five different methods that were used. Methanol extract is su- The aim of this study was also to evaluate antibacte- perior to the chloroform extract in the content of phenols and rial/antifungicidal activity of chloroform and methanol ex- antioxidant activity. Chloroform extract has a higher content tracts of S. minor subsp. muricata aerial parts. Both extracts of flavonoids. Methanol extract was stronger than the chlo- exhibited considerable antimicrobial activity against all ex- roform extract against all investigated bacterial strains and it amined strains of bacteria (B. cereus, E. faecalis, S. aureus, is also stronger against the Gram-positive bacteria. S. minor E. coli, P. aeruginosa, E. aerogenes, P. mirabilis, K. pneu- subps. muricata is active mostly against Staphylococcus au- moniae, S. enteritidis) but not against Candida albicans. All reus. The obtained data indicate that S. minor subsp. muri- tested strains, except C. albicans, were more susceptible to cata has evidence-based potential for further more compre- the methanol extract than the chloroform extract (Table 2). hensive studies. Methanol extract exhibited the stronger bacteriostatic and bactericidal activity than the chloroform extract, with MIC ACKNOWLEDGMENTS range 0.1-3.13 mg/mL, and MBC range 0.39-3.13 mg/mL (Table 2). Our research confirms previous reports on San- This work was supported by the projects: No. MFVMA guisorba genus plants which demonstrated that methanol ex- 04/19-21 of the University of Defense, Serbia and No. OI tracts were more active than chloroform extracts (16, 26). 172051, No. OI 172047, No. OI 175014 of the Ministry of Our investigated extracts of S. minor were more active Education, Science and Technological Development of Ser- against the Gram-positive than Gram-negative bacteria. The bia. most sensitive was the Staphylococcus aureus (MIC=0.10 mg/mL, MBC=0.39mg/ml). Since there has not been any re- search on antimicrobial activity of S. minor species, our re- REFERENCES sults represent the first data of this kind. Therefore, in the following discussion we assume as relevant the comparison 1. Viano J, Masotti V, Gaydou EM. Nutritional value of of antimicrobial activity of S. minor to S. officinalis, since Mediterranean sheep's burnet (Sanguisorba minor ssp. 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