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Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract
SAJB-01995; No of Pages 5 South African Journal of Botany xxx (2018) xxx–xxx
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Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract N. Eruygur a,b,⁎, U.M. Koçyiğit c, P. Taslimi d, M. Ataş e, M. Tekin f, İ. Gülçin d a
Cumhuriyet University, Faculty of Pharmacy, Department of Pharmacognosy, Sivas, Turkey Selçuk University, Faculty of Pharmacy, Department of Pharmacognosy, Konya, Turkey c Cumhuriyet University, Vocational School of Health Services, Sivas, Turkey d Ataturk University, Faculty of Sciences, Department of Chemistry, Erzurum, Turkey e Cumhuriyet University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Sivas, Turkey f Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Edirne, Turkey b
a r t i c l e
i n f o
Article history: Received 10 January 2018 Received in revised form 9 March 2018 Accepted 2 April 2018 Available online xxxx Edited by Kannan Ragupathi Raja Rengasamy Keywords: Achillea cucullata Antioxidant activity Antimicrobial activity Anticholinesterase activity Antidiabetic activity
a b s t r a c t The Achillea genus belongs to the Asteraceae family, which is mostly found in the northern hemisphere and is comprised of 115 species in the world. In Turkish flora, there are 52 species and 58 taxa, among them half of which are recorded as endemic. To the best of our knowledge, there has been no biological activity studied in this species until now, with the exception of one study of the antimicrobial activity of certain essential oils. This study focused primarily on the determination of antioxidant, antimicrobial, and enzyme-inhibition activity of aqueous ethanol extract of Turkish endemic Achillea cucullata by in vitro methods. The extract exhibited DPPH radical scavenging activity with an IC50 value of 132.55 ± 0.026 μg/mL, the total phenol content was 53.807 ± 0.059 (mg GAE/g), and the total flavonoid content was 21.372 ± 0.026 (mg QE/g), on the dry-weight basis. Antimicrobial activity was evaluated by a micro-dilution method focused on five microorganisms; two Gram-positive [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)], two Gram-negative [Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)], and one fungal strain [Candida albicans (ATCC 10231)]. Results show that the MIC value for the tested microorganism was higher than 5 mg/mL. In this work, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase enzymes were strongly inhibited by the A. cucullata extract, and the IC50 values for these enzymes were 2.4 μg/mL, 0.26 μg/mL, and 24.75 μg/mL, respectively. Certain acetylcholinesterase inhibitors have been used for treatment of Alzheimer's disease in the past. α-Glucosidase inhibitors are strong drug candidates, as well as potential functional food agents, for deferring the postprandial absorbency of glucose. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Plants are able to synthesize different secondary metabolites, and those metabolites possess the potential for significant biological activity. In traditional medicine, plants are used in many different ways to treat various ailments. Through the use of modern techniques, the traditional use of the plants can be analyzed scientifically, and may have the potential to uncover promising compounds which can be used in the treatment of different diseases (Gülçin, 2012). Due to its unique phytogeographical features, Turkey is very rich in terms of medicinal and aromatic plants. Most of the plants native to Turkey contain medicinal qualities and are widely used by the local ⁎ Corresponding author at: Cumhuriyet University, Faculty of Pharmacy, Department of Pharmacognosy, 58140 Sivas, Turkey. E-mail address: [email protected]. (N. Eruygur).
population as a curative for minor (and sometimes even major) diseases. The genus Achillea L., belonging to the Asteraceae (Compositae) family, is represented by 52 different species and 58 taxa in Turkey, most of which are used in Turkish folk medicine (Davis, 1982). These species are known as “Civanperçemi” locally, and have traditional medicinal uses ranging from topical healing ointments to diuretics, carminatives, and weight-gaining supplements (Arabacı, 2012; Aytaç et al., 2016). In Turkish and Iranian traditional medicine, different species of the genus Achillea are widely used for various carminative, tonic, diaphoretic and diuretic purposes (Mohammadhosseini et al., 2017). These species are also recommended for rheumatic pain, hemorrhage, pneumonia and emphysema, in addition to their analgesic, antipyretic, digestive, anthelmintic, antiulcerative, and antinociceptive uses . As reported in previous studies, the essential oil of Achillea cucullata (Hausskn) Bornm. contains camphor, 1,8-cineole and isoborneol (Toker et al., 2003). The chemical composition of the essential oil of
https://doi.org/10.1016/j.sajb.2018.04.001 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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N. Eruygur et al. / South African Journal of Botany xxx (2018) xxx–xxx
A. cucullata have been reported (Toncer et al., 2010), but despite intensive research on the biological activity of the Achillea species, there was no information regarding the biological activity of the A. cucullata extract included. The objective of our present study is to determine the in vitro antioxidant, antimicrobial, anti-AChE, anti-BChE, and antidiabetic activities of the aqueous ethanol extract prepared from the aerial part of A. cucullata. Alzheimer's Disease (AD) is a neurodegenerative disturbance characterized primarily by advancing memory loss and other cognitive disorders (Turan et al., 2016). AD is associated with oxidative stress, inflammation, and a deficiency in acetylcholine (ACh) levels in particular parts of the brain (Farlow and Cummings, 2007; Seebaluck-Sandoram et al., 2017). Therefore, the use of antioxidants can potentially slow down AD progression and neurodegeneration (Amoo et al., 2011). The therapy of AD currently depends on the Cholinergic Theory, which hypothesizes that the reduction in ACh can have a significant effect on lowering the number of incidences of AD (Bartus et al., 1982; Garibov et al., 2016). Acetylcholinesterase (AChE), a serine protease which can hydrolyze ACh generation, leading to a reduction in ACh and disruption of the nervous signal transmission, contributes to the creation of AD (Aksu et al., 2016; Taslimi et al., 2017). Therefore, AChE is defined as a goal of AD therapy. AChE inhibitor (AChEI) compounds can prevent the activity of AChE and reduce the degradation of ACh, as well as some AChEI drugs, such as rivastigmine, tacrine, and donepezil, which have been recorded by the Food and Drug Administration (FDA) (Aktaş et al., 2017). These drugs, however, also have several negative factors, such as low activity, low selectivity, and toxicity (Bayrak et al., 2017). Thus, novel drugs with low toxicity, high selectivity, and high activity are essential to treat AD. Butyrylcholinesterase (BChE) is another serine hydrolase that depends on AChE. For BChE, butyrylcholine (BCh) is the best substrate—although, physiologically, neither exists naturally essential for humans (Öztaşkın et al., 2015; Kocyigit et al., 2017). The function of BChE has not yet been entirely explained. Definitive selective BChE inhibitors (BChEIs) have been defined to enhance ACh concentrations and to reduce the production of abnormal amyloid that originates in AD (Gül et al., 2017). Diabetes mellitus (DM) is a metabolic disturbance caused by hyperglycemia, the result of either impaired insulin secretion (insulin deficiency) or impaired insulin action (insulin resistance) (Choi et al., 2005). According to the International Diabetes Federation (IDF), around 435 million people globally suffered from type II diabetes mellitus in 2016 (T2DM) (Inyushkina et al., 2007). In this regard, plants used in traditional medicines have proven to be precious sources of chemical factors and phytotherapeutic preparations for the extension of novel antidiabetic drugs (Kang et al., 2012). Another section of our work is to record the effect of selected plants for treating diabetic disease and to discover novel α-glucosidase inhibitors (AGIs), beneficial for the extension of novel antidiabetic therapies, which is recorded through the evaluation of A. cucullata. In this study we aimed to determine the antimicrobial and antioxidant activity and BChE, AChE, and α-glucosidase enzyme inhibition properties of A. cucullata. 2. Materials and methods 2.1. Chemicals 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), quercetin, gallic acid, ascorbic acid, acetylcholinesterase, butyrylcholinesterase, ɑ-amylase, and α-glucosidase were obtained from Sigma Aldrich Co., St. Louis, USA. All other chemicals used were of analytical grade.
of Sivas, Turkey at its flowering season on June 22, 2016. Collection and taxonomic identification of the plant were performed by Dr. Mehmet Tekin, and herbarium materials were deposited in the Herbarium of Cumhuriyet University, Faculty of Science (CUFH) under herbarium code M. Tekin 1734. 2.3. Preparation of the extract The extraction was performed at room temperature after samples were air-dried and finely powdered using a grinder. One hundred gram of aerial parts of A. cucullata were macerated in 80% ethanol (1000 mL) for 48 h with intermittent shaking. Then extract was filtered through Whatman filter paper No.1. and then concentrated under vacuum on a rotary evaporator (Buchi R-100 equipped with Vacuum Pump V-300 and Control unit I-300) at 40 °C to constant dryness (yield: 22.18%, w/w), and stored at −20 °C for further use. 2.4. Antioxidant activities 2.4.1. DPPH free radical scavenging activity The free radical scavenging activity of the extracts was evaluated with the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) by the method of Blois (Blois, 1958). Three milliliters of sample solution or standard solution in various concentrations was added to 1 mL of 0.1 mM DPPH solution prepared in methanol. The mixture was shook vigorously, then was allowed to stand in the dark for 30 min and optical density was measured at 517 nm using a UV–VIS Spectrophotometer. Methanol (3 mL) with DPPH solution (0.1 mM, 1 mL) was used as blank. Methanol was used for base line corrections in absorbance of sample. The optical density was recorded and % inhibition was calculated by the formula given below: Percent ð%Þ inhibition of DPPH activity ¼ ðAbsorbance of Blank−Absorbance of TestÞ∕Absorbance of Blank 100 2.4.2. Determination of total phenolic contents The determination of total phenolic content was performed by the Folin–Ciocalteu method with slight modifications (Ainsworth and Gillespie, 2007). A dilute solution of A. cucullata extract or gallic acid was mixed with Folin–Ciocalteu reagent previously diluted with water ten times and 7% Na2CO3 solution. The mixture was allowed to stand for 2 h and the samples were read at 730 nm in a spectrophotometer. The total phenolic content was expressed in milligrams equivalents of gallic acid (GAE) per gram of extract. The equation obtained for the calibration curve of gallic acid the range of 0.1–1 mg/mL. 2.4.3. Determination of total flavonoid contents Aluminum chloride colorimetric method was used for determination of total flavonoids according to the Chang method (Chang et al., 2002). 0.5 mL of A. cucullata extract (2 mg/mL in ethanol) was mixed separately with 1.5 mL ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M sodium acetate, and 2.8 mL of distilled water. The mixture was kept at room temperature for 30 min. The absorbance of the reaction mixture was determined by a spectrophotometer at 415 nm. Ethanol was used as a blank. The experiments were conducted in triplicate. The calibration curve of quercetin was obtained in the range of 0.0625–1.0 mg/mL. The content of flavonoids was established as quercetin mg/g dry extract. 2.5. Antimicrobial activities
2.2. Plant materials The aerial parts of A. cucullata were collected from the gypseous area of Akkaya village, 1402 m, 39° 32′ 41,3″ N; 37° 03′ 47,5″ E, Ulaş district
2.5.1. Micro-well dilution assay In order to determine the minimum inhibitory concentration (MIC) of A. cucullata extract, the broth micro-dilution method was used as
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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recommended by NCCLS (Nccls, 2002; CLSI, 2012). The antimicrobial activity of the extract was tested against bacterial (Gram positive: Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212), Gram-negative: Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)) and fungal strain: Candida albicans (ATCC 10231). Plant extracts were dissolved in 8% DMSO to prepare 20 mg/mL of stock solutions. A serial two-fold dilution was prepared in a 96-well microtiter plate (concentration range 5.00 to 0.02 μg/mL), positive control was Gentamicin for bacteria and Fluconazole for Candida respectively. 11th well was added 2% DMSO used as growth control and 12th well was added 50 μL Mueller Hinton Broth (sterility control). Final inoculum size was 5 × 105 CFU/mL for bacteria and 0.5–2.5 × 103 CFU/mL for Candida in each well. Mueller Hinton Broth and Saboraud Dextrose Broth was used for dilution bacteria and Candida culture's, respectively. Microtiter plates were incubated at 37 °C for bacteria and 35 °C for Candida between 16 and 24 h. Afterwards, 50 μL 2 mg/mL of 2,3,5-Triphenyltetrazolium chloride (TTC) (Merck, Germany) was added to each well to observe microbial growth. The microplates were further incubated at 37 °C for 2 h. Reduction in density of red color of formazan after incubation was accepted as MIC value. The experiment was performed in duplicate and the standard deviation was zero.
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Table 1 Antioxidant activities of A. cucullata aerial part ethanol extract. Extract/positive control
A. cucullata Gallic acid
DPPH
Total phenol content (TPC)
Total flavonoid content (TFC)
IC50 (μg/mL)
mg GAE/g
mg QE/g
132.55 ± 0.026 7.548 ± 0.047
53.807 ± 0.059 –
21.372 ± 0.026 –
radical scavenging activity had an IC50 value of 132.55 ± 0.026 μg/mL. In a previous report on Achillea schischkinii Sosn. ex. Grossh. and Achillea teretifolia Wild., it was found that methanol extract showed 68% and 69.2% activity at 100 μg doses (Turkoglu et al., 2010). Flavonoids are phenolic compounds isolated from a wide range of plants, acts as antioxidant, antimicrobial, feeding repellants and for light screening. Much interest has been focused on the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals (Pietta, 2000). The total phenol content was found as 53.807 ± 0.059 (mg GAE/g) and total flavonoid content was calculated as 21.372 ± 0.026 (mg QE/g) on the dry weight basis.
2.6. AChE/BChE activity determination 3.2. Antimicrobial activity The BChE/AChE inhibitory effect of A. cucullata was determined by the procedure of Ellman et al. (1961). Butyrylthiocholine iodide and Acetylthiocholine iodide (BChI/AChI) were used as substrates for both reactions. Briefly, 100 μL of Tris/HCl buffer (1 M, pH 8.0), 750 μL of sample solution dissolved in deionized water at different concentrations and 50 μL BChE/AChE (5.32 × 10−3 U) solution were mixed and incubated for 8 min at 20 °C (Alzheimer's Association, 2015). Then 50 μL of DTNB (0.5 mM) was added. 5,5´-Dithio-bis(2nitro-benzoic) acid (DTNB) was used for the measurement of the AChE/BChE activities. The reaction was then initiated by the addition of 50 μL of BChI/AChI (Giacobini, 2003) The hydrolysis of these substrates of BChI/AChI was monitored at a wavelength of 412 nm (Silman and Sussman, 2005). 2.7. α-Glucosidase inhibition assay α-Glucosidase inhibitory efficacy of A. cucullata was performed using p-nitrophenyl-D-glycopyranoside (P-NPG) as the substrate, according to the procedure of Tao et al. (2013). Samples were prepared by dissolving 10 mg in 10 mL (EtOH:H2O). First, 100 μL of phosphate buffer was mixed with 20 μL of the enzyme solution in phosphate buffer (0.15 U/mL, pH 7.4) and 10–100 μL of the sample. Multiple solutions in phosphate buffer were prepared in case of getting full enzyme inhibition. Then it was pre-incubated at 35 °C for 12 min previous by adding the PNPG to the initiation of the reaction. Also, 50 μL of p-NPG in phosphate buffer (5 mM, pH 7.4) after preincubation was added and again incubated at 37 °C (Madigan et al., 2000). The absorbance was spectrophotometrically measured at 405 nm. The IC50 amount was calculated from activity (%) versus plant concentration plots (Athar et al., 2007). 3. Results and discussion 3.1. Antioxidant activity Scavenging activity for free radicals of DPPH has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources due to its shortness in analysis time compared to other methods (Soares et al., 1997). Free radical scavenging activity of ethanolic extract from the herbs of A. cucullata was quantitatively determined using DPPH assay (Table 1). The scavenging ability of the samples showed a concentration dependent activity profile. DPPH
Agar diffusion techniques are widely used to determine plant extracts for antimicrobial activity, but there are problems with this method in terms of extracts with undefined components, leading to false positive and negative results (Eloff, 1998). In this study, a serial dilution technique using 96-well microplates was used to evaluate antimicrobial activity of A. cucullata ethanol extract. MIC values were detected with broth micro-dilution assay. According to the results, A. cucullata ethanol extract showed weak inhibitory effects toward most tested bacteria and fungi, the MIC values of the extracts against all the tested microorganisms were higher than 5 mg/mL (Table 2). Nevertheless, the strong antimicrobial activity was observed in other reports about 11 Achillea species flower head extract, most of which showed antimicrobial activity; the MIC values were lower than 250 μg/mL (Karaalp et al., 2009). 3.3. Enzymes results In our study, AChE, BChE, and α-glucosidase enzymes were efficiently inhibited by A. cucullata plant (Fig. 1 and Table 3). AChEIs, such as huperzine A, donepezil, rivastigmine, and galantamine have been obviously considered as symptomatic treatments for AD. By enhancing the amounts of ACh in the brain cells via its cholinesterase inhibitor (ChEI) activity, the studied plant in the present paper and tacrine (TAC) are postulated to reverse the cholinergic shortage related to AD (Sujayev et al., 2016). In addition, although there are many potential drug purposes for AD therapy, AChEIs are now defined as the significant therapeutic factors applied clinically for AD (Ozbey et al., 2016). Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a reversible inhibitor of BChE and AChE and the first drug to be authorized for the placative treatment of AD (Taslimi et al., 2017). IC50 values obtained for these enzymes were: 2.4 μg/mL for AChE, 0.26 μg/mL for BChE, and 24.75 μg/mL for α-glucosidase. In addition,
Table 2 Antimicrobial activities of A. cucullata aerial part ethanol extract (MIC values, mg/mL). Ethanol extract
E. coli
S. aureus
P. aeruginosa
E. faecalis
C. albicans
A. cucullata
N5
N5
N5
N5
N5
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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4. Conclusion Phenolics contain the identical general structure comprised of an aromatic hydroxyl core and include an approximated number of 8000 in nature (Köksal et al., 2016; Hamad et al., 2017). So far, plant phenolic compounds create one of the main groups of molecules acting as primary free radical terminators or antioxidants (Kalın et al., 2015; Bae et al., 2016; Tohma et al., 2016; Topal et al., 2016). Plant polyphenols are multifunctional in the way that they can perform as hydrogen atom donators, reducing agents, and singlet oxygen scavengers (Işik et al., 2015). Antioxidant properties (particularly radical scavenging activities such as DPPH· and ABTS) are significant due to the disadvantageous role of free radicals in biological systems and foods (Cakmakcı et al., 2015; Sehitoglu et al., 2015). Extreme generations of free radicals accelerate the oxidation of lipid molecules in foods and reduce consumer acceptance and food quality (Bursal et al., 2013; Topal et al., 2016). The results of this work recorded that A. cucullata can be used as a significant source of natural antioxidants, as well as an important food supplement and a potentially significant advantage in the pharmaceutical industry. αGlucosidase inhibitors are also strong drug candidates, as well as potential functional food factors for deferring postprandial absorbency of glucose. Based on the diverse causative agents of AD, various hypotheses, including tau-based therapies (AChEI, amyloid, cholinergic, amyloid targeted strategies, anti-inflammatory therapy) have been used in the treatment of AD (Moretto, 1998). Thus, antioxidant capability is extensively used as a main parameter to characterize food or pharmaceutical plants and their bioactive components. AChEIs, induced by various plants contain with natural compounds, leads to ACh agglomeration, muscarinic receptors, and hyper stimulation of nicotinic, and disordered neurotransmission. As such, AChEIs – interacting with the enzyme as their significant purpose – are applied as relevant toxins and drugs (AGIs). As drugs help lower blood sugar amounts in the body by disturbing the hydrolyzation of starchy foods like potatoes and bread in the intestine (and thus delaying the absorption of carbohydrates, conforming to the American Diabetes Association), they also slow down the breakdown of some sugars. Because they work to slow digestion, AGIs are taken at the beginning of a meal. The results from this work show that the ethanol extract from aerial parts of A. cucullata exhibited strong antioxidant, anticholinesterase, antidiabetic and moderate antimicrobial activities. It indicates that the data obtained from this study could serve as basic scientific research data for possible use of this plant as a source of natural antidiabetic, anti-AD and antioxidant agents in the future. Fig. 1. The IC50 graphs of ethanol extract of A. cucullata against AChE (A), BChE (B) and α-glucosidase (C) enzymes.
tacrine (TAC) was used as positive standard BChE and AChE inhibitor and it had IC50 values of 19.11 μmol/L and 12.36 μmol/L, respectively. The α-glucosidase inhibitors acting against enzymes in the intestine cells have been defined to efficiently reprieve the blood glucose level, reducing glucose absorption (Noh et al., 2011).
Table 3 Half maximal inhibition concentration (IC50, μg/mL) of A. cucullata (Ethanol extract) and standard compounds (Tacrine and Acarbose) against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase. Compounds
A. cucullata Tacrinea Acarboseb a b
α-Glucosidase
AChE
BChE
IC50 (μg/mL)
R2
IC50 (μg/mL)
R2
IC50 (μg/mL)
R2
24.75 – 22.8
0.9778 – –
2.40 12.36 –
0.990 0.958 –
0.26 19.11 –
0.975 0.981 –
Tacrine was used as positive control for AChE and BChE and determined as μM levels. Acarbose was used as positive control for α-glucosidase and determined as μM levels.
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Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract N. Eruygur a,b,⁎, U.M. Koçyiğit c, P. Taslimi d, M. Ataş e, M. Tekin f, İ. Gülçin d a
Cumhuriyet University, Faculty of Pharmacy, Department of Pharmacognosy, Sivas, Turkey Selçuk University, Faculty of Pharmacy, Department of Pharmacognosy, Konya, Turkey c Cumhuriyet University, Vocational School of Health Services, Sivas, Turkey d Ataturk University, Faculty of Sciences, Department of Chemistry, Erzurum, Turkey e Cumhuriyet University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Sivas, Turkey f Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Edirne, Turkey b
a r t i c l e
i n f o
Article history: Received 10 January 2018 Received in revised form 9 March 2018 Accepted 2 April 2018 Available online xxxx Edited by Kannan Ragupathi Raja Rengasamy Keywords: Achillea cucullata Antioxidant activity Antimicrobial activity Anticholinesterase activity Antidiabetic activity
a b s t r a c t The Achillea genus belongs to the Asteraceae family, which is mostly found in the northern hemisphere and is comprised of 115 species in the world. In Turkish flora, there are 52 species and 58 taxa, among them half of which are recorded as endemic. To the best of our knowledge, there has been no biological activity studied in this species until now, with the exception of one study of the antimicrobial activity of certain essential oils. This study focused primarily on the determination of antioxidant, antimicrobial, and enzyme-inhibition activity of aqueous ethanol extract of Turkish endemic Achillea cucullata by in vitro methods. The extract exhibited DPPH radical scavenging activity with an IC50 value of 132.55 ± 0.026 μg/mL, the total phenol content was 53.807 ± 0.059 (mg GAE/g), and the total flavonoid content was 21.372 ± 0.026 (mg QE/g), on the dry-weight basis. Antimicrobial activity was evaluated by a micro-dilution method focused on five microorganisms; two Gram-positive [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)], two Gram-negative [Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)], and one fungal strain [Candida albicans (ATCC 10231)]. Results show that the MIC value for the tested microorganism was higher than 5 mg/mL. In this work, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase enzymes were strongly inhibited by the A. cucullata extract, and the IC50 values for these enzymes were 2.4 μg/mL, 0.26 μg/mL, and 24.75 μg/mL, respectively. Certain acetylcholinesterase inhibitors have been used for treatment of Alzheimer's disease in the past. α-Glucosidase inhibitors are strong drug candidates, as well as potential functional food agents, for deferring the postprandial absorbency of glucose. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Plants are able to synthesize different secondary metabolites, and those metabolites possess the potential for significant biological activity. In traditional medicine, plants are used in many different ways to treat various ailments. Through the use of modern techniques, the traditional use of the plants can be analyzed scientifically, and may have the potential to uncover promising compounds which can be used in the treatment of different diseases (Gülçin, 2012). Due to its unique phytogeographical features, Turkey is very rich in terms of medicinal and aromatic plants. Most of the plants native to Turkey contain medicinal qualities and are widely used by the local ⁎ Corresponding author at: Cumhuriyet University, Faculty of Pharmacy, Department of Pharmacognosy, 58140 Sivas, Turkey. E-mail address: [email protected]. (N. Eruygur).
population as a curative for minor (and sometimes even major) diseases. The genus Achillea L., belonging to the Asteraceae (Compositae) family, is represented by 52 different species and 58 taxa in Turkey, most of which are used in Turkish folk medicine (Davis, 1982). These species are known as “Civanperçemi” locally, and have traditional medicinal uses ranging from topical healing ointments to diuretics, carminatives, and weight-gaining supplements (Arabacı, 2012; Aytaç et al., 2016). In Turkish and Iranian traditional medicine, different species of the genus Achillea are widely used for various carminative, tonic, diaphoretic and diuretic purposes (Mohammadhosseini et al., 2017). These species are also recommended for rheumatic pain, hemorrhage, pneumonia and emphysema, in addition to their analgesic, antipyretic, digestive, anthelmintic, antiulcerative, and antinociceptive uses . As reported in previous studies, the essential oil of Achillea cucullata (Hausskn) Bornm. contains camphor, 1,8-cineole and isoborneol (Toker et al., 2003). The chemical composition of the essential oil of
https://doi.org/10.1016/j.sajb.2018.04.001 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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A. cucullata have been reported (Toncer et al., 2010), but despite intensive research on the biological activity of the Achillea species, there was no information regarding the biological activity of the A. cucullata extract included. The objective of our present study is to determine the in vitro antioxidant, antimicrobial, anti-AChE, anti-BChE, and antidiabetic activities of the aqueous ethanol extract prepared from the aerial part of A. cucullata. Alzheimer's Disease (AD) is a neurodegenerative disturbance characterized primarily by advancing memory loss and other cognitive disorders (Turan et al., 2016). AD is associated with oxidative stress, inflammation, and a deficiency in acetylcholine (ACh) levels in particular parts of the brain (Farlow and Cummings, 2007; Seebaluck-Sandoram et al., 2017). Therefore, the use of antioxidants can potentially slow down AD progression and neurodegeneration (Amoo et al., 2011). The therapy of AD currently depends on the Cholinergic Theory, which hypothesizes that the reduction in ACh can have a significant effect on lowering the number of incidences of AD (Bartus et al., 1982; Garibov et al., 2016). Acetylcholinesterase (AChE), a serine protease which can hydrolyze ACh generation, leading to a reduction in ACh and disruption of the nervous signal transmission, contributes to the creation of AD (Aksu et al., 2016; Taslimi et al., 2017). Therefore, AChE is defined as a goal of AD therapy. AChE inhibitor (AChEI) compounds can prevent the activity of AChE and reduce the degradation of ACh, as well as some AChEI drugs, such as rivastigmine, tacrine, and donepezil, which have been recorded by the Food and Drug Administration (FDA) (Aktaş et al., 2017). These drugs, however, also have several negative factors, such as low activity, low selectivity, and toxicity (Bayrak et al., 2017). Thus, novel drugs with low toxicity, high selectivity, and high activity are essential to treat AD. Butyrylcholinesterase (BChE) is another serine hydrolase that depends on AChE. For BChE, butyrylcholine (BCh) is the best substrate—although, physiologically, neither exists naturally essential for humans (Öztaşkın et al., 2015; Kocyigit et al., 2017). The function of BChE has not yet been entirely explained. Definitive selective BChE inhibitors (BChEIs) have been defined to enhance ACh concentrations and to reduce the production of abnormal amyloid that originates in AD (Gül et al., 2017). Diabetes mellitus (DM) is a metabolic disturbance caused by hyperglycemia, the result of either impaired insulin secretion (insulin deficiency) or impaired insulin action (insulin resistance) (Choi et al., 2005). According to the International Diabetes Federation (IDF), around 435 million people globally suffered from type II diabetes mellitus in 2016 (T2DM) (Inyushkina et al., 2007). In this regard, plants used in traditional medicines have proven to be precious sources of chemical factors and phytotherapeutic preparations for the extension of novel antidiabetic drugs (Kang et al., 2012). Another section of our work is to record the effect of selected plants for treating diabetic disease and to discover novel α-glucosidase inhibitors (AGIs), beneficial for the extension of novel antidiabetic therapies, which is recorded through the evaluation of A. cucullata. In this study we aimed to determine the antimicrobial and antioxidant activity and BChE, AChE, and α-glucosidase enzyme inhibition properties of A. cucullata. 2. Materials and methods 2.1. Chemicals 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), quercetin, gallic acid, ascorbic acid, acetylcholinesterase, butyrylcholinesterase, ɑ-amylase, and α-glucosidase were obtained from Sigma Aldrich Co., St. Louis, USA. All other chemicals used were of analytical grade.
of Sivas, Turkey at its flowering season on June 22, 2016. Collection and taxonomic identification of the plant were performed by Dr. Mehmet Tekin, and herbarium materials were deposited in the Herbarium of Cumhuriyet University, Faculty of Science (CUFH) under herbarium code M. Tekin 1734. 2.3. Preparation of the extract The extraction was performed at room temperature after samples were air-dried and finely powdered using a grinder. One hundred gram of aerial parts of A. cucullata were macerated in 80% ethanol (1000 mL) for 48 h with intermittent shaking. Then extract was filtered through Whatman filter paper No.1. and then concentrated under vacuum on a rotary evaporator (Buchi R-100 equipped with Vacuum Pump V-300 and Control unit I-300) at 40 °C to constant dryness (yield: 22.18%, w/w), and stored at −20 °C for further use. 2.4. Antioxidant activities 2.4.1. DPPH free radical scavenging activity The free radical scavenging activity of the extracts was evaluated with the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) by the method of Blois (Blois, 1958). Three milliliters of sample solution or standard solution in various concentrations was added to 1 mL of 0.1 mM DPPH solution prepared in methanol. The mixture was shook vigorously, then was allowed to stand in the dark for 30 min and optical density was measured at 517 nm using a UV–VIS Spectrophotometer. Methanol (3 mL) with DPPH solution (0.1 mM, 1 mL) was used as blank. Methanol was used for base line corrections in absorbance of sample. The optical density was recorded and % inhibition was calculated by the formula given below: Percent ð%Þ inhibition of DPPH activity ¼ ðAbsorbance of Blank−Absorbance of TestÞ∕Absorbance of Blank 100 2.4.2. Determination of total phenolic contents The determination of total phenolic content was performed by the Folin–Ciocalteu method with slight modifications (Ainsworth and Gillespie, 2007). A dilute solution of A. cucullata extract or gallic acid was mixed with Folin–Ciocalteu reagent previously diluted with water ten times and 7% Na2CO3 solution. The mixture was allowed to stand for 2 h and the samples were read at 730 nm in a spectrophotometer. The total phenolic content was expressed in milligrams equivalents of gallic acid (GAE) per gram of extract. The equation obtained for the calibration curve of gallic acid the range of 0.1–1 mg/mL. 2.4.3. Determination of total flavonoid contents Aluminum chloride colorimetric method was used for determination of total flavonoids according to the Chang method (Chang et al., 2002). 0.5 mL of A. cucullata extract (2 mg/mL in ethanol) was mixed separately with 1.5 mL ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M sodium acetate, and 2.8 mL of distilled water. The mixture was kept at room temperature for 30 min. The absorbance of the reaction mixture was determined by a spectrophotometer at 415 nm. Ethanol was used as a blank. The experiments were conducted in triplicate. The calibration curve of quercetin was obtained in the range of 0.0625–1.0 mg/mL. The content of flavonoids was established as quercetin mg/g dry extract. 2.5. Antimicrobial activities
2.2. Plant materials The aerial parts of A. cucullata were collected from the gypseous area of Akkaya village, 1402 m, 39° 32′ 41,3″ N; 37° 03′ 47,5″ E, Ulaş district
2.5.1. Micro-well dilution assay In order to determine the minimum inhibitory concentration (MIC) of A. cucullata extract, the broth micro-dilution method was used as
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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recommended by NCCLS (Nccls, 2002; CLSI, 2012). The antimicrobial activity of the extract was tested against bacterial (Gram positive: Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212), Gram-negative: Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)) and fungal strain: Candida albicans (ATCC 10231). Plant extracts were dissolved in 8% DMSO to prepare 20 mg/mL of stock solutions. A serial two-fold dilution was prepared in a 96-well microtiter plate (concentration range 5.00 to 0.02 μg/mL), positive control was Gentamicin for bacteria and Fluconazole for Candida respectively. 11th well was added 2% DMSO used as growth control and 12th well was added 50 μL Mueller Hinton Broth (sterility control). Final inoculum size was 5 × 105 CFU/mL for bacteria and 0.5–2.5 × 103 CFU/mL for Candida in each well. Mueller Hinton Broth and Saboraud Dextrose Broth was used for dilution bacteria and Candida culture's, respectively. Microtiter plates were incubated at 37 °C for bacteria and 35 °C for Candida between 16 and 24 h. Afterwards, 50 μL 2 mg/mL of 2,3,5-Triphenyltetrazolium chloride (TTC) (Merck, Germany) was added to each well to observe microbial growth. The microplates were further incubated at 37 °C for 2 h. Reduction in density of red color of formazan after incubation was accepted as MIC value. The experiment was performed in duplicate and the standard deviation was zero.
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Table 1 Antioxidant activities of A. cucullata aerial part ethanol extract. Extract/positive control
A. cucullata Gallic acid
DPPH
Total phenol content (TPC)
Total flavonoid content (TFC)
IC50 (μg/mL)
mg GAE/g
mg QE/g
132.55 ± 0.026 7.548 ± 0.047
53.807 ± 0.059 –
21.372 ± 0.026 –
radical scavenging activity had an IC50 value of 132.55 ± 0.026 μg/mL. In a previous report on Achillea schischkinii Sosn. ex. Grossh. and Achillea teretifolia Wild., it was found that methanol extract showed 68% and 69.2% activity at 100 μg doses (Turkoglu et al., 2010). Flavonoids are phenolic compounds isolated from a wide range of plants, acts as antioxidant, antimicrobial, feeding repellants and for light screening. Much interest has been focused on the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals (Pietta, 2000). The total phenol content was found as 53.807 ± 0.059 (mg GAE/g) and total flavonoid content was calculated as 21.372 ± 0.026 (mg QE/g) on the dry weight basis.
2.6. AChE/BChE activity determination 3.2. Antimicrobial activity The BChE/AChE inhibitory effect of A. cucullata was determined by the procedure of Ellman et al. (1961). Butyrylthiocholine iodide and Acetylthiocholine iodide (BChI/AChI) were used as substrates for both reactions. Briefly, 100 μL of Tris/HCl buffer (1 M, pH 8.0), 750 μL of sample solution dissolved in deionized water at different concentrations and 50 μL BChE/AChE (5.32 × 10−3 U) solution were mixed and incubated for 8 min at 20 °C (Alzheimer's Association, 2015). Then 50 μL of DTNB (0.5 mM) was added. 5,5´-Dithio-bis(2nitro-benzoic) acid (DTNB) was used for the measurement of the AChE/BChE activities. The reaction was then initiated by the addition of 50 μL of BChI/AChI (Giacobini, 2003) The hydrolysis of these substrates of BChI/AChI was monitored at a wavelength of 412 nm (Silman and Sussman, 2005). 2.7. α-Glucosidase inhibition assay α-Glucosidase inhibitory efficacy of A. cucullata was performed using p-nitrophenyl-D-glycopyranoside (P-NPG) as the substrate, according to the procedure of Tao et al. (2013). Samples were prepared by dissolving 10 mg in 10 mL (EtOH:H2O). First, 100 μL of phosphate buffer was mixed with 20 μL of the enzyme solution in phosphate buffer (0.15 U/mL, pH 7.4) and 10–100 μL of the sample. Multiple solutions in phosphate buffer were prepared in case of getting full enzyme inhibition. Then it was pre-incubated at 35 °C for 12 min previous by adding the PNPG to the initiation of the reaction. Also, 50 μL of p-NPG in phosphate buffer (5 mM, pH 7.4) after preincubation was added and again incubated at 37 °C (Madigan et al., 2000). The absorbance was spectrophotometrically measured at 405 nm. The IC50 amount was calculated from activity (%) versus plant concentration plots (Athar et al., 2007). 3. Results and discussion 3.1. Antioxidant activity Scavenging activity for free radicals of DPPH has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources due to its shortness in analysis time compared to other methods (Soares et al., 1997). Free radical scavenging activity of ethanolic extract from the herbs of A. cucullata was quantitatively determined using DPPH assay (Table 1). The scavenging ability of the samples showed a concentration dependent activity profile. DPPH
Agar diffusion techniques are widely used to determine plant extracts for antimicrobial activity, but there are problems with this method in terms of extracts with undefined components, leading to false positive and negative results (Eloff, 1998). In this study, a serial dilution technique using 96-well microplates was used to evaluate antimicrobial activity of A. cucullata ethanol extract. MIC values were detected with broth micro-dilution assay. According to the results, A. cucullata ethanol extract showed weak inhibitory effects toward most tested bacteria and fungi, the MIC values of the extracts against all the tested microorganisms were higher than 5 mg/mL (Table 2). Nevertheless, the strong antimicrobial activity was observed in other reports about 11 Achillea species flower head extract, most of which showed antimicrobial activity; the MIC values were lower than 250 μg/mL (Karaalp et al., 2009). 3.3. Enzymes results In our study, AChE, BChE, and α-glucosidase enzymes were efficiently inhibited by A. cucullata plant (Fig. 1 and Table 3). AChEIs, such as huperzine A, donepezil, rivastigmine, and galantamine have been obviously considered as symptomatic treatments for AD. By enhancing the amounts of ACh in the brain cells via its cholinesterase inhibitor (ChEI) activity, the studied plant in the present paper and tacrine (TAC) are postulated to reverse the cholinergic shortage related to AD (Sujayev et al., 2016). In addition, although there are many potential drug purposes for AD therapy, AChEIs are now defined as the significant therapeutic factors applied clinically for AD (Ozbey et al., 2016). Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a reversible inhibitor of BChE and AChE and the first drug to be authorized for the placative treatment of AD (Taslimi et al., 2017). IC50 values obtained for these enzymes were: 2.4 μg/mL for AChE, 0.26 μg/mL for BChE, and 24.75 μg/mL for α-glucosidase. In addition,
Table 2 Antimicrobial activities of A. cucullata aerial part ethanol extract (MIC values, mg/mL). Ethanol extract
E. coli
S. aureus
P. aeruginosa
E. faecalis
C. albicans
A. cucullata
N5
N5
N5
N5
N5
Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001
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4. Conclusion Phenolics contain the identical general structure comprised of an aromatic hydroxyl core and include an approximated number of 8000 in nature (Köksal et al., 2016; Hamad et al., 2017). So far, plant phenolic compounds create one of the main groups of molecules acting as primary free radical terminators or antioxidants (Kalın et al., 2015; Bae et al., 2016; Tohma et al., 2016; Topal et al., 2016). Plant polyphenols are multifunctional in the way that they can perform as hydrogen atom donators, reducing agents, and singlet oxygen scavengers (Işik et al., 2015). Antioxidant properties (particularly radical scavenging activities such as DPPH· and ABTS) are significant due to the disadvantageous role of free radicals in biological systems and foods (Cakmakcı et al., 2015; Sehitoglu et al., 2015). Extreme generations of free radicals accelerate the oxidation of lipid molecules in foods and reduce consumer acceptance and food quality (Bursal et al., 2013; Topal et al., 2016). The results of this work recorded that A. cucullata can be used as a significant source of natural antioxidants, as well as an important food supplement and a potentially significant advantage in the pharmaceutical industry. αGlucosidase inhibitors are also strong drug candidates, as well as potential functional food factors for deferring postprandial absorbency of glucose. Based on the diverse causative agents of AD, various hypotheses, including tau-based therapies (AChEI, amyloid, cholinergic, amyloid targeted strategies, anti-inflammatory therapy) have been used in the treatment of AD (Moretto, 1998). Thus, antioxidant capability is extensively used as a main parameter to characterize food or pharmaceutical plants and their bioactive components. AChEIs, induced by various plants contain with natural compounds, leads to ACh agglomeration, muscarinic receptors, and hyper stimulation of nicotinic, and disordered neurotransmission. As such, AChEIs – interacting with the enzyme as their significant purpose – are applied as relevant toxins and drugs (AGIs). As drugs help lower blood sugar amounts in the body by disturbing the hydrolyzation of starchy foods like potatoes and bread in the intestine (and thus delaying the absorption of carbohydrates, conforming to the American Diabetes Association), they also slow down the breakdown of some sugars. Because they work to slow digestion, AGIs are taken at the beginning of a meal. The results from this work show that the ethanol extract from aerial parts of A. cucullata exhibited strong antioxidant, anticholinesterase, antidiabetic and moderate antimicrobial activities. It indicates that the data obtained from this study could serve as basic scientific research data for possible use of this plant as a source of natural antidiabetic, anti-AD and antioxidant agents in the future. Fig. 1. The IC50 graphs of ethanol extract of A. cucullata against AChE (A), BChE (B) and α-glucosidase (C) enzymes.
tacrine (TAC) was used as positive standard BChE and AChE inhibitor and it had IC50 values of 19.11 μmol/L and 12.36 μmol/L, respectively. The α-glucosidase inhibitors acting against enzymes in the intestine cells have been defined to efficiently reprieve the blood glucose level, reducing glucose absorption (Noh et al., 2011).
Table 3 Half maximal inhibition concentration (IC50, μg/mL) of A. cucullata (Ethanol extract) and standard compounds (Tacrine and Acarbose) against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase. Compounds
A. cucullata Tacrinea Acarboseb a b
α-Glucosidase
AChE
BChE
IC50 (μg/mL)
R2
IC50 (μg/mL)
R2
IC50 (μg/mL)
R2
24.75 – 22.8
0.9778 – –
2.40 12.36 –
0.990 0.958 –
0.26 19.11 –
0.975 0.981 –
Tacrine was used as positive control for AChE and BChE and determined as μM levels. Acarbose was used as positive control for α-glucosidase and determined as μM levels.
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Please cite this article as: Eruygur, N., et al., Screening the in vitro antioxidant, antimicrobial, anticholinesterase, antidiabetic activities of endemic Achillea cucullata (Asteraceae) ethanol extract..., South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.04.001