87 urn:lsid:arphahub.com:pub:A116C711-4C18-5A38-8F1E-5E97753A8A64 Folia Medica FM 0204-8043 1314-2143 Plovdiv Medical University 10.3897/folmed.65.e79094 79094 Original Article Chemistry & biophysics Antioxidant properties and antibacterial activity of water extracts from Sambucus nigra L. under different conditions Milkova-Tomova Iliana 1 Kazakova Zornica 1 Buhalova Dragomira 1 Gentscheva Galia 2 Nikolova Krastena Krastena.Nikolova@mu-varna.bg 3 Minkova Stefka 3 Department of Catering and Tourism, University of Food Technologies, Plovdiv, Bulgaria University of Food Technologies Plovdiv Bulgaria Department of Chemistry and Biochemistry, Medical University, Pleven, Bulgaria Medical University Pleven Bulgaria Department of Physics and Biophysics, Medical University, Varna, Bulgaria Medical University Varna Bulgaria

Corresponding author: Krastena Nikolova, Department of Physics and Biophysics, Medical University, Varna, Bulgaria; Email: Krastena.Nikolova@mu-varna.bg

 2023 30 04 2023 65 2 295 300 35672E42-3949-5D77-A697-4919471C7098 08 12 2021 18 02 2022 Iliana Milkova-Tomova, Zornica Kazakova, Dragomira Buhalova, Galia Gentscheva, Krastena Nikolova, Stefka Minkova This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction: In folk medicine, dried white flowers of Sambucus nigra L. are used to make infusions, decoctions, and juices.

Aim: The present article aims to study and compare the antioxidant activity of aqueous solutions of leaves and flowers of Sambucus nigra L obtained at different exposure times and assess the antibacterial activity of these solutions against Escherichia coli ATCC 8739, Salmonella NCTC 6017, Listeria monocytogenes NCTC 11994, and Staphylococcus aureus ATCC 25093.

Materials and methods: We studied the physicochemical properties of aqueous extracts of leaves (fresh) and flowers (fresh and dry) of Sambucus nigra L collected from the Rhodope region of Bulgaria. The samples from Sambucus nigra L were analyzed to determine their total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The diameters (in millimeters) of the growth inhibition zones of four pathogens were measured, and a comparative assessment of their antibacterial activity was made.

Results: The infusions of fresh blossoms and fresh leaves of Sambucus nigra L had the highest antioxidant activity at the total contact time of 30 minutes (82.7 mmol TE/100 ml) and 35 minutes (36.5 mmol TE/100 ml), respectively. The phenol-richest infusions were those made from dried flowers of Sambucus nigra L after a 30-minute contact time (86.7 mg GAE/ml). Of the four pathogens we studied, we found that the extracts affected partially only the pathogenic bacteria of Salmonella.

Conclusions: The highest content of bioactive components was obtained from dried blossoms of Sambucus nigra L. for infusions with a total contact time of 30 minutes and for decoctions at a contact time of 45 minutes.

 antioxidant activity antibacterial activity decoctions flavonoids infusion Sambucus nigra L Citation

Milkova-Tomova I, Kazakova Z, Buhalova D, Gentscheva G, Nikolova K, Minkova S. Antioxidant properties and antibacterial activity of water extracts from Sambucus nigra L. under different conditions. Folia Med (Plovdiv) 2023;65(2):295-300. doi: 10.3897/folmed.65.e79094.

 Introduction

Black elderberry is a member of the family Caprifoliaceae Vent, genus Sambucus. The genus has about forty species, but the fruits of only three (Sambucus nigra L, Sambucus canadensis Hasse, and Sambucus cerulean Ral.) are edible. On the Balkan Peninsula, one of the most common plants is the black elderberry (Sambucus nigra L.). Folk medicine uses all parts of this plant. Its flower, bark, leaves, and fruits are high in carbohydrates, lipids, terpenoids, flavonoids, phenolic acids, alkaloids, and other compounds.[1] Dried white flowers of Sambucus nigra L are used for preparation of infusions, decoctions, and juices. Water extracts and decoctions of the flowers are recommended to use to relieve the symptoms of colds, runny nose, sore throat, cough, inflammation of the urinary tract, and some more.[2–4] The water-soluble substances contained in the blossoms of Sambucus nigra L can directly induce insulin secretion and increase glucose metabolism.[5,6] Elder blossoms also have antimicrobial activity.[7] Standardized plant fruit extracts inhibit the reproduction of influenza B virus[8] and influenza A virus[9]. Ethanolic extracts of Sambucus nigra L. blooms and fruits have been shown to inhibit 13 pathogens, including Staphylococcus sp., Bacillus cereus, Salmonella poona, Staphylococcus aureus, and Pseudomonas aeruginosa.[10]

Water extracts are frequently used in a variety of food, cosmetic[3], and pharmaceutical products[11,12] due to their high concentration of biologically active components.

The majority of Sambucus nigra L research is focused on the preparation of extracts using hydrophobic solvents, with only a few studies focusing on water extracts.

 Aim

The present study aimed to study and compare (I) the antioxidant activity of water extracts of leaves and flowers of Sambucus nigra L obtained at different exposure times and temperatures about 100°C and (II) the antibacterial effect of these water extracts on Escherichia coli ATCC 8739, Salmonella NCTC 6017, Listeria monocytogenes NCTC 11994, and Staphylococcus aureus ATCC 25093.

 Materials and methods Samples

Black elder (Sambucus nigra L.) blossoms and leaves were taken from the ground at an altitude of 600–900 m in the Rhodope region during peak flowering (May-June). The blossoms were shade dried for 10 days by turning.

 Methods for obtaining water extracts

Infusions

A quantity of 5 g of chopped material (fresh or dry leaves or blossoms) was soaked in a porcelain volume graduated vessel of hot water (98°–100°C) at a hydromodule of 1:20 (product: water, w/v). The dish was placed in a water bath at the indicated temperature for 15 minutes. After the specified time, the vessel was taken out of the water bath, and the samples were taken at 10, 15, and 20 minutes.

 Decoctions

Five grams of chopped material were boiled in water (98°–100°C) at a hydromodule of 1:20 (product: water, w/v) for 30 minutes. Then, the decoctions were left at room temperature, and the samples were taken at 10, 15, and 20 minutes.

The obtained decoctions and infusions were filtered and stored at 4°C for measurement.

 Determination of total phenolic (<abbrev xlink:title="total phenolic content" id="ABBRID0EONAC">TPC</abbrev>) and total flavonoid contents (<abbrev xlink:title="total flavonoid content" id="ABBRID0ESNAC">TFC</abbrev>)

The total phenolic content of the investigated samples was determined using the method of Folin-Ciocalteu.‌[6] Folin-Ciocalteu reagent (1 ml) diluted five times was mixed with a 0.2-ml sample and 0.8 ml of 20% Na2CO3 (Sigma-Aldrich, Germany). After staying for one hour in darkness, the absorbance was measured at 750 nm. TFC was determined spectrophotometrically using Al(NO3)3 in water extracts.[18]

 The <abbrev xlink:title="1,1-diphenyl-2-picrylhydrazyl" id="ABBRID0EWOAC">DPPH</abbrev> radical-scavenging ability

The analyzed sample (0.15 ml) was mixed with 2.85 ml freshly prepared 0.1 mM solution of DPPH in methanol. The sample was incubated for 15 minutes at 37°C in darkness. The reduction of absorbance at 517 nm was measured by spectrophotometer in comparison to the blank containing methanol, and the inhibition percentage was calculated.[13]

 Ferric reducing antioxidant power (<abbrev xlink:title="ferric reducing antioxidant power" id="ABBRID0ELPAC">FRAP</abbrev>) assay

The assay was performed according to Benzie and Strain[13] with slight modification. The reaction was started by mixing 3.0 ml FRAP reagent with 0.1 ml of investigated extract. The reaction time was 10 minutes at 37°С in darkness, and the absorbance was measured at 593 nm against blank prepared with methanol.

 Determination of antibacterial activity

The strains of microorganisms (Listeria monocytogenes NCTC 11994, Escherichia coli ATCC 8739, Salmonella enterica subsp., Enterica serovar Abony NCTC 6017, and Staphylococcus aureus ATCC 25093) were supplied by the National Bank for Industrial Microorganisms and Cell Cultures. Selective bacteriological media were used for the microbiological test, respectively: Listeria Oxford Agar Base with an additive containing cycloheximide (Biolife); ENDO agar (Merck); LEIFSON Agar (Merck); Baird Parker Agar Base (Biolife) with yolk-tellurite supplement, and Plate Mount Agar (Merck), which were inoculated with pathogen suspensions prepared from a 24-hour culture. The antibacterial activity of the water extracts was assessed. The experiments were performed by using 24-h old bacterial suspensions. The extracts were tested using sterilized metal rings 5 mm in diameter. The discs were impregnated with 15 μl of the extract, kept until dry under laminar airflow and then placed into previously inoculated Petri dishes. Subsequently, the plates were incubated for 24 hours at 37°C. Comparative assessment of their antibacterial activity was made. For this purpose, the diameters of inhibition zones of pathogen growth were measured around the metal rings.

 Statistical analysis

All measurements were repeated five times. The presence of reliable variance between the types of the samples in the analyzed indicators has been determined by the two-factor analysis of variance (ANOVA) and evaluation of averages according to Duncan’s method.[14]

 Results

The antioxidant activity measured by DPPH and FRAP methods, the total phenolic and flavonoid content of infusions and decoctions from fresh and dried blossoms or leaves were evaluated. The results are presented in Table 1.

The results from the antibacterial activity are presented in Fig. 1.

Total phenols, flavonoids, and antioxidant activity of infusions and decoctions from leaves and blossom from Sambucus nigra L.

Samples Total phenolic content Total flavonoids Antioxidant activity mmol TE/100 ml±SD
Contact time mg GAE/ml±SD mg QE/ml±SD DPPH method FRAP method
Infusions from fresh leaves
25 min 1.6±0.1f 0.7±0.1e 13.4±1.1f 12.8±0.3f
30 min 2.0±0.1e 0.7±0.1e 23.3±0.7e 16.5±0.2e
35 min 4.1±0.1d 1.4±0.1d 36.5±1.3d 34.3±1.4d
Infusions from fresh blossom
25 min 13.5±0.1d* 6.8±0.2e* 64.9±1.3e* 80.7±1.1d*
30 min 13.0±0.1d* 5.5±0.1f* 82.7±2.1d* 70.8±0.9e*
35 min 13.0±0.1d* 8.7±0.2d* 64.5±1.3e* 74.4±1.6e*
Decoctions from fresh leaves
40 min 22.2±0.1c 10.2±0.1b 181.3±0.8c 146.6±0.6c
45 min 36.8±0.2b 8.5±0.1c 344.6±1.1b 265.6±0.8b
50 min 55.1±0.1a 14.6±0.1a 442.9±0.7a 372.9±0.5a
Decoctions from fresh blossom
40 min 21.4±0.1f 15.7±0.2f 136.7±0.1f 133.9±0.5f
45 min 34.0±0.1e 16.8±0.1e 180.2±0.1e 165.7±0.9e
50 min 43.5±0.2d 17.1±0.1d 249.2±1.0d 234.3±0.9d
Infusions from dry blossom
25 min 45.6±0.4c* 23.3±0.1c* 324.4±6.2c* 286.6±6.3c*
30 min 86.7±0.5a* 48.2±0.3a* 648±9.7a* 566.9±5.2a*
35 min 78.8±0.3b* 46.4±0.3b* 596.6±6.7b* 522.2±5.4b*
Decoctions from dry blossom
40 min 129.5±0.3c 66.0±0.1b 707.5±1.3b 662.4±1.4c
45 min 164.9±0.2a 81.0±0.2a 986.2±1.3a 832.1±1.1a
50 min 134.2±0.3b 64.3±0.1c 693.3±2.1c 722.5±0.9b

a, b, c - indexes for Duncan test of infusion and decoctions from fresh leaves; level of significance 0.5

 4AB8D654-128B-583E-9F45-A70C1D3C5609

Zones of inhibition of the growth of pathogenic bacteria (mm) in selective media from various extracts of Sambucus nigra L.

https://binary.pensoft.net/fig/848092 Discussion

All plants have different antioxidant potential which also depends on many external factors[15] such as soil type, climate, variety, wild or cultivated plants, storage, etc.[16]. Significant differences in concentrations of total phenols, flavonoids, and antioxidant activity were observed between infusions obtained from fresh leaves and blossoms depending on the time of extraction. The total phenol concentrations and antioxidant activity by both methods increased with the contact time, only the concentrations of total flavonoids required longer treatment to change their values of 0.7 mg QE/ml /25 and 30 min/twice at 35 min.

Total flavonoids in an infusion from fresh blossoms were 6 to 10 times greater than the content in the infusion from fresh leaves made under the same conditions. Dawidowicz et al. have obtained similar results.[17] There were no significant differences between the content of total flavonoids in decoctions from fresh leaves and blossoms. The highest concentrations of phenolic compounds were found in the decoctions of fresh leaves exposed to temperature for 50 minutes (55.1 mg GAE/ml), and the poorest infusions of fresh leaves at 25 minutes of treatment temperature (1.6 mg GAE/ml). The trend is similar for flavonoids in infusions and decoctions of fresh elder leaves.[18] The content of phenols in fresh blossom infusions is not affected by the duration of heat treatment. In infusions prepared from dry blossoms, the phenol content decreases with increasing time factor. The highest concentrations of phenols were found in the infusions of dried flowers of Sambucus nigra L, subjected to a 30-minute (86.7 mg GAE/ml) and 35-minute (78.8 mg GAE/ml) contact. Both the duration and the infusion of fresh/dry material had a proven effect on the content of flavonoids, and in the case of those of fresh flowers, they were many times lower. The greater the content of flavonoids, the higher the antioxidant ability of the extracts. The antioxidant activity determined by the DPPH method in most of the extracts of Sambucus nigra L was higher than that determined by the FRAP method. The antioxidant activity of fresh leaf decoctions during the total contact time of 50 minutes was 1.8 and 1.6 times higher than that of fresh flower decoctions determined by DPPH and FRAP methods, respectively. Infusions of fresh blossom Sambucus nigra L had the highest antioxidant activity during the total contact time of 30 minutes (82.7 mmol TE/100 ml). Fresh leaf infusions had the highest antioxidant activity at 35 minutes of total contact time (36.5 mmol TE/100 ml). Higher value of antioxidant activity determined by DPPH method for blossoms and leaves, respectively, was also reported by Dawidowicz et al.[4] The authors reported antioxidant activity of 94.15 mmol TE/100 ml for extracts from blossoms and 16.76 mmol TE/100 ml for extracts of leaves, respectively. This was most likely caused by the use of water-ethanol extracts. The change in antioxidant activity depends on the temperature, extraction time, and the raw material type. A significant difference was observed in decoctions and infusions of dry blossoms.

The study of phenols and flavones in infusions and decoctions is important because of their redox properties preventing the decomposition of hydroperoxides into free radicals.[5,19]Sambucus nigra L. dry blossoms infusions prepared at total contact time of 30 and 35 minutes are characterized by the highest content of total flavonoids. The trend is similar for decoctions. The total phenolic content for decoctions for dry blossoms is similar to that determined from Stoeva et al. (194.0 mg GAE/100 ml).[20] The antioxidant activity of decoctions and infusions is mainly related to the presence of flavonoids. Linear relationships are obtained between FRAP and TFC, as well as between DPPH and TFC, DPPH and TPC, FRAP and TPC with correlation coefficients greater than 0.91.

Of the four pathogens studied, only the pathogenic bacteria of Salmonella were partially affected by the extracts. Infusions of fresh leaves and dried and fresh flowers have an inhibitory effect, while decoctions do not show such an effect. The fresh blossom infusions with total contact times of 30 min and 35 minutes were the most effective against the Gram-negative bacteria of Salmonella NCTC 6017 (18 mm inhibition zone, IZ). The inhibition zones of dry blossom infusion with total contact times of 25 and 30 minutes were quite similar (10 mm IZ), but the largest inhibition zone was found for the dry blossom infusions with a 35-min total contact time (22 mm). The inhibition zones of fresh leaves infusion with 35 minutes and 30 minutes had the same size (10 mm IZ). On the other hand, the decoctions of blossoms or leaves did not inhibit the test cultures.

Fig. 1 illustrates the growth inhibition zones of Salmonella from Sambucus nigra L. extracts. Similar results are reported by Hearst et al.[10] In this study, the aqueous extracts from Sambucus nigra L. demonstrated a notable inhibition of Salmonella (7 mm). It is known that the activity on the main components of aromatic products (essential oils, extracts) is arranged in the following sequence: phenols > alcohols > aldehydes > ketones > esthers > hydrocarbons.[21] It can be concluded that the antimicrobial activity of different part of the plants is influenced by the chemical composition of these plant parts and the conditions under which the extracts were obtained.

We also determined the total number of mesophilic aerobic and facultative anaerobic microorganisms, molds, and yeasts in fresh flowers and elder leaves. It was found that the total number of microorganisms in fresh flowers (8.4×105 CFU/g) was 2.6 times higher than that in fresh leaves (3.2×105 CFU/g). The content of mold and yeast in the leaves (2.5×105 CFU/g) was significantly higher than that in the flowers (5.3×104 CFU/g).

 Conclusions

The highest content of bioactive components was obtained from as follows:

from fresh leaves and fresh blossoms - infusions at 35 min and decoctions at 50 min of contact time

from dry blossoms - infusions at 30 minutes and decoctions at 45 minutes of treatment

The extracts of Sambucus nigra L. show antibacterial activity against the pathogenic bacteria of Salmonella. The results obtained for antioxidant activity, total phenolic content and total flavonoids can be used to select parts of the plant (leaves, blossoms) and method of preparation (infusion, decoction) depending on the desired application.

 References 1. Agalar H. Elderberry (Sambucus nigra L.). Nonvitamin and nonmineral nutritional supplements. Acad Press 2019; 211–5. doi: 10.1016.C2016-0-03546-5 2. Cejpek K, Maloušková I, Konečný M, et al. Antioxidant activity in variously prepared elderberry foods and supplements. Czech J Food Sci 2009; 27:S45–8. 3. Christensen LP, Kaack K, Fretté XC. Selection of elderberry (Sambucus nigra L.) genotypes best suited for the preparation of elderflower extracts rich in flavonoids and phenolic acids. Eur Food Res Technol 2008; 227:293–305. 4. Dawidowicz AL, Wianowska D, Baraniak B. The antioxidant properties of alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts). LWT Food Sci Technol 2006; 39:308–15. 5. Lu B, Li M, Yin R. Phytochemical content, health benefits, and toxicology of common edible flowers: a review (2000–2015). Crit Rev Food Sci Nutr 2016; 56:S130–48. 6. Ivanov I. Polyphenols content and antioxidant activities of Taraxacum officinale F.H. Wigg (dandelion) leaves. Int J Pharmacognosy and Phytoch Res 2014; 6(4):889–93. 7. Arjoon A, Saylor C, May M. In vitro efficacy of antimicrobial extracts against the atypical ruminant pathogen, Mycoplasma mycoides subsp. Capri BMC Complement Altern Med 2012; 12:169. 8. Krawitz C, Mraheil MA, Stein M, et al. Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses. BMC Complement Altern Med 2011; 11:16. 9. Roschek B, Fink Jr RC, McMichael MD, et al. Elderberry flavonoids bind to and prevent H1N1 infection in vitro. Phytochemistry 2009; 70(10):1255–61. 10. Hearst C, McCollum G, Nelson D, et al. Antibacterial activity of elder (Sambucus nigra L.) flower or berry against hospital pathogens. J Med Plant Res 2010; 4(17):1805–9. 11. Fernandes L, Casal S, Pereira JA, et al. Edible flowers: A review of the nutritional, antioxidant, antimicrobial properties and effects on human health. J Food Compos 2017; 60:38–50. 12. Kaltsa O, Lakka A, Grigorakis S, et al. A green extraction process for polyphenols from elderberry (Sambucus nigra) flowers using deep eutectic solvent and ultrasound-assisted pretreatment. Molecules 2020; 25:921. 13. Kivrak I, Duru ME, Öztürk M, et al. Antioxidant, anticholinesterase, and antimicrobial constituents from the essential oil and ethanol extract of Salvia potentillifolia. Food Chem 2009; 116:470–9. 14. Roychev V, Tzanova M, Keranova N, et al. Antioxidant content and antioxidant activity in raisins from seedless hybrid vine varieties with colored grape juice. Czech J Food Sci 2020; 38:410–6. 15. Contessa C, Mellano MG, Beccaro GL, et al. Total antioxidant capacity and total phenolic and anthocyanin contents in fruit species grown in Northwest Italy. Sci Horticul 2013; 160:351–7. 16. Buřičivá L, Réblová Z. Czech medicinal plants as possible sources of antioxidants. Czech J Food Sci 2008; 26:132–8. 17. Dawidowicz AL, Wianowska D, Baraniak B. The antioxidant properties of alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts). LWT - Food Sci Technol 2006; 39:308–15. 18. Mikulic-Petkovsek M, Samoticha J, Eler K, et al. Traditional elderflower beverages: A rich source of phenolic compounds with high antioxidant activity. J Agric Food Chem 2015; 63:1477–87. 19. Liebert K. Elder: Myth, healing effects and recipes of elder. Berlin: Demmler Verlag Gmbh; 2017: 20–2. 20. Stoilova I, Wilker M, Stoyanova A, et al. Antioxidant activity of elderberry extract (Sambucus nigra L.). Herba Pol 2007; 53(1):45–54. 21. Baser KHC, Buchbauer G, editors. Handbook of essential oils: science, technology, and applications. Boca Raton, FL, USA: Taylor and Francis Group; 2010: 34-50.