Multidirectional approaches on autofermented chamomile ligulate
flowers: Antioxidant, antimicrobial, cytotoxic and enzyme
inhibitory effects
A. Cvetanovi
ć
a,⁎
, Z. Zekovi
ć
a, G. Zengin
b, P. Ma
šković
c, M. Petronijevi
ć
d, M. Radojkovi
ć
aaFaculty of Technology, University of Novi Sad, Department of Pharmaceutical Engineering and Biotechnology, Bulevar cara Lazara 1, 21 000 Novi Sad, Serbia b
Department of Biology, Faculty of Science, Selcuk University, Campus/Konya, Konya, Turkey
c
Faculty of Agronomy, Cara Dušana 34, 32000 Čačak, Serbia
d
Faculty of Science, University of Novi Sad, Dositeja Obradovica 3, 21 000 Novi Sad, Serbia
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 10 October 2017
Received in revised form 27 November 2017 Accepted 6 January 2018
Available online 1 February 2018
In the frame of the present paper the enzymatic transformation of apigenin-glucosides into free aglycone was achieved by autofermentation of chamomile ligulateflowers (CLF). Antioxidant properties of the autofermented CLF (A-CLF) extract were evaluated by their radical scavenging activity against hydroxyl radicals and inhibition of lipid peroxidation. Obtained results showed that A-CLF extract in a concentration of 0.84 mg/mL was able to inhibit 50% of hydroxyl radicals, while IC50 value in the case of inhibition of lipid peroxidation was
5.21 mg/mL. Antimicrobial activity was done by measuring minimal inhibitory concentration (MIC) values for eight microbial strains. Obtained MIC values (9.75–156.25 μg/mL) confirmed high antibacterial and antifungal activities of the extract. Cytotoxic activity was done by using three histological different cell lines: Hep2C; RD and L2OB. Obtained IC50values for these cell lines were: 28.72; 17.31 and 10.92, respectively. Furthermore,
in vitro investigation of the A-CLF ability to inhibit selected enzymes (α-amylase, α-glucosidase, tyrosinase) was done as well. Determined activities against α-amylase and α-glucosidase were 0.94 and 3.24 mmol ACAE/g, respectively. Further, measured activity against tyrosinase was 0.69 mg KAEs/g indicating high enzyme-inhibitory activity of examined sample. Results demonstrated that A-CLF extracts showed consid-erable pharmacological activity.
© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords: Chamomile Autofermentation Biological activity Enzyme-inhibitions Natural agents 1. Introduction
Recently, there has been a growing need for medicines and treatment approaches that are inexpensive, effective, and that are not associated with the high side effect burden that often limits conventional pharmaco-logical therapy (Ngemakwe et al., 2017). Although numerous technolog-ical and chemtechnolog-ical methods for the synthesis of various compounds have been developed, medicinal plants still remain one of the most important sources of new drugs, new drug leads and new chemical entities (Newman et al., 2000; Butler, 2004). Moreover, medicinal plants are con-sidered natural and therefore safer than conventional synthetic pharma-ceuticals, and the beneficial effects of phytochemicals on human health have gained a major interest (Mocan et al., 2016, 2017; Mollica et al., 2017). One of the most popular and well documented medicinal plants over the world is Chamomilla recutita (L.) or simply chamomile. Its flower-heads are used both internally and externally to alleviate or even cure a vast list of health conditions (Rotblatt, 2000). Although it is used
in different pharmaceutical formulations (Craker and Simon, 1986; Maiche et al., 1990; Fidler et al., 1996; Li et al., 1996), chamomile is mostly consumed as infusion for sedative and anxiolytic purposes (Viola et al., 1995; Cauffield and Forbes, 1999; Larzelere and Wiseman, 2002), as a digestive, and to treat gastrointestinal disturbances, especially for babies and children (Weizman et al., 1993; De la Motte et al., 1997; Presibella et al., 2006). Bioactivity and potential health benefits of chamomile are associated with essential oil andflavonoid fraction. Although chamo-mile represents a rich source of different classes of phytochemicals, researchers focus special attention on phenols andflavonoids. The various mechanisms of biological effects of these compounds have been demonstrated by laboratory experiments, including antioxidant, induction of detoxification enzymes, estrogenic and anti-estrogenic activity (Shukla and Gupta, 2010). Among otherflavonoids, apigenin (Fig. 1) is considered as one of the most important bioactive molecules due to its high bioactivity and huge biopotential. Thisflavonoid belongs to theflavone structural class while chemically is known as 4′,5,7-trihydroxyflavone. It is recognized as the molecule with the lowest level of toxicity in comparison to other flavonoids (quercetin, kaempferol, luteolin, and others). Different studies have confirmed that ⁎ Corresponding author.
E-mail address:amod@uns.ac.rs(A. Cvetanović).
https://doi.org/10.1016/j.sajb.2018.01.003
0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Contents lists available atScienceDirect
South African Journal of Botany
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s a j bapigenin possesses: antioxidant, antimutagenic, anticarcinogenic, anti-inflammatory, antineoplastic proliferation and antineoplastic progres-sion properties (Birt et al., 1986). Moreover, over the years, thisflavon has been increasingly recognized as inhibitor of intestinal motility.
Although apigenin is synthetized by many plant species, its most im-portant source is chamomile where it is presented in its free form and in the form of its glucoside and various acylated forms. Pharmacological studies suggest that spasmolytic activity of chamomile is mainly due to the presence of apigenin and apigenin-7-O-glucoside (Ap-7-Glc). The spasmolytic action of apigenin is 7–9 times higher than its glucoside and 3.29 times higher than papaverine, an official spasmolytic com-pound. Therefore, when considering the use of chamomile spasmolytic activity, it is evident that the content of apigenin in chamomile or in its preparation is very important factor. However, the content of Ap-7-Glc (3–9%) in chamomile is much higher than of its aglycone-apigenin (0–0.5%) (Pekic et al., 1989). In order to increase the amount of agly-cone, hydrolysis of its glucosides into aglycone could be done by several ways (Maier et al., 1991; Pekić et al., 1994; Pekić and Zeković, 1994). One of the most suitable methods implies the use ofβ-glucosidase, nor-mally present in chamomile. The action of this enzyme breaks glycoside links between aglycone and sugar component in the molecule of Ap-7-Glc, releasing a free aglycone— apigenin.
In this study, apigenin was liberated from its glycosides by enzy-matic biotransformation, i.e. autofermentation. Since the highest apigenin content is found in chamomile ligulateflowers (CLF), they were served as a starting plant material. Autofermentation was per-formed with chamomile native enzymes under conditions that allowed optimum enzyme activity. Extraction process was performed with autofermented (A-CLF) plant material. In order to explore bio-logical potential of A-CLF extracts, in vitro antioxidant, antimicrobial, cytotoxic and enzyme inhibition effects were determined. According to our knowledge, there is no available literature about the influence of autofermentation of enzyme-inhibitory activity of the chamomile. This paper offers thefirst data.
2. Material and methods 2.1. Chemicals and reagents
Solution of amylase (ex-porcine pancreas, EC 3.2.1.1), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20),L
-glutathione (0.5 mg/mL),β-carotene, tyrosinase solution (200 u/mL), 3,4-dihydroxy-L-phenylalanine (L-DOPA), kojic acid, starch (0.05%), acarbose (2 mg/mL), linoleic acid, chlorogenic acid, Folin–Ciocalteu reagent, Tween-80, thiobarbituric acid (TBA), 2-deoxyribose and β-carotene, were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Iron (II) sulfate was obtained from Zorka (Šabac, Serbia). Cirsimarin, amaricin, nystatin, Mueller–Hinton broth, Sabouraud dextrose and cis-diamminedichloroplatinum (cis-DDP) were purchased from Tedia Company (USA). Sodium carbonate was obtained from Merck (Darmstadt, Germany). Ethylenediaminetetraacetic acid (EDTA) was purchased from Centrohem (Stara Pazova, Serbia). All chemicals and reagents were of analytical reagent grade.
2.2. Plant material
Chamomile ligulateflowers (CLF) used in this study were produced at the locality of Bački Petrovac, the Institute of Field and Vegetable
Crops, Novi Sad, Serbia. Chamomileflowers were dried at 40 °C in a solar dryer. The layer thickness of plant material during the process of drying was 5 cm, and the process was performed until the moisture con-tent of about 12%. Thereafter, the CLF were separated from the tubular parts by sieving. Separated CLF were packed into paper bags and stored in the dark place until further use.
The autofermentation process was performed according to the procedure previously described byZekovic (1993). The process was car-ried out using sodium-acetate buffer (0.1 mol/dm3, pH = 5.5) at 37 °C for 72 h. The plant:buffer ratio was 1:5 (w/v). Dry plant material was moisturized with the buffer and the mixture was stirredfive times per day. The sample was further dried at room temperature for 5 days. A-CLF plant samples were extracted by maceration extraction technique.
2.3. Extraction of the plant material
A-CLF samples were extracted using ethanol (70%) as a solvent. A sample (5 g) was placed in a volumetricflask and 250 mL of solvent was added. Extraction was performed at room temperature for three days. The obtained extracts werefiltrated and evaporated using a vacuum evaporator (Devarot, Elektromedicina, Slovenia). Drying was performed at 40 °C in a conventional laboratory dryer. Obtained dry extracts were stored in a dark place at 4 °C until analysis.
2.4. Determination of total phenolic content
The total phenolic content (TPC) in obtained extracts was deter-mined by the Folin–Ciocalteu procedure (Singleton and Rossi, 1965; Kähkönen et al., 1999) using chlorogenic acid as a standard. Absorbance was measured at 750 nm. Content of phenolic compounds was expressed as mg of chlorogenic acid equivalent (CAE) per g of dry extract (mg CAE/g). All experiments were performed in triplicate.
2.5. Determination of antioxidant activity of extracts
Dry extracts of A-CLF were diluted to obtain a series of dilutions in the concentration range from 0.10 to 10.0 mg/mL (mg of dry extract per mL). The diluted solutions of each extract were analyzed for antioxidant activity using two different in vitro assay systems: lipid peroxidation assay and assay for the scavenging effect on hydroxyl radicals.
2.5.1. Lipid peroxidation assay
Lipid peroxidation assay was performed in the lipid model system based on the oxidative degradation ofβ-carotene in the β-carotene– linoleic acid emulsion (Moure et al., 2000). 2 mg ofβ-carotene was dissolved in 10 mL of chloroform. After that 2 mL of this solution was pipetted into a round-bottomedflask, and chloroform was re-moved by evaporating on a rotary evaporator.β-Carotene (2 mg) in a round-bottomed flask was dissolved with 100 mL of emulsion made from linoleic acid (40 mg), Tween 80 (400 mg) and distillated water.
The reaction mixture was prepared by mixing 5 mL of emulsion with 0.2 mL of the extract. Immediately after preparation absorption was measured at 470 nm for each test-tube (τ = 0 min). The tubes with reaction mixture were placed in a water bath at 50 °C until the total decolorization ofβ-carotene (about 2 h). After that, absorption was measured again (τ = 120 min). A control sample was prepared with distilled water instead of extract.
The degradation rate ofβ-carotene was calculated by using the following equation:
%AOA ¼ Vcontrol−Vsample
Vcontrol 100
where Vcontroland Vsamplewere calculated according to the following equation: V¼ ln A0 min A120 min 1 120
2.5.2. Assay for the scavenging effect on hydroxyl radicals
Assay for the scavenging effect on hydroxyl radicals was done according to the Gutteridge method (Gutteridge, 1987) with some modification. Various concentrations of the extracts were mixed with 100 mL of 2-deoxyribose (0.05 mmol/L), 100 mL H2O2(0.0147%),
10 mL FeSO4(100 mmol/L) and 2.7 mL of phosphate buffer (pH 7.4).
Control samples contained 80% methanol instead of extracts. Solution which was used for a correction of test solution contained 3 mL of phosphate buffer and extract. Correction of control was prepared with 3 mL of phosphate buffer and 20–40 mL of 80% methanol. All prepared solutions were incubated for 60 min at 37 °C. Further, 200 mL of EDTA (3.72%) and 2 mL of TBA reagents were added to mixtures and heated at 100 °C for 10 min. Immediately after cooling to room temperature, ab-sorbance was measured spectrophotometrically at 532 nm. Scavenging rate (RSC) was calculated by the following equation:
RSCð Þ ¼ 1−% Asample−Acorr:
Acontr:−Acorr:control
100
The results were expressed as inhibitory concentration at 50% (IC50),
which is the concentration of the test solution for achieving 50% of the radical scavenging capacity. All analyses were run in triplicate. 2.6. Determination of antibacterial and antifungal activity
The antibacterial activity was tested against the Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus vulgaris ATCC 13315, Proteus mirabilis ATCC 14153 and Bacillus subtilis ATCC 6633. The antifungal activity was tested against the Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231. Antibacterial and antifungal activities were estimated by measuring their minimum inhibitory concentrations (MIC). MICs of the extracts and cirsimarin against the test bacteria were determined by microdilution method in 96 multi-well microtiter plates (Sarker et al., 2007). All tests were performed in Mueller–Hinton broth (MHB) with the exception of the yeast, in which case Sabouraud dextrose broth was used. A volume of 100μL stock solutions of extracts (in methanol, 200μL/mL) and cirsimarin (in 10% DMSO, 2 mg/mL) were pi-petted into thefirst row of the plate. Fifty microliters of Mueller Hinton or Sabouraud dextrose broth (supplemented with Tween 80 to afinal concentration of 0.5% (v/v)) were added to other wells. A volume of 0.05 mL from thefirst test wells was pipetted into the second well of each microtiter line, and then 0.05 mL of scalar dilution was transferred from the second to the twelfth well. Ten microliters of resazurin indica-tor solution (prepared by dissolving 270-mg tablet in 40 mL of sterile distilled water) and 0.03 mL of nutrient broth were added to each well. Finally, 0.01 mL of bacterial suspension (106CFU/mL) and yeast spore
suspension (3 × 104CFU/mL) were added to each well. For each strain,
the growth conditions and the sterility of the medium were checked. Standard antibiotic amracin was used to control the sensitivity of the tested bacteria, whereas nystatin was used as a control against the tested yeast. Plates were wrapped loosely with clingfilm to prevent dehydration and prepared in triplicate. The plates were placed in an incubator at 37 °C for 24 h for the bacteria and at 28 °C for 48 h for the yeast. Subsequently, color change was assessed visually. Any color change from purple to pink or colorless was recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of 3 values was calculated, and the obtained value was taken as the MIC for the tested sample and a standard drug.
2.7. Measurement of cytotoxic activity by MTT assay
The influence of extracts on the growth of malignantly transformed cell lines was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The following cell lines were used: RD (cell line derived from human rhabdomyosarcoma), Hep2c (cell line derived from human cervix carcinoma— HeLa derivative) and L2OB (cell line derived from murinefibroblast). Cells were seeded (2 × 105cell/mL; 100μL/well) in 96-well cell culture plates in nutrient medium (Minimum Essential Medium (MEM) Eagle supplemented with 5% of Hep2c, RD and L2OB) and grown at 37 °C in humidified atmo-sphere for 24 h. Then, corresponding extract (stock solution: 5 mg of extract dissolved in 1 mL of absolute ethanol) and control (absolute ethanol) diluted with nutrient medium to desired concentrations were added (100μL/well) and cells were incubated at 37 °C in humidi-fied atmosphere for 48 h. Pure nutrient medium (100 μL) represented positive control for each cell line. After incubation period, supernatants were discarded and MTT (dissolved in Dulbecco's modification of Eagle's medium (D-MEM) in concentration of 500μg/mL) was added in each well (100μL/well). Immediately after, all wells were incubated at 37 °C in humidified atmosphere for 4 h. Reactions were halted by adding 100μL of sodium dodecyl sulfate (SDS) (10% in 10 mM HCl). After overnight incubation at 37 °C, absorbance was measured at 580 nm using a spectrophotometer (Ascent 6-384 [Suomi], MTX Lab Systems Inc., Vienna, VA 22182, USA). The number of viable cells per well (NVC) was calculated from a standard curve plotted as cell numbers against A580. Corresponding cells (grown inflasks), after cell
count by a hemocytometer, were used as standards. Standard suspen-sions were plated in serial dilution, centrifuged at 800 rpm for 10 min and then treated with MTT/D-MEM and 10% SDS/10 mM HCl solutions in the same way as the experimental wells (ut supra). The number of vi-able cells in each well was proportional to the intensity of the absorbed light, which was then read in an ELISA plate reader at 580 nm. Absor-bance (A) at 580 nm was measured 24 h later. Cell survival (%) was calculated by dividing the absorbance of a sample with cells grown in the presence of various concentrations of the investigated extracts with control optical density (the A of control cells grown only in nutri-ent medium), and multiplying by 100. The blank absorbance was always subtracted from the absorbance of the corresponding sample with target cells. IC50concentration was defined as the concentration of an
agent inhibiting cell survival by 50%, compared with a vehicle-treated control. The results of the measurements were expressed as the percentage of positive control growth taking the cis-DDP determined in positive control wells as the 100% growth (Baviskar et al., 2012). All experiments were done in triplicate.
2.8. Enzyme inhibitory activity
The enzyme inhibitory activity of chamomile extracts was evaluated by measuringα-amylase, α-glucosidase as well as tyrosinase inhibitory activity. α-Amylase inhibitory activity was performed using the Caraway–Somogyi iodine/potassium iodide (IKI) method (Zengin et al., 2014). Sample solution (2 mg/mL; 0.025 mL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (0.05 mL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After pre-incubation, the reaction was initiated with the addition of starch solution (0.05 mL, 0.05%). Similarly, a blank was prepared by adding sample so-lution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated for 10 min at 37 °C. The reaction was then stopped with the addition of HCl (0.025 mL, 1 M). This was followed by addition of the iodine–potassium iodide solution (0.1 mL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalent (mg ACAE/g extract).
α-Glucosidase inhibitory activity was performed by the previous method (Zengin et al., 2014). Sample solution (2 mg/mL;0.05 mL) was mixed with glutathione (0.05 mL),α-glucosidase solution (from S. cerevisiae, EC 3.2.1.20, Sigma) (0.05 mL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside) (0.05 mL) in a 96-well microplate and incubated for 15 min at 37 °C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (0.05 mL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalent (mg ACAE/g extract).
Tyrosinase inhibitory activity was measured using the modified dopachrome method with 10 mML-DOPA (3,4-dihydroxy-L -phenylala-nine) as substrate, as previously reported (Zengin, 2016) with slight modification. Sample solution (2 mg/mL; 0.025 mL) was mixed with tyrosinase solution (0.04 mL) and phosphate buffer (0.1 mL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition ofL-DOPA (0.04 mL). Similarly, a
blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absor-bances were read at 492 nm after a 10 min incubation at 25 °C. The ab-sorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalent (mg KAE/g extract).
3. Results and discussion 3.1. Antioxidant activity of extracts
Quantification of apigenin in A-CLF has already been reported in the literature and the results suggest that the content of apigenin increased in plant materials after fermentation (Pekic et al., 1989; Zekovic, 1993; Cvetanović et al., 2015b). Taking into account that thisflavone expresses good antioxidant activity it could be expected that extracts prepared from the A-CLF express high level of antioxidant activity. Several studies have been conducted in order to determine the antioxidant activity of this importantflavon. Its ability to act as free radical scavenger is related to its chemical structure. The compound itself may scavenge free radi-cals and prevent biological systems from reactive oxygen species (ROS). Apigenin expresses effects on the expression and activity of antioxidant enzymes that modulate H2O2 levels, such as catalase
(CAT), superoxide dismutase (CuZnSOD and MnSOD), glutathione per-oxidase (GPx), and glutathione reductase (Gred) (Galati et al., 1999; Valdameri et al., 2011). The free radical scavenging property of apigenin might be attributed to the OH groups possessed in the 4′, 5 and 7 posi-tions of its structure (Noroozi et al., 1998). Further, the antioxidant abil-ity of the apigenin may be due to the double bond between carbon atoms 2 and 3 of the C ring (Ratty and Das, 1988). Additionally, the 4 and 5 OH groups in A and C rings are giving maximum radical scaveng-ing potency to apigenin (Fig. 1).
In this study, conventional antioxidant methodologies were used to determine the antioxidant profile of autofermented chamomile extracts (Fig. 2). Also, content of total phenols was determined due to the fact that polyphenols play an important role in antioxidant protection (Barroso et al., 2011).
Results showed that the amount of total phenolic compounds in A-CLF extracts was 180.52 mg CAE/g indicating their quite high content. Data from literature suggest that TPC content in native chamomile could be in the range from 117.31 to 141.41 mg CAE/g, depending on extrac-tion methods. Obtained TPC values in this study may further influence high antioxidant potential of the sample.
Thefirst antioxidant assay was conducted in order to determine the ability of the sample to inhibit the process of lipid peroxidation. In this method theβ-carotene reacts with radicals formed by linoleic acid
oxidation in an emulsion. The rate ofβ-carotene bleaching can be slowed down in the presence of antioxidants (Kulisic et al., 2004). Obtained re-sult is expressed as a half maximal inhibitory concentration i.e. IC50
value. As it can be seen fromFig. 2, IC50value for A-CLF extracts was
5.21 mg/mL. This value shows high potential of A-CLF which means that chamomile can inhibit the process of lipid peroxidation. This property is mainly due to the presence of chamazulene as well as other components of its essential oil (Rekka et al., 1996; Owlia et al., 2007).
The second consecutive method which was used in order to make more accurate consideration of antioxidant potential of the extracts was determination of hydroxyl radical scavenging activity. This type of free radicals is considered to be the most reactive among others. In bio-logical systems these hydroxyl radicals could be generated in the Fenton reaction in the presence of reduced transition metals (such as Fe2+) and
H2O2. These radicals could cause different cell damages in vivo and
con-sequently they can be considered as powerful causers of numerous dis-eases. Scavenging of hydroxyl radicals is a very important task for antioxidant molecules for the protection of living systems. In this study the inhibitory activity of obtained extracts against hydroxyl radi-cals was determined by measuring the level of oxidation of 2-deoxy-D -ribose by hydroxyl radicals with subsequent measurement of the prod-ucts by their reaction with thiobarbituric acid (TBA). The results show that A-CLF extract in a concentration of 0.84 mg/mL was able to inhibit 50% of hydroxyl radicals in the system (Fig. 2).
Obtained results clearly demonstrated high activity and great anti-oxidant potential of autofermented samples, indicating the possible high health benefits of such samples.
3.2. Antimicrobial activity of extracts
The anti-microbial activity of the A-CLF extracts was studied in six different concentrations (9.75, 19.53, 39.10, 78.125, 156.25 and 312.50 μg/mL) against six bacterial strains, two Gram-positive (S. aureus, B. subtilis) and four Gram-negative (E. coli, K. pneumoniae, P. vulgaris, P. mirabilis), as well as two fungal strains (C. albicans, A. niger). Measured IC values for investigated extract are presented in
Table 1. Data fromTable 1show that extract exhibits antimicrobial activ-ity against all used strains but with different degrees of effectiveness. Antimicrobial activity of standard compounds (amracin in the case of bac-teria and nystatin in the case of fungi) was also reported inTable 1.
E. coli is bacteria normally present in the intestinalflora of humans and can cause disease especially in immunocompromised and debilitated individuals. In case of this strain obtained MIC value was 9.75μg/mL, indicating strong antimicrobial activity of observed extract. This high sensitivity of E. coli on activity of the extract is very important especially if taking into account that this bacterium is already known to be multi-resistant to drugs. The highest MIC values (156.25μg/mL) were achieved in the case of B. subtilis. Notwithstanding, it can be
considered that the extract possesses quite well antimicrobial activity. In terms of antifungal activity, A-CLF expressed different effectiveness in the case of two different fungal strains. Namely, in the case of A. niger, the obtained MIC value was 78.12μg/mL. Such values were in ac-cordance with literature data (Cvetanović et al., 2015a, 2015b). On the other hand, better activity was noticed in the case of C. albicans (MIC = 78.12μg/mL).
Investigated extracts showed higher level of activity in comparison to those ones from literature (Cvetanović et al., 2015a). Namely, accord-ing to the literature, A-CLF extracts of chamomile obtained by sonification show very good antimicrobial activity, but still lower than extracts prepared in this research. More preciously, literature data shows that in the case of S. aureus sonified extracts of A-CLF expressed an MIC value of 156.25μg/mL. Further, in the case of K. pneumoniae, P. vulgaris and P. mirabilis observed values were 78.12, 78.12 and 156.25μg/mL, respectively. On the other side, MIC values obtained in current study were lower (Table 1). However, in the case of B. subtilis extract obtained in this study expresses lower activity that the sample in the abovementioned study.
Such strong biological activity implies that autofermented chamomile extracts could serve as a good source of components with antimicrobial activity. Thus the extract can be incorporated into a myriad of food and cosmetic products, or nutritional supplements. For example, obtained ex-tracts could serve for exploring and developing a functional food with improved preservation. This is very important considering a huge ten-dency of consumers to choose minimally processed foods instead of processed ones, or with minimum incorporation of synthetic additives.
3.3. Cytotoxic activity of extracts
Cytotoxic activity of A-CLF extract was investigated in the panel of three different cell lines in a wide range of mass concentrations from 1 to 100μg/mL. Results of tested cytotoxicity of the extract are shown in
Table 2.
Treatment of cell lines with extract resulted in a considerable dose-dependent inhibition of cell growth. The most effective cell growth inhi-bition activity and extremely low IC50value of 10.92μg/mL were
ob-served in cell line derived from murinefibroblast (L2OB). Slightly higher IC50value was achieved in the case of RD cells (17.31μg/mL),
while in the case of Hep2C cells the highest value of 28.72μg/mL was achieved. Inhibitory concentrations for cis-DDP were in the range from 0.94 to 1.4μg/mL. Bearing in mind that the criterion for cytotoxic activ-ity for plant extracts (according to the American National Cancer
Institute) is IC50b30 μg/mL, it is evident that A-CLF extracts are
cyto-toxic for all three cell lines, indicating antitumor properties of autofermented chamomile constituents.
Cytotoxic activity of A-CLF ultrasonic extracts was represented in the literature (Cvetanović et al., 2015a, 2015b) and this is thefirst evidence of cytotoxic potential of A-CLF samples obtained by maceration. In com-parison with sonified samples A-CLF extract examined in the current study suggests quite higher cytotoxic potential. It is well known that content of bio-active constituents of extracts is influenced by different factors and one of them is extraction method. Ultrasound extraction could produce a series of effects, one of them is tendency of some analytes to reabsorb into the sample matrix or their aggregation during extraction. Also, ultrasound extraction can lead to the degradation of some organic species under the influence of high energy processes (Švarc-Gajić, 2012).Thus it could be assumed that higher cytotoxic po-tential could be a consequence of mild conditions and extended extrac-tion time during maceraextrac-tion process.
3.4. Enzyme-inhibitory activity of extracts 3.4.1. Anti-diabetic activity
Diabetes mellitus (DM) is one of the major chronical problems worldwide and about 400 million people are affected with it. In this sense, new therapeutic strategies are needed to control of DM. Among the strategies, the inhibition of key enzymes (amylase and α-glucosidase) involved in carbohydrate metabolism is considered as one of the most effective ways. With this in mind, several compounds (acarbose, voglibose, etc.) have been chemically produced for this pur-pose but they have some unpleasant side effects including gastrointes-tinal disturbances and toxicity (Etxeberria et al., 2012). Therefore, natural enzyme inhibitors have great potential for designing novel anti-diabetic drugs (Katanić et al., 2017; Kocak et al., 2017). To the best of our knowledge, there is no evidence in the literature about enzyme-inhibitory activity of fermented chamomileflowers. Thus, this study offersfirst data (Table 3).
For the abovementioned reasons, the studied extract was tested to-wardsα-amylase and α-glucosidase. The extract was more effective on α-glucosidase compared to α-amylase. Observed results may be linked to the presence of apigenin in the extract. Several studies were con-ducted on anti-diabetic ability of apigenin. For example, the inhibition mechanism of apigenin on glucosidase was reported in a recent paper byZeng et al. (2016). Also, thesefinding were supported by in vivo an-imal assays, which found that apigenin is an effective natural agent for reducing blood glucose level in DM patients (especially type II) (Babu et al., 2013). In accordance with our results, chamomile has been sug-gested as a preventive agent in managing DM.
Table 1
Antifungal and antibacterial activity of autofermented chamomile extracts (MIC values,μg/mL).
Candida albicans Aspergillus niger Staphylococcus aureus Klebsiella pneumoniae Escherichia coli Proteus vulgaris Proteus mirabilis Bacillus subtilis
Extracts of A-CLF 39.10 78.12 78.12 39.10 9.75 39.10 39.10 156.25
Amracin 0.97 0.49 0.97 0.49 0.49 0.24
Nystatin 1.95 1.95
Table 2
Cytotoxic activity of autofermented chamomile extracts. Cell lines IC50values (μg/mL)
Sample cis-DDPe Hep2c cellsa 28.72 ± 1.32d 0.94 ± 0.55d RD cellsb 17.31 ± 1.02d 1.4 ± 0.97d L2OBcellsc 10.92 ± 0.99d 0.72 ± 0.14d
aCell line derived from human cervix carcinoma. b Cell line derived from human rhabdomyosarcoma. c
Cell line derived from murinefibroblast.
d
Mean value ± 2SD.
e
Cis-diamminedichloroplatinum.
Table 3
Enzyme-inhibitory activity of A-CLF extracts. α-Amylase
(mmol ACAE/g)a α-Glucosidase (mmol ACAE/g) Tyrosinase (mg KAEs/g)b Extracts of A-CLF 0.94 ± 0.02c 3.24 ± 0.08c 0.69 ± 0.01c a
mmol acarbose equivalent per g of dry extract;
b
Kojic acid equivalent per g of dry extract.
c
3.4.2. Tyrosinase-inhibitory activity
Tyrosinase is a key enzyme in the synthesis of melanin, which is the main pigment in skin and hair. However, various factors (genetic or en-vironmental) could lead to excessive melanin production and then it re-sults in hyperpigmentation problems. From this point, the inhibition of tyrosinase is considered as a therapeutic tool for controlling hyperpig-mentation. Kojic acid and some hydroquinones are chemically pro-duced for novel cosmetic products. However, the uses of these synthetics have some concerns such as toxicity. Therefore, natural com-pounds for the treatment of hyperpigmentation events are gaining the at-tention of the scientific community (Kim and Uyama, 2005). In this context, we tested A-CLF against tyrosinase. The extract exhibited mild in-hibition on the enzyme with the value of 0.69 mg KAE/g. Observed results can be attributed to the presence of apigenin in the extracts. Several stud-ies shown that apigenin could be considered as a natural inhibitor for ty-rosinase. For example,Ye et al. (2010)tested 35 botanical compounds on tyrosinase and apigenin is one of the most effective compounds in their study. Similar to our approach,Takekoshi et al. (2014)reported that apigenin exerts good action on tyrosinase in both in vivo and in vitro ex-periments. Also, a recent review of the literature on this topic showed that severalflavonoids including apigenin have very good potential in the treatment of pigmentation disorders (Kim et al., 2006). Taken to-gether, our finding could be useful for discovering novel natural cosmeceuticals in the treatment of hyperpigmentation problems.
4. Conclusion
Natural products and their extraction procedures are gaining inter-est for designing new applications in the scientific area. With this in mind, chamomile ligulateflowers were subjected under the process of enzymatic transformation prior to its extraction. The process leads to obtaining phenol-rich extracts with improved biological activity. Deter-mined IC50values in the case of inhibition of lipid peroxidation and
in-hibition of hydroxyl radicals were 5.21 and 0.84 mg/mL, respectively. Depending on microbial strains, antimicrobial activity was in the range from 9.75 to 156.25μg/mL. Obtained extracts showed to be rich in compounds with cytotoxic and antidiabetic effects. The extracts exerted remarkable inhibitory effects onα-amylase, glucosidase and ty-rosinase, which are targets for controlling global health problems. In this direction, autofermented chamomile extracts could be considered as a potential candidate for designing not only new functional foods but also cosmetic and pharmaceutical ingredients.
Acknowledgment
The present work was carried out within the projects of the Ministry of Education, Science and Technological Development of the Republic of Serbia, (Projects No. TR31013). The authors are grateful to Dr. Dušan Adamović, Institute of Field and Vegetable Crops, Bački Petrovac, Serbia, for his support in supplying plant material.
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