J Food Biochem. 2020;44:e13128. wileyonlinelibrary.com/journal/jfbc | 1 of 11 https://doi.org/10.1111/jfbc.13128
© 2019 Wiley Periodicals, Inc.
1 | INTRODUCTION
The bioactive compounds obtained from plants have been used as the main resource of natural products recently (Babotă et al., 2018;
Bahadori, Kirkan, & Sarikurkcu, 2019; Korga et al., 2017). The herbal products become very popular especially because the uses of syn-thetic drugs have several concerns (Zengin et al., 2018).
For the abovementioned reasons, the aromatic plants are utilized to treat many diseases (Gonçalves et al., 2017). It is known that the high levels Received: 8 October 2019
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Revised: 12 November 2019|
Accepted: 2 December 2019DOI: 10.1111/jfbc.13128
F U L L A R T I C L E
An assessment of phenolic profiles, fatty acid compositions,
and biological activities of two Helichrysum species: H. plicatum
and H. chionophilum
Tuba Acet
1| Kadriye Ozcan
2| Gokhan Zengin
31Department of Genetic and Bioengineering, Faculty of Engineering and Natural Sciences, Gumushane University, Gumushane, Turkey 2Department of Genetic and Bioengineering, Engineering Faculty, Giresun University, Giresun, Turkey 3Deparment of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey Correspondence Gokhan Zengin, Deparment of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey. Email: gokhanzengin@selcuk.edu.tr
Abstract
In the present study, we aimed to search and compare the biological activities of the ethanol (EtOH), methanol (MeOH), and ethylacetate (EtOAc) solvent extracts of the flower, stem, and root parts of two Helichrysum plants (H. chionophilum (Hc) and H.plicatum subsp. plicatum (Hp)). The antioxidant properties were determined by using
(2,2-diphenyl-1-picrylhydrazyl) (DPPH) and ABTS (2,2′-azino-bis(3-ethylbenzothia-zoline-6-sulfonic acid) (ABTS) assays. The enzyme inhibitory effects of the extracts were investigated on butyrylcholinesterase (BChE), acetylcholinesterase (AChE), α-glucosidase, and α-amylase. Palmitic acid (C 16:0) was also determined as major fatty acids in the tested oils (31.21%–67.68%). In both plants, it was found that the EtOAc extracts of the flowers had a strong antioxidant and enzyme inhibitory effect. In conclusion, the results obtained in the present study showed that H. chionophilum and H. plicatum can be seen as a promising source for the natural bioactive com-pounds that can be used in therapeutic applications.
Practical applications
The members of the genus Helichrysum have been widely taken for therapeutic pur-poses in traditional medicine as well as food. In this context, we investigated the chemical characterization and biological activities of two Helichrysum species ex-tracts (H. chionophilum and H. plicatum subsp. plicatum). Antioxidant capacity, enzyme inhibition and anti-microbial effects were tested for biological activities. Chemical characterization was identified by high performance liquid chromatography (HPLC) (for phenolic) and gas chromatography-flame ioanization detector (GC-FID) (for fatty acids). Based on our findings, the species may be valuable for designing novel food products. K E Y W O R D S bioactive compounds, enzymes, Helichrysum, natural agents, phenolics
of free radicals especially reactive oxygen species cause several diseases such as cancer, cardiovascular and neurological diseases, diabetes, inflam-mation, aging, and respiratory problems (Fu et al., 2011; Lee et al., 2010; Maritim, Sanders, & Watkins, 2003; Tepe, Sokmen, Akpulat, & Sokmen, 2005; Tsao & Deng, 2004). The control of production of these radicals, which are harmful to human health, is of vital importance. In this context, the antioxidant compounds in aromatic plants such as phenolic content and flavonoids are useful in scavenging these harmful radicals (Aktumsek, Zengin, Guler, Cakmak, & Duran, 2013; Ebrahimzadeh & Tavassoli, 2015). In Turkish flora, the genus Helichrysum (Asteraceae family) is repre-sented by 27 taxa and 15 of them are endemic to Turkey (Davis, Mill, & Tan, 1988; Guner, Ozhatay, & Ekim, 2000; Sümbül, Göktürk, & Düşen, 2003). These species are mostly known as “altın otu,” “yayla çiçeği,” or “ölmez çiçek” and are substantially used as herbal tea (Baytop, 1997). The members of the genus Helichyrsum are mostly used in traditional medicine for antimicrobial, antioxidant, and anti-inflammatory thera-pies, as well as removing the kidney stone and supporting the wound healing process (Albayrak, Aksoy, Sağdiç, & Budak, 2010; Sala et al., 2002; Sezik et al., 2001). Furthermore, many studies have been car-ried out on the biological activities and phytochemicals of Helichrysum species, including the antioxidant, antimicrobial, anti-inflammatory, cytotoxic, enzyme inhibitory activities related with the diabetes and Alzheimer diseases, as well as the anti-aging properties (Aslan, Orhan, Orhan, Sezik, & Yesilada, 2007; Gonçalves et al., 2017; Gouveia-Figueira et al., 2014; Ozcan & Acet, 2018a; Popoola, Marnewick, Rautenbach, Iwuoha, & Hussein, 2015, Tepe et al., 2005).
In the light of literature search, a few studies were observed on the tested Helichyrsum species. Therefore, the objective of this study is to reveal the phenolic components and biological activities of the Helichyrsum species. At this point, the presented work is the first comprehensive report on the Helichyrsum species. The obtained re-sults will be a springboard for designing new studies on the genus.
2 | MATERIAL AND METHODS
2.1 | Plant specimens
Helichrysum chionophilum Boiss. & Bal. and Helichrysum plicatum DC. subsp. plicatum specimens were collected from 2000-2600 m altitude in Gümüşhane-Turkey in July 2017. Plants were identified by using “Flora of Turkey and the East Aegean Island” (Davis et al., 1988). Identification and confirmation of plant material, as well as issuing of voucher specimen, was done by botanist Dr. Tuba Acet from the Gumushane University (Gumushane, Turkey). Voucher specimens were registered in the Gümüşhane University, Turkey (Herbarium number TA1601 and TA1705, respectively).
2.2 | Preparation of the plant extracts
The plants (the aerial parts) were harvested and after dried pow-dered by using miller (Fritsch P-15, Germany). Then, 20g plants
powder were extracted using approximately 500 mL solvent (EtOH, MeOH, EtOAc) for 1 day at till maximum 40°C and 125 rpm. After filtration of the plant extracts, the solvent was evaporated, and the residuals were stored at −20°C until future tests.
2.3 | Total phenolic (TPC) and flavonoid content
(TFC)
Total phenolicand flavonoid content (TFC) of the plant extracts were evaluated by using colorimetric methods such as the Folin-Ciocalteu (Ozcan & Acet, 2018b; Slinkard & Singleton, 1977) and Aluminum Chloride (Moreno, Isla, Sampietro, & Vattuone, 2000), respectively. While TPC was expressed as gallic acid (mg GAE g−1 extract), TPC
was given as quercetin equivalents (mg QE g-1 extract.
2.4 | Phenolic compound analyses
The phenolic compounds of in the extracts (methanol extracts from aerial parts as mix) were analyzed by using using high performance liquid chromatography (HPLC) (Shimadzu, Japan). Separation was conducted at 30°C by using a reversed phase column (Eclipse XDB C-18) with the length of 250 × 4.6 mm and with 5 m particle size, (United State of America) and the determination at 278 nm.The broad standards that are used are chlorogenic acid, gallic acid, cat-echin, rutin, epicatechin,ferulic acid, syringic acid, trans-cinnamic acid, vanillic acid, caffeic acid, p-coumaric acid, kaempferol, hes-peridin, luteolin, and apigenin. The phenolic substance examinations and the quantitative analyses were realized under the same chroma-tographic conditions by doing few modifications in the method of Caponio, Alloggio, and Gomes (1999). The results of phenolic com-pound examinations were given as mg/g extract.
2.5 | Antioxidant activity
2.5.1 | DPPH method
2,2-diphenyl-1-picrylhydrazyl radical scavenging activities of the extracts were measured by doing some minor modifications to the described by Kirby and Schmidt (1997). Shortly, 125 μL of the extract was mixed with equal volume 0.1 mM DPPH, and then measured at 490 nm after 45 min. The activity of each extract was evaluated as Trolox equivalents (mg TE g−1 extract) (Ozcan & Acet, 2018a).
2.5.2 | ABTS method
2,2′-azino-bis method was realized by using a spectrophotometric method with minor mofification as expressed in Re et al. (1999). Concisely, 160 μl ABTS solution and 80 μL sample were mixed and waited for 6–7 min. The mixture was then analyzed at 750 nm. The
activity of extracts were expressed as Trolox equivalents (mg TE g−1
extract) (Ozcan & Acet, 2018b).
2.6 | Enzyme inhibitory activity
2.6.1 | α-amylase inhibition
Enzyme inhibition analysis of the extracts was performed by using the method prescribed by (Zengin, Locatelli, Carradori, Mocan, & Aktumsek, 2016). All solutions used in the reaction were prepared at equal volumes (25 µL). First, the specimens and α-amylase solutions were gently mixed in a microplate and incubated for 10 min at under 37°C. After this, starch solution (0.05%) was put into the mixture. In the control group, no enzyme was added to the mixture and the mix-ture was incubated for 10 min at 37°C. Later, 1 Molar HCl was inserted for the final reaction. Finally, the plate was added with 100 µL of io-dine-potassium iodide solution. Then, both control and the samples were examined for absorbance by using microplate reader (ELISA) at 595 nm. The results of enzyme inhibition were expressed as millimoles of acarbose equivalents (mmolACE g−1 extract).
2.6.2 | α-glucosidase inhibition
The inhibition activity of extracts was analyzed by employing the method described by (Zengin, Locatelli, et al., 2016) with small modifications. Shortly, 50 µL equal volumes of the sample solution, α-glucosidase solution (pH 6.8), glutathione, and PNPG solution (10 mM) were mixed together and incubated for 15 min at 37°C. One sample was also prepared without adding the enzyme as a blank. The reaction was finished with 50 µL of sodium carbonate (0.2 M). After this, both the control and study specimens were checked for absorbance at 415 nm. However, the blank reading was excluded. Enzyme inhibition analysis was calculated as millimoles of acarbose equivalents (mmolACE g−1 extract).
2.6.3 | Cholinesterase inhibition
Inhibitory activity of cholinesterase (ChE) was measured by using Elman’s method, as previously described (Zengin, Nithiyanantham, et al., 2016) with minor modifications. Sample solution (50 μL) was mixed with 5,5′-Dithiobis(2-nitrobenzoic acid (125 μL) and acetylcholinester-ase (AChE) (or butyrylcholinesterase (BChE)) solution (0.26 U/mL, 25 μL) in Tris-HCl buffer (pH 8.0) in a microplate and then incubated for 15 min at 25°C. The reaction was started by putting 25 μL of acetylthi-ocholine iodide or butyrylthiocholine. Identically, a blank sample was arranged by adding sample solution to all reaction reagents without any ChE enzyme. The samples and blank absorbance were read at 405 nm after 10 min of incubation at room temperature. ChE inhibition analysis was expressed as galantamine equivalents (mg GALE g−1 extract).
2.7 | Antimicrobial activity
2.7.1 | Microbial strains
The strains used are Enterococcus faecalis ATCC 29212, MRSA ATCC 43300, Enterococcus faecium DSMZ 13590, Staphylococcus epider-midis ATCC 12228, Bacillus cereus RSKK 709, Enterococcus hirae ATCC 10541, Listeria monocytogenes, Klebsiella pneumoniae ATCC 13883, Vibrio parahaemolyticus ATCC 17802, Salmonella typhimu-rium CCM 5445, Candida albicans DSMZ 5817, Yersinia enterocolitica ATCC 27729.
2.7.2 | Disc diffusion assay
The antimicrobial potential of the extracts were analyzed by using the disc diffusion method (Ozcan & Acet, 2018b). Briefly, the fresh microbial strains were prepared at 0.5 MacFarland tur-bidity and then transferred to Mueller-Hinton agar plates. Later, the sterile empty paper discs were placed on plates with 20 μL extracts. After 24 h of incubation at 37°C and room temperature for bacteria and yeasts, respectively, the clear zone diameters were measured.
2.7.3 | Minimum inhibitory concentrations (MIC)
The minimum inhibitory concentrations (MIC) values of the extracts were analyzed using the broth dilution method with 96-well microti- ter plates (CLSI, 2017). The samples were prepared with dimethyl sul-foxide, the resultant serial dilution concentrations ranged between 1.024 and 0.002 mg/mL, and the MIC values were determined. The test microorganisms with 0.5 MacFarland turbidity were inoculated to the wells. After 48h of incubation, the microbial growth was de-tected by using a microplate absorbance reader. The MIC value was taken as the lowest concentrations of plant extracts in order to pre-vent microbial growth. The chloramphenicol, novobiocin, nalidixic acid, and nystatin were used as positive controls.2.8 | Fatty acid analysis
2.8.1 | Fatty acid extraction and fatty acids methyl
esters (FAMEs) preparation
About 10 g plant samples (flowers and stems) were extracted for oil, using petroleum ether for 6 hr in a Soxhlet apparatus. Then, the solvent was evaporated by using rotary evaporator. The obtained oil was esterified to determine fatty acid composition. The fatty acids in the oil were esterified into methyl esters by saponification with 0.5 mol/L methanolic NaOH and transesterified with 14% BF3 (v/v) in methanol (MeOH) (IUPAC 1979).
2.8.2 | Gas chromatographic analysis
Fatty acid extraction and fatty acids methyl esters were analyzed on a HP (Hewlett Packard) Agilent 6890N model gas chromatograph (GC), equipped with a flame ionization detector (FID) and fitted to a HP-88 capillary column (100 m, 0.25 mm i.d., and 0.2 µm). The ana-lytical conditions were as described by Demirci Kayiran et al. (2019).
2.9 | Statistical analysis
The statistical analyses were conducted after the experiments and the results were expressed in mean values ± SD of the triplicated measurements. ANOVA test was used in order to identify the varia-tions between various extracts (p < .05). The statistical calculations were conducted by SPSS version 20.0 program.
3 | RESULTS AND DISCUSSION
3.1 | Total bioactive contents
Then, the TPC and TFC of the extracts (Helichrysum chionophilum (Hc) and Helichrysum plicatum subsp. plicatum (Hp) were investigated with spectrophotometric methods and the results are presented in Figure 1. According to the obtained data, the ethylacetate (EtOAc) ex-tracts contained more TPC and TFCs with compared to MeOH and ethanol (EtOH) extracts. The highest phenolic and TFCs were noted in the ethylacetae extract of Hp stem bark (823.8 mg GAE g−1 extract) and of Hc flower (102.4 mg QE g−1 extract), respectively. In a similar study was performed on 16 Helichrysum species, the TPC of MeOH extracts were found to range between 73.70 and 160.63 mg GAE g−1 extract (Albayrak et al., 2010). In the same study, the highest amount of phenolic content was found in H. noeanum and the lowest in H. ori-entale. Moreover, the MeOH extract of Hc collected from Sivas region was examined in this study as well, and the TPC value of the plant was determined to be 100.97 mg GAE g-1 extract. In another study, four different Helichrysum species were investigated and TPC and TFC val-ues were found as 0.04–121.4 mg GAE g−1 extract and 0.02–8.2 mg RUE g−1 extract, respectively (Gouveia-Figueira et al., 2014). However, Ebrahimzadeh and Tavassoli (2015) reported the TPC (22.7 mg GAE g−1 extract) and TFC (9.6 mg QE g−1 extract) of MeOH extract of H. pseudoplicatum. However, in the present study, the EtOAc extracts of the flower and stem parts of plants (Hc and Hp) were found to have higher TPC and TFC values when compared with the earlier reports. High performance liquid chromatography analysis was performed by using 16 standard compounds in order to elucidate the phenolic components in these extracts and the results are shown in Table 1. In the analyses, the phenolic component profiles of plants showed sim- ilarities; however, the amounts of components showed some differ-ences in each plant. In both plants, the amount of chlorogenic acid was found to be the highest (8.9, 15.3 mg/g extract), followed by apigenin (4.4, 4.7 mg/g extract), luteolin (2.6, 1.9 mg/g extract), and ferulic acid (2.5, 1.2 mg/g extract). However, catechin, epicatechin, p-coumaric
F I G U R E 1 Total phenolic and flavonoid contents of the plants. (A,B) H. chionophilum. (C,D) H. plicatum. Values expressed are means ± SD of three different measurements. GAE, gallic acid equivalents; QE, quercetin equivalents. The data shown with different letters in the same column refer to statistically significant differences between the extracts of each species (p < .05)
acid, and rutin were not detected. Kaempferol was detected only in Hp (0.9 mg/g extract). Except for these, other standard compounds were determined at small amounts (0.06–0.6 mg/g extract) in both plants. In many studies, the phenolic compounds were reported to be responsible for the bioactivity. According to the literature, the chloro-genic acid and its derivatives are the most commonly found phenolic acids in the analyzed herbal samples and, in the present study, they were found at high amounts in both plants (Mattila & Hellström, 2007; Nicolle et al., 2004).
3.2 | Antioxidant properties
The antioxidants inhibit or delay the oxidation caused by free radicals (Aguilera, Martin-Cabrejas, & Mejia, 2016; Popoola et al., 2015). The radical scavenging activities of the plant extracts were determined by using the in vitro assays (ABTS and DPPH). Trolox equivalents and IC50values of extracts are shown in Table 2. In ABTS assay, the highest activity was found in EtOAc extract of the Hc flower and the ethyl ace- tate extract of Hp stem parts. However, in DPPH assay, the highest ac-tivity was found in the MeOH extract of the Hc stem and EtOH extract of the Hp flower parts. Similarly, the antioxidant capacity of the meth-anol extracts of Hc, Hp, and H. arenarium collected from the region around Sivas was reported by using DPPH assay (IC50 = 40.5, 48 and 47.6 µg/mL, respectively) (Tepe et al., 2005). Moreover, Ebrahimzadeh and Tavassoli (2015) investigated the antioxidant effects of H. pseu-doplicatum by using the same assay and they reported that the MeOH extract of the plant exhibited lower activity (IC50 = 438 µg/mL value) than in the present study.
3.3 | Enzyme inhibitory activities
Diabetes mellitus (type-II diabetes) is a metabolic sickness related with blood glucose level and it is considered a major health problem (Pari & Srinivasan, 2010). It is estimated that approximately 250 million people will suffer from this disorder by the year 2030 (Hwang, Han, Zabetian, Ali, & Narayan, 2012). From this point, the disorder has to manage and thus carbohydrate-hydrolyzing enzymes are main target in the pre-ventation strategies. These enzymes are amylase and glucosidase (Hu, Wang, & Kong, 2013). For this reason, the inhibition of these enzymes could be alleviating pathologies of diabetes mellitus. To this end, sev-eral compounds have been produced such as acarbose (Chiasson et al., 2002). Although this kind of synthetic products is still used, they cause TA B L E 1 Phenolic compounds of the plants No Phenolic compound Amount (mg/g extract) H. plicatum H. chionophilum 1 Gallic acid 0.05 ± 0.001 0.06 ± 0.002 2 Catechin * * 3 Chlorogenic acid 8.9 ± 0.4 15.3 ± 0.8 4 Epicatechin * * 5 Caffeic acid 0.60 ± 0.01 0.27 ± 0.03 6 Syringic acid 0.23 ± 0.01 0.15 ± 0.01 7 p-Coumaric acid * * 8 Ferulic acid 2.5 ± 0.1 1.2 ± 0.09 9 Rutin * * 11 Hesperidin 0.09 ± 0.001 0.33 ± 0.025 12 trans-Cinnamic acid 0.03 ± 0.02 0.06 ± 0.01 13 Luteolin 2.6 ± 0.01 1.9 ± 0.12 14 Kaempferol 0.9 ± 0.006 * 15 Apigenin 4.4 ± 0.02 4.7 ± 0.03 16 Vanillic acid 0.27 ± 0.02 0.24 ± 0.01
Note: Values expressed are means ± SD of three different measurements.
*not detected.
TA B L E 2 Antioxidant properties of the plants
Plant Part Extract ABTS mgTE/g extract
ABTS IC50 value (µg/mL) DPPH mg TE/g extract DPPH IC50 value (µg/mL)
H. chionophilum Flower ethanol 66.2 ± 1.5e 51.4 ± 0.9b 17.7 ± 0.3e 87.1 ± 1.5a
methanol 32.6 ± 2.9f 64.2 ± 2.6a 20.8 ± 0.1c 72.3 ± 0.4c
ethylacetate 445.2 ± 1.5a 25.9 ± 0.1f 19.7 ± 0.3d 77.1 ± 1.3b
Stem ethanol 355.3 ± 2.9b 34.8 ± 2.1e 22.0 ± 0.2b 67.9 ± 0.6d
methanol 105.5 ± 2.1c 41.1 ± 0.8d 22.8 ± 0.4a 65.2 ± 1.3e
ethylacetate 74.9 ± 2.6d 48.0 ± 1.3c 20.6 ± 0.4c 72.9 ± 1.3c
H. plicatum Flower ethanol 89.5 ± 0.5c 44.6 ± 0.2c 7.9 ± 0.2a 234.8 ± 6.5e
methanol 85.8 ± 5.2c 46.3 ± 2.4c 6.8 ± 0.1b 289.7 ± 1.2d,e
ethylacetate 328.9 ± 5.6b 36.1 ± 0.5d 4.6 ± 0.3d 572.0 ± 6.2c
Stem ethanol 52.6 ± 5.6d 57.4 ± 4.0b 5.8 ± 0.6c 381.6 ± 6.1d
methanol 43.2 ± 4.0d 61.2 ± 3.7a,b 3.7 ± 0.1e 917.7 ± 8.5a
ethylacetate 372.5 ± 6.5a 31.6 ± 1.1e 3.9 ± 0.3e 766.5 ± 7.9b
Note: Values expressed are means ± SD of three different measurements. The data shown with different letters in the same column refer to statistically significant differences between the extracts of each species. (p < .05).
damage in many tissues and organs among humans; hence, the natural inhibitors recently became popular (Lasano et al., 2019).
The enzyme inhibitory results of the plants are shown in Table 3. The highest inhibitory activities were determined in the stem EtOH (Hc) (for amylase) and flower ethyl acetate (Hp) (for glucosidase).
In the literature, there are studies about glucosidase and amylase inhibitory activities of the Asteraceae family (Aktumsek et al., 2013; Spínola & Castilho, 2017); however, a few studies were found on the enzyme inhibitory activities of Helichrysum species. For instance, while Gonçalves et al. (2017) investigated H. italicum, Orhan, Hoçbaç, Orhan, Asian, and Ergun (2014) studied the H. graveolens and Hp. In previous studies, phenolic compounds were shown to be effective in inhib-iting the α-glucosidase and α-amylase (Ani & Naidu, 2008; Shobana, Sreerama, & Malleshi, 2009). Of these compounds, chlorogenic acid has been reported to exhibit antidiabetic activity in many studies (Hunyadi, Martins, Hsieh, Seres, & Zupkó, 2012; Naveed et al., 2018).Therefore, the strong anti-diabetic activities of two plants we investigated in this study may arise from the chlorogenic acid. Alzheimer’s disease (AD) is one of the most dangerous diseases in the era. The disease develops as a result of the damage in the nerve cells or in the connections them and it progresses to worse. Although it is also seen among the young individuals, it is seen in one of every five people aged ≥ 65 years. ChE inhibitors are often used in order to control the disease or slow down the course of progression, resulting in increased levels of acetylcholine and increased cholinergic function (Howes & Houghton, 2003).
Cholinesterase inhibition results were calculated as galantamine equivalence and inhibition % (Table 4). Among Hc flower extracts, EtOAc extract showed the highest AChE (18.10% inhibition) and BChE (35.91% inhibition) activity. Butyrylcholine inhibitory activities were found to be higher than the acetylcholine inhibitory activities. As a result, the flow- er’s ethyl acetate extract of both plants showed the highest ChE inhi-bition. When all the extracts were evaluated together, Hc exhibited a higher inhibition activity of acetylcholine and butyrylcholine than Hp.
There are few studies carried out on the ChE activity of Helichrysum species: H. italicum (only AChE) (Gonçalves et al., 2017). However, there are many studies carried out on the ChE inhibition activities of Asteraceae members. For example Dittrichia viscosa (Trimech et al., 2014), Trichocline reptans, Eupatorium viscidum, Achyrocline tomentosa (Carpinella, Andrione, Ruiz, & Palacios, 2010), Centaurea saligna, C. depressa, C. kotschyi var. per-sica, C. urvillei subsp. hayekiana, C. triumfettii, C. tchihatcheffi, C. pulchella, C. patula (Zengin, Locatelli, et al., 2016), C. antalyanse, C. pyrrhoblephara, C. polypodiifolia var. pseudobehen (Aktumsek et al., 2013).
3.4 | Antimicrobial activity
The antimicrobial activity of the plants was investigated by using four Gram (−), seven Gram (+) and one yeast. The activity of the extracts de-termined by disc diffusion and MIC tests (Tables 5 and 6). According to the results, all extracts of two plants showed poor activity against Gram (−) organisms. However, the plant extracts exhibited equal or higher ac-tivity than positive control novobiocin. In addition, Hc extracts did not show any activity against K. pneumoniae (Table 5). Both plants showed similar strong activity against all Gram (+) test organisms (Tables 6 and 7). In addition, the plant extracts were found to have strong anti-candidal activity (Table 8). The results are in corroboration with the literature. Furthermore, the antimicrobial activity of the plants examined in the present study was found to be more effective when compared to the relative species (Albayrak et al., 2010). It is thought that the high level of effect might be because of the conditions, under which the plant grows.
3.5 | Fatty acid composition
Fatty acid profiles of the Helichrysum oils were determined by gas chromatography-flame ioanization detector (GC-FID) and the results TA B L E 3 α-amylase and α-glucosidase inhibitory activity of the plants
Plant Part Extract α-amylase (mmolACAE/g extract) α-glucosidase (mmolACAE/g extract)
H. chionophilum Flower ethanol 156.53 ± 1.35d 7.31 ± 0.05e
methanol 173.01 ± 1.84b 7.43 ± 0.02d
ethylacetate 158.98 ± 0.53c 25.42 ± 0.09a
Stem ethanol 193.36 ± 0.67a 7.61 ± 0.04c
methanol 158.15 ± 0.82c,d 3.77 ± 0.03f
ethylacetate 149.16 ± 0.91e 8.92 ± 0.06b
H. plicatum Flower ethanol 105.35 ± 0.65e 17.84 ± 0.02d
methanol 170.02 ± 0.99b 11.70 ± 0.05f ethylacetate 197.12 ± 0.94a 22.33 ± 0.03a Stem ethanol 105.12 ± 0.95e 18.83 ± 0.05b methanol 162.55 ± 1.39c 15.25 ± 0.04e ethylacetate 146.18 ± 0.8 d 18.73 ± 0.02c Note: Values expressed are means ± SD of three different measurements. ACAE, acarbose equivalents. The data shown with different letters in the same column refer to statistically significant differences between the extracts of each species (p < .05).
are summarized in Table 9. Palmitic acid (C 16:0) was major fatty acid in the oils and its level varied from 31.21% (in H. plicatum flowers) to 67.68% (in H. chionophium stems). In addition to palmitic acid, myristic (C 14:0), lauric (C 12:0), and stearic (C 18:0) acids had high levels in saturated fatty acids. Regarding monounsaturated fatty acids (MUFA), oleic (C 18:1 ω9), myristoleic (C 14:1 ω5), and palmi-toleic (C 16:1 ω7) acids were main fatty acids and, H. plicatum oils (11.50%–11.85%) had higher levels of MUFA when compared with H. chionophilum oils (2.17%–3.43%). In polyunsaturated fatty acids, two fatty acids were identified. Linolenic acid (C 18:3 ω3) was found
to be main PUFA in H. chionophilum oils, whereas H. plicatum oils were were the richest in terms of linoleic acid ( 18:2 ω6). Linoleic and linolenic acid are not synthesized by animal cells and thus must be provided by exogenous sources. In this context, the tested oils could be considered as good sources of essential oils and the highest level of these fatty acids was observed to be H. plicatum flowers oil. The present study is the first attempt to determine fatty acid composition of the Helichyrsum species and thus could provide valuable informa-tions in scientific platform to desing further studies on the members of the Helichrysum genus.
TA B L E 4 Cholinesterase inhibitory activity of the plants
Plant Part Extract
AChE inhibition
mgGALE/g extract AChE inhibition%
BChE inhibition
mgGALE/g extract BChE inhibition %
H. chionophilum Flower ethanol 1.10 ± 0.12c 16.14 ± 0.02c 17.89 ± 0.11c 32.28 ± 0.08c
methanol nd Nd 9.77 ± 0.04e 27.12 ± 0.03e
ethylacetate 1.48 ± 0.10a 18.10 ± 0.08a 23.60 ± 0.36a 35.91 ± 0.22a
Stem ethanol 0.94 ± 0.04d 15.92 ± 0.02d 17.18 ± 0.05d 31.82 ± 0.09d
methanol nd nd 6.67 ± 0.10f 25.14 ± 0.07f
ethylacetate 1.24 ± 0.08b 17.60 ± 0.09b 21.34 ± 0.29b 34.47 ± 0.19b
H. plicatum Flower ethanol 0.81 ± 0.02d 15.53 ± 0.07d 16.63 ± 0.06d 31.47 ± 0.04d
methanol nd nd 10.71 ± 0.05e 27.72 ± 0.04e
ethylacetate 1.35 ± 0.10a 17.75 ± 0.08a 22.37 ± 0.23a 35.12 ± 0.15a
Stem ethanol 1.30 ± 0.06a 17.70 ± 0.09a 22.38 ± 0.03a 35.13 ± 0.02a
methanol 1.14 ± 0.03b 16.82 ± 0.06b 19.78 ± 0.03b 33.48 ± 0.02b
ethylacetate 1.08 ± 0.04c 16.39 ± 0.03c 18.52 ± 0.45c 32.68 ± 0.29c
Note: Values expressed are means ± SD of three different measurements.
Abbrevioations: AChE, Acetylcholinesterase; BChE, Butrylcholinesterase; GALE, galantamine equivalents. The data shown with different letters in the same column refer to statistically significant differences between the extracts of each species (p < .05).
TA B L E 5 Antimicrobial activity on Gram (−) bacteria (inhibition zone/MIC value)
Plant Part Extract Y. enterecolitica K.pneumoniae V. paraheomolyticus S. typhimurium
H. chionophilum Flower E M 8/512 EA Stem E 9/512 M EA 9/256 8/512 H.plicatum Flower E 8/512 M 8/512 9/512 9/512 EA Stem E 9/128 8/512 8/512 7/512 M 8/512 EA Chloramphenicol 20/2 18/4 11/8 18/1 Novobiocin 15/512 30/2 11/512 13/512 Nalidixic acid 22/4 28/1 20/256 18/8 Note: First value: inhibition zone (200 µg/disc). Second value: MIC value (µg/mL).
TA B L E 6 Antimicrobial activity of H. chionophilum extracts on Gram (+) bacteria (inhibition zone/MIC value)
Plant Part Extract E. hirae MRSA E. faecium E. feacalis S. epidermidis L. monocytogenes B. cereus
H. chionophilum Flower E 15/128 18/128 – 12/512 9/512 20/64 20/64 M 20/64 14/128 10/512 14/128 13/128 18/64 EA Stem E M 15/128 8/512 8/512 16/128 EA 10/256 20/64 16/128 17/256 12/256 28/64 21/64 Chloramphenicol 18/8 15/32 20/4 15/8 17/16 18/16 20/2 Novobiocin 25/1 30/1 20/1 24/4 25/4 25/2 21/1 Nalidixic acid 15/256 23/64 nd/256 nd/128 12/4 12/256 23/4 Note: First value: inhibition zone (200 µg/disc). Second value: MIC value (µg/mL).
TA B L E 7 Antimicrobial activity of H. plicatum on Gram (+) bacteria (inhibition zone/MIC value)
Plant Part Extract E. hirae MRSA E. faecium E. feacalis S. epidermidis L. monocytogenes B. cereus
H. plicatum Flower E 18/64 18/128 21/128 20/64 22/256 21/64 M 18/128 25/32 28/32 24/64 20/64 23/32 EA Stem E 25/256 26/32 21/64 24/64 21/128 18/256 26/32 M 18/256 20/64 21/64 21/128 19/128 21/512 21/32 EA Chloramphenicol 18/8 15/32 20/4 15/8 17/16 18/16 20/2 Novobiocin 25/1 30/1 20/1 24/4 25/4 25/2 21/1 Nalidixic acid 15/256 23/64 nd/256 nd/128 12/4 12/256 23/4 Note: First value: inhibition zone (200 µg/disc). Second value: MIC value (µg/mL).
Plant Part Extract
Inhibition zone
(mm) 200 µg/disc MIC value (µg/mL)
H. chionophilum Flower ethanol 20 64
methanol 15 128
ethylacetate nd nd
Stem ethanol
methanol 18 64
ethylacetate nd nd
H. plicatum Flower ethanol 22 64
methanol nd nd ethylacetate nd nd Stem ethanol 21 64 methanol nd nd ethylacetate nd nd nystatin 15 16 TA B L E 8 Anti-candidal activity of the extracts (Candida albicans DSMZ 5817)
4 | CONCLUSION
In the present study, H. chionophilum and H. plicatum, which are widely used in folk medicine in Gümüşhane, were analyzed in terms of the phenolic compounds and several biological activities. For both plants, the ethyl acetate extracts of the flowers showed strong antioxidant and antimicrobial effects. The phytochemical analysis of the MeOH extracts from both plants showed that the chlorogenic acid was the major phenolic component. Furthermore, the plants examined in the present study showed a high level of inhibitory activity against α-glucosidase and α-amylase. It was suggested that these activities might be related with the high amount of phenolic content. In previ-ous studies, it was already confirmed that the phenolic compounds have positive effects on human health regarding the prevention of the development or progression of many diseases. In addition, the Helichyrsum oils had significant levels of essential oils. In conclusion, our results showed that H. chionophilum and H. plicatum could be re-garded as a novel and alternative natural bioactive agents in nutraceu-tical and pharmaceutical areas. However, further studies are need to explain their in vivo actions, toxicities and bioaccessibilities.
ACKNOWLEDGMENTS
This work was supported by Gümüşhane University Scientific Research Project Unit [grant number 18.F5119.03.01].
CONFLIC T OF INTEREST
The authors declare that there are no conflicts of interest.
ORCID
Gokhan Zengin https://orcid.org/0000-0001-6548-7823 REFERENCES
Aguilera, Y., Martin-Cabrejas, M. A., & de Mejia, E. G. (2016). Phenolic compounds in fruits and beverages consumed as part of the mediterra-nean diet: Their role in prevention of chronic diseases. Phytochemistry Reviews, 15, 405–423. https ://doi.org/10.1007/s11101-015-9443-z Aktumsek, A., Zengin, G., Guler, G. O., Cakmak, Y. S., & Duran, A. (2013).
Antioxidant potentials and anticholinesterase activities of methano-lic and aqueous extracts of three endemic Centaurea L. species. Food and Chemical Toxicology, 55, 290–296.
Albayrak, S., Aksoy, A., Sağdiç, O., & Budak, Ü. (2010). Phenolic com-pounds and antioxidant and antimicrobial properties of Helichrysum species collected from eastern Anatolia, Turkey. Turkish Journal of Biology, 34, 463–473.
Ani, V., & Naidu, K. A. (2008). Antihyperglycemic activity of polyphe-nolic components of black/bitter cumin Centratherum anthelminti-cum (L.) Kuntze seeds. European Food Research and Technology, 226, 897–903.
Aslan, M., Orhan, D. D., Orhan, N., Sezik, E., & Yesilada, E. (2007). In vivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp. plicatum capitulums in streptozotocin-induced-diabetic rats. Journal of Ethnopharmacology, 109, 54–59.
Babotă, M., Mocan, A., Vlase, L., Crișan, O., Ielciu, I., Gheldiu, A.-M., … Păltinean, R. (2018). Phytochemical analysis, antioxidant and an-timicrobial activities of Helichrysum arenarium (L.) Moench. and Antennaria dioica (L.) Gaertn. flowers. Molecules, 23, 409.
Bahadori, M. B., Kirkan, B., & Sarikurkcu, C. (2019). Phenolic ingredients and therapeutic potential of Stachys cretica subsp. smyrnaea for the management of oxidative stress, Alzheimer’s disease, hyperglycemia, and melasma. Industrial Crops and Products, 127, 82–87.
Fatty acids H. chionophilum flowers H. chionophilum stem H. plicatum flowers H. plicatum stem C 10:0 2.11 ± 0.01* 0.94 ± 0.02 1.18 ± 0.01 1.72 ± 0.05 C 12:0 1.45 ± 0.04 1.47 ± 0.03 1.77 ± 0.04 14.44 ± 0.04 C 14:0 18.09 ± 0.01 7.74 ± 0.25 16.98 ± 0.12 10.96 ± 0.15 C 15:0 1.86 ± 0.29 1.47 ± 0.18 1.73 ± 0.13 1.04 ± 0.04 C 16:0 44.16 ± 0.47 67.68 ± 0.11 31.21 ± 0.02 40.04 ± 0.46 C 17:0 1.39 ± 0.11 1.61 ± 0.01 1.01 ± 0.06 0.90 ± 0.25 C 18:0 10.47 ± 0.43 2.73 ± 0.71 6.73 ± 0.10 5.20 ± 0.97 ΣSFA 78.44 ± 0.95 83.63 ± 0.13 60.60 ± 0.06 74.28 ± 0.02 C 14:1 ω5 1.04 ± 0.08 0.27 ± 0.04 1.00 ± 0.03 4.99 ± 0.01 C 15:1 ω5 0.06 ± 0.01 0.27 ± 0.04 0.28 ± 0.08 0.15 ± 0.05 C 16:1 ω7 0.23 ± 0.08 0.41 ± 0.13 1.00 ± 0.04 0.23 ± 0.03 C 17:1 ω8 0.20 ± 0.02 1.45 ± 0.07 0.98 ± 0.23 0.53 ± 0.11 C 18:1 n9 0.66 ± 0.04 1.03 ± 0.07 8.25 ± 0.05 5.96 ± 0.11 ΣMUFA 2.17 ± 0.18 3.43 ± 0.06 11.50 ± 0.19 11.85 ± 0.25 C 18:2 ω6 7.16 ± 0.64 6.28 ± 0.13 23.73 ± 0.16 11.03 ± 0.54 C 18:3 ω3 11.16 ± 0.27 6.66 ± 0.21 4.19 ± 0.29 2.86 ± 0.28 ΣPUFA 18.32 ± 0.37 12.93 ± 0.07 27.92 ± 0.13 13.89 ± 0.25 ΣUFA 20.49 ± 0.55 16.36 ± 0.13 39.41 ± 0.06 25.74 ± 0.01 Abbreviations: SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
*Values are reported as mean ± SD of three parallel analysis.
TA B L E 9 Fatty acid composition of
Baytop, T. (1997). Turkce bitki adlari sozlugu (A dictionary ofvernacular names of wild plants of Turkey) (p. 578). Ankara: Publication of the Turk Dil Kurumu (The Turkish Language Society).
Caponio, F., Alloggio, V., & Gomes, T. (1999). Phenolic compounds of virgin olive oil: Influence of paste preparation techniques. Food Chemistry, 64, 203–209.
Carpinella, M. C., Andrione, D. G., Ruiz, G., & Palacios, S. M. (2010). Screening for acetylcholinesterase inhibitory activity in plant ex-tracts from Argentina. Phytotherapy Research, 24, 259–263. Chiasson, J.-L., Josse, R.G., Gomis, R., Hanefeld, M., Karasik, A., Laakso, M. & Group, S.-N.T.R. (2002). Acarbose for prevention of type 2 dia-betes mellitus: The STOP-NIDDM randomised trial. The Lancet, 359, 2072–2077. https ://doi.org/10.1016/S0140-6736(02)08905-5 CLSI. (2017). Performance standards for antimicrobial susceptibility test-ing; 27th informational supplement. CLSI/NCCLS (27th ed). Wayne, PA, USA: Clinical and Laboratory Standards Institute.
Davis, P., Mill, R., & Tan, K. (1988). Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburgh University Press.
Demirci Kayiran, S., Eroglu Ozkan, E., Mataraci Kara, E., Yilmaz, M. A., Zengin, G., & Boga, M. (2019). Comprehensive analysis of an uninves-tigated wild edible medicinal garlic species from Turkey: Allium macro-chaetum Boiss. & Hausskn. Journal of Food Biochemistry, 43(7), e12928. Ebrahimzadeh, M. A., & Tavassoli, A. (2015). Antioxidant properties of
Helichrysum pseudoplicatum Nab. Pharmaceutical and Biomedical Research, 1, 37–43.
Fu, L., Xu, B.-T., Xu, X.-R., Gan, R.-Y., Zhang, Y., Xia, E.-Q., & Li, H.-B. (2011). Antioxidant capacities and total phenolic contents of 62 fruits. Food Chemistry, 129, 345–350.
Gonçalves, S., Moreira, E., Grosso, C., Andrade, P. B., Valentão, P., & Romano, A. (2017). Phenolic profile, antioxidant activity and enzyme inhibitory activities of extracts from aromatic plants used in Mediterranean diet. Journal of Food Science and Technology, 54, 219–227.
Gouveia-Figueira, S., Gouveia, C., Carvalho, M., Rodrigues, A., Nording, M., & Castilho, P. (2014). Antioxidant capacity, cytotoxicity and an-timycobacterial activity of madeira archipelago endemic Helichrysum dietary and medicinal plants. Antioxidants, 3, 713–729. https ://doi. org/10.3390/antio x3040713
Guner, A., Ozhatay, N., & Ekim, T. (2000). Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburgh University Press.
Howes, M.-J.- R., & Houghton, P. J. (2003). Plants used in Chinese and Indian traditional medicine for improvement of memory and cogni-tive function. Pharmacology Biochemistry and Behavior, 75, 513–527. Hu, X.-J., Wang, X.-B., & Kong, L.-Y. (2013). α-Glucosidase inhibitors via
green pathway: Biotransformation for bicoumarins catalyzed by Momordica charantia peroxidase. Journal of Agricultural and Food Chemistry, 61, 1501–1508.
Hunyadi, A., Martins, A., Hsieh, T.-J., Seres, A., & Zupkó, I. (2012). Chlorogenic acid and rutin play a major role in the in vivo anti-dia-betic activity of Morus alba leaf extract on type II diaChlorogenic acid and rutin play a major role in the in vivo anti-dia-betic rats. PLoS ONE, 7, e50619. https ://doi.org/10.1371/journ al.pone.0050619 Hwang, C. K., Han, P. V., Zabetian, A., Ali, M. K., & Narayan, K. V. (2012).
Rural diabetes prevalence quintuples over twenty-five years in low- and middle-income countries: A systematic review and meta-analy-sis. Diabetes Research and Clinical Practice, 96, 271–285.
IUPAC. (1979). Determination of the saponification value (S.V.). In C. Paquot (Ed.), Standards methods for analysis of oils, fats and derivatives (pp. 59–66). Oxford: Pergamon Press. Kirby, A. J., & Schmidt, R. J. (1997). The antioxidant activity of Chinese herbs for eczema and of placebo herbs—I. Journal of Ethnopharmacology, 56, 103–108. Korga, A., Józefczyk, A., Zgórka, G., Homa, M., Ostrowska, M., Burdan, F., & Dudka, J. (2017). Evaluation of the phytochemical composition and protective activities of methanolic extracts of Centaurea borysthen-ica and Centaurea daghestanborysthen-ica (Lipsky) Wagenitz on cardiomyocytes treated with doxorubicin. Food & Nutrition Research, 61, 1344077.
Lasano, N. F., Hamid, A. H., Karim, R., Dek, M. S. P., Shukri, R., & Shazini Ramli, N. (2019). Nutritional composition, anti-diabetic properties and identification of active compounds using UHPLC-ESI-orbitrap-MS/ MS in Mangifera odorata L. peel and seed kernel. Molecules, 24, 320. https ://doi.org/10.3390/molec ules2 4020320 Lee, H. P., Zhu, X., Casadesus, G., Castellani, R. J., Nunomura, A., Smith, M. A., … Perry, G. (2010). Antioxidant approaches for the treat-ment of Alzheimer’s disease. Expert Review of Neurotherapeutics, 10, 1201–1208.
Maritim, A., Sanders, A., & Watkins, J., Iii. (2003). Diabetes, oxida-tive stress, and antioxidants: A review. Journal of Biochemical and Molecular Toxicology, 17, 24–38.
Mattila, P., & Hellström, J. (2007). Phenolic acids in potatoes, vegetables, and some of their products. Journal of Food Composition and Analysis, 20, 152–160. Moreno, M. A. I. N., Isla, M. A. I., Sampietro, A. R., & Vattuone, M. A. (2000). Comparison of the free radical-scavenging activity of prop-olis from several regions of Argentina. Journal of Ethnopharmacology, 71, 109–114. Naveed, M., Hejazi, V., Abbas, M., Kamboh, A. A., Khan, G. J., Shumzaid, M., … Modarresi-Ghazani, F. (2018). Chlorogenic acid (CGA): A pharmacological review and call for further research. Biomedicine & Pharmacotherapy, 97, 67–74.
Nicolle, C., Carnat, A., Fraisse, D., Lamaison, J. L., Rock, E., Michel, H., … Remesy, C. (2004). Characterisation and variation of antioxidant micronutrients in lettuce (Lactuca sativa folium). Journal of the Science of Food and Agriculture, 84, 2061–2069.
Orhan, N., Hoçbaç, S., Orhan, D. D., Asian, M., & Ergun, F. (2014). Enzyme inhibitory and radical scavenging effects of some antidiabetic plants of Turkey. Iranian Journal of Basic Medical Sciences, 17, 426.
Ozcan, K., & Acet, T. (2018a). Antimicrobial and antioxidant screen-ing, synergy studies of Helichrysum chionophilum extracts against to resistant microbial strains. Fresenius Environmental Bulletin, 27, 5045–5052.
Ozcan, K., & Acet, T. (2018b). In vitro antioxidant and antimicrobial ac-tivities of the five different solvent extracts of Centaurea pulcher-rima var. freynii from Turkey. Fresenius Environmental Bulletin, 27, 4047–4051.
Pari, L., & Srinivasan, S. (2010). Antihyperglycemic effect of diosmin on he- patic key enzymes of carbohydrate metabolism in streptozotocin-nic-otinamide-induced diabetic rats. Biomedicine & Pharmacotherapy, 64, 477–481.
Popoola, O., Marnewick, J., Rautenbach, F., Iwuoha, E., & Hussein, A. (2015). Acylphloroglucinol derivatives from the South African Helichrysum niveum and their biological activities. Molecules, 20, 17309–17324.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. https ://doi.org/10.1016/S0891-5849(98)00315-3 Sala, A., Recio, M. d. C., Giner, R. M., Máñez, S., Tournier, H., Schinella, G.
and Ríos, J. L. (2002). Anti-inflammatory and antioxidant properties of Helichrysum italicum. Journal of Pharmacy and Pharmacology, 54(3), 365–371.
Sezik, E., Yeşilada, E., Honda, G., Takaishi, Y., Takeda, Y., & Tanaka, T. (2001). Traditional medicine in Turkey X. Folk medicine in central Anatolia. Journal of Ethnopharmacology, 75, 95–115.
Shobana, S., Sreerama, Y., & Malleshi, N. (2009). Composition and en-zyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: Mode of inhibition of α-glucosidase and pancreatic amylase. Food Chemistry, 115, 1268–1273. https ://doi.org/10.1016/ j.foodc hem.2009.01.042
Slinkard, K., & Singleton, V. L. (1977). Total phenol analysis: Automation and comparison with manual methods. American Journal of Enology and Viticulture, 28, 49–55.
Spínola, V., & Castilho, P. C. (2017). Evaluation of Asteraceae herbal extracts in the management of diabetes and obesity. Contribution of caffeoylquinic acids on the inhibition of digestive enzymes ac-tivity and formation of advanced glycation end-products (in vitro). Phytochemistry, 143, 29–35.
Sümbül, H., Göktürk, R. S., & Düşen, O. D. (2003). A new endemic species of Helichrysum Gaertn. (Asteraceae-Inuleae) from south Anatolia. Botanical Journal of the Linnean Society, 141, 251–254.
Tepe, B., Sokmen, M., Akpulat, H. A., & Sokmen, A. (2005). In vitro anti-oxidant activities of the methanol extracts of four Helichrysum spe-cies from Turkey. Food Chemistry, 90, 685–689.
Trimech, I., Weiss, E. K., Chedea, V. S., Marin, D., Detsi, A., Ioannou, E., … Kefalas, P. (2014). Evaluation of anti-oxidant and acetylcholinester-ase activity and identification of polyphenolics of the invasive weed Dittrichia viscosa. Phytochemical Analysis, 25, 421–428.
Tsao, R., & Deng, Z. (2004). Separation procedures for naturally occur-ring antioxidant phytochemicals. Journal of Chromatography B, 812, 85–99.
Zengin, G., Locatelli, M., Carradori, S., Mocan, A. M., & Aktumsek, A. (2016). Total phenolics, flavonoids, condensed tannins content of eight Centaurea species and their broad inhibitory activities against
cholinesterase, tyrosinase, α-amylase and α-glucosidase. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 44, 195–200.
Zengin, G., Nithiyanantham, S., Locatelli, M., Ceylan, R., Uysal, S., Aktumsek, A., … Maskovic, P. (2016). Screening of in vitro antioxidant and enzyme inhibitory activities of different extracts from two uninvestigated wild plants: Centranthus longiflorus subsp. longiflorus and Cerinthe minor subsp. auriculata. European Journal of Integrative Medicine, 8, 286–292. Zengin, G., Zheleva-Dimitrova, D., Gevrenova, R., Nedialkov, P., Mocan,
A., Ćirić, A., … Mahomoodally, M. F. (2018). Identification of pheno-lic components via LC–MS analysis and biological activities of two Centaurea species: C. drabifolia subsp. drabifolia and C. lycopifolia. Journal of Pharmaceutical and Biomedical Analysis, 149, 436–441.
How to cite this article: Acet T, Ozcan K, Zengin G. An assessment of phenolic profiles, fatty acid compositions, and biological activities of two Helichrysum species: H. plicatum and H. chionophilum. J Food Biochem. 2020;44:e13128. https :// doi.org/10.1111/jfbc.13128
