Phenolic composition, antioxidant and enzyme inhibitory activities of ethanol and water extracts of Chenopodium botrys

Abstract

In this study, we aimed to evaluate the phenolic composition, antioxidant, and enzyme inhibitory activities of ethanol and water extracts of Chenopodium botrys L. In the ethanol extract, the amounts of flavonoids, saponins, and condensed tannins were found to be higher than those of the water extract (4.54 mg of rutin equivalent (RE) per g of dry plant, 25.45 mg of quillaja equivalent (QAE) per g of dry plant, and 59.20 mg of catechin equivalent (CE) per g of dry plant, respectively). On the other hand, a total phenolic assay showed a superiority of the water extract (3.85 mg of gallic acid equivalent (GAE) per g of dry plant). The extracts were also subjected to screening for the quantification of selected compounds. Among the compounds, benzoic acid was found to be the most abundant one in the extracts (59.93 and 2974.24 μg per g of dry plant, respectively). In general, antioxidant activity assays showed the superiority of the water extract. C. botrys extracts were also evaluated for their inhibitory activities on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase, and α-glucosidase. The water extract exhibited the highest inhibitory activity on AChE, tyrosinase, α-amylase, and α-glucosidase (113.69 μg of galantine equivalent (GALAE) per g of dry plant, 165.56 μg of kojic acid equivalent (KAE) per g of dry plant, 7.16 mg of acarbose equivalent (ACE) per g of dry plant, and 6.47 mg of ACE per g of dry plant, respectively). On the other hand, BChE inhibitory activity of the ethanol extract was found to be higher than that of the water extract (60.90 μg of GALAE per g of dry plant). According to the results of the antioxidant and enzyme inhibitory activity assays, C. botrys may have the potential to prevent diseases associated with oxidative stress and to prevent AD, pigmentation diseases, hyperglycaemia and its associated complications.

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1. Introduction

Reactive oxygen species (ROS) are formed as a result of normal metabolic reactions. They are kept under control by means of enzymatic and non-enzymatic metabolic reactions. Any disturbance disrupting the balance of these reactions can lead to an increase in the amounts of ROS in the body. Oxidative stress plays an important role in the pathogenesis of numerous diseases such as cardiovascular diseases, diabetes mellitus, Alzheimer's disease, inflammatory diseases, carcinogenesis, neurodegenerative diseases, pulmonary, and hematological diseases.^1–5 Antioxidant substances prevent and/or reduce the hazardous effects of free radicals by neutralizing them. In recent years, researchers have focused on finding natural antioxidants in order to support the antioxidant defense system of the human body. The natural antioxidants can be used as alternative agents in several areas such as food, pharmaceutical and cosmetic industries.^6–8

Since there is an increase in the prevalence of degenerative diseases such as Alzheimer's disease (AD) and cancer, they have become a global health problem. AD is characterized by the loss of memory and eventually incapacitates the patient. Cholinergic hypothesis is one of the most accepted key approaches used in the management of this disease.⁹ According to this hypothesis, low level of acetylcholine (ACh) leads to the impairment of memory. Hydrolytic cholinesterases such as acetylcholinesterase (AChE) and butyrylcholinestrase (BChE) terminate the cholinergenic signal transfer by acting on ACh in the synaptic cleft.^10,11 In the presence of AChE and BChE inhibitory agents, the half-life of ACh can be prolonged and thus these agents can been used as the effective strategies for treatment of AD.^12–14

Melanin is the major pigment secreted by the melanocytes located in the basal layer of human skin. Melanin can be overproduced in the case of hyperpigmentation diseases and overexposure to the sunlight.¹⁵ The synthesis of melanin is catalyzed by tyrosinase in the melanocytes. In recent years, the researchers have identified a number of tyrosinase inhibitory agents from both natural and synthetic sources. These compounds can be used as the therapeutic agent in the treatment of skin disorders associated with melanin hyperpigmentation. Since they have skin-whitening effect, the tyrosinase inhibitors can be used in cosmetic industry as well.¹⁶ By screening the natural sources, new and alternative tyrosinase inhibitors such as phenolics and flavonoids can be explored.^17,18

In diabetes mellitus patients, free glucose is accumulated in the blood in excessive amounts. The accumulation of this molecule leads to a number of vascular diabetic complications and oxidation-related damage in various organs.¹⁹ Carbohydrates are digested in small intestine by the key enzymes, α-amylase and α-glucosidase.²⁰ In diabetic patients, the breakdown of starch can be delayed by using α-amylase and α-glucosidase inhibitors. The inhibitory agents can lower the postprandial blood glucose levels as well. To control the hyperglycemia effectively in type II diabetic patients, synthetic inhibitors are frequently used. However, the synthetic inhibitors have been proven to have side effects in human body and they should be replaced with the safer alternatives.²¹ Until now, a number of phenolic compounds have been shown to have α-amylase and α-glucosidase inhibitory activities.^22–27

The Chenopodiaceae family consists of perennial herbs and widely distributed in the temperate and subtropical saline zones of the world.²⁸ Chenopodium genus, which is a member of this family, contains more than one hundred species.²⁹ Chenopodium species have long been used in folk medicine for the treatment of several ailments.^30,31 According to Gennadios,³¹ C. botrys is used to keep away the moths instead of lavender. In Serbian traditional medicine, the dried aerial parts of C. botrys have been used for the preparation of infusions or liquid extracts. These preparations can be used as diuretic, antispasmodic, carminative and antidiarrheal agents; sometimes as a spice.³² According to an ethnobotanical survey carried out by Khan and Khatoon,³³ whole plant is used as antiseptic agent. It can also be used for the treatment of abdominal problems, uterus problems, tumors, swellings, bleeding, and regulating the disturbed menstruation.³³ According to another ethnobotanical survey, the young leaves and branches of C. botrys are used for their wound healing effect.³⁴ C. botrys contains flavonoids, alkaloids and several terpenoids. According to the pharmacological reports, C. botrys can be used as a source material for the development of new drugs.³⁵

Although the people have used C. botrys traditionally for a long time for the treatment of several disorders, the antioxidant and enzyme inhibitory activities of this species have not previously been reported elsewhere. The aim of this study was to evaluate the antioxidant and enzyme inhibitory activities of the ethanol and water extracts of C. botrys along as a quantification of the selected compounds. Antioxidant activities of the extracts were determined by using several complementary test systems named as free radical scavenging [on 1,1-diphenyl-2-picrylhydrazyl (DPPH˙), 2,2-azino-bis(3-ethylbenzothiazloine-6-sulphonic acid) (ABTS˙⁺), superoxide anion (O₂˙⁻), nitric oxide (˙NO), and hydroxyl (HO˙) radicals], reducing power [cupric ion reducing (CUPRAC), ferric reducing antioxidant power (FRAP), and iron(III) to iron(II) reduction], phosphomolybdenum, and metal chelating assays. Enzyme inhibitory activities of the extracts were tested on AChE, BChE, tyrosinase, α-amylase, and α-glucosidase. In addition to the determination of the biological activities, the extracts were also screened for their total phenolics, flavonoids, saponins, and condensed tannins as well as the quantification of selected compounds [gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, (−)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapic acid, benzoic acid, o-coumaric acid, rutin, naringin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, naringenin, luteolin, kaempferol, and apigenin].

2. Materials and methods

2.1. Chemicals

Ferric chloride, Folin–Ciocalteu's reagent and methanol were purchased from Merck (Darmstadt, Hessen, Germany). DPPH, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), AChE (Electric ell acetylcholinesterase, Type-VI-S, EC 3.1.1.7), BChE (horse serum butyrylcholinesterase, EC 3.1.1.8), 3,4-dihydroxy-L-phenylalanine (L-DOPA), tyrosinase, acetylthiocholine iodide (ATCI), butyrylthiocholine chloride (BTCl), and phenolic standards were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Höxter, North Rhine-Westphalia, Germany). All other chemicals and solvents were of analytical grade.

2.2. Plant material

Aerial parts of C. botrys were collected from Senirkent-Şuhut highway, Senirkent-Isparta-Turkey on 30 June 2013 (15–20 km, 38° 11′ 44′′ N 30° 42′ 44′′ E, 955 m). Taxonomic identification of the plant material was made by Dr Olcay Ceylan, who is from the Department of Biology, Mugla Sitki Kocman University, Mugla-Turkey.

2.3. Preparation of the extracts

To prepare the ethanol extract, dried plant samples (5 g) were macerated with 100 mL of ethanol at room temperature for 24 h. Another portion of the dried sample (5 g) was extracted in boiling deionized water (100 mL) for 15 min to obtain the water extract. The ethanol was then removed using a rotary evaporator at 40 °C. The water extract was freeze-dried. All of the extracts were stored at +4 °C until analyzed. Yields of the ethanol and water extracts were determined as 7.05 and 18.58% (w/w) respectively.

2.4. Antioxidant activity

Antioxidant activity of the extracts obtained from C. botrys was evaluated by using the following assays: phosphomolybdenum, ferrous ion chelating, reducing power (CUPRAC, FRAP, and potassium ferricyanide), free radical scavenging (on DPPH˙, ABTS˙⁺, O₂˙⁻, ˙NO, and HO˙). All of the analyses were carried out by using the same conditions reported by Zengin et al.³⁶

2.5. Enzyme inhibitory activity

Inhibitory activities of C. botrys extracts on AChE, BChE, tyrosinase, α-amylase, and α-glucosidase were evaluated according to the method of Zengin et al.³⁶

2.6. Determination of total bioactive components

Total phenolic, flavonoid, saponin and condensed tannin contents were determined by employing the method given in the literature.³⁶

2.7. Quantification of the phenolic compounds by RP-HPLC

Phenolic compounds were evaluated by using a Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) system (Shimadzu Scientific Instruments, Kyoto, Honshu, Japan). Detection and quantification were carried out with an LC-10ADvp pump, a Diode Array Detector, a CTO-10Avp column heater, SCL-10Avp system controller, DGU-14A degasser, and SIL-10ADvp auto sampler (Shimadzu Scientific Instruments, Columbia, MD, USA). Separations were conducted at 30 °C on an Agilent® Eclipse XDB C-18 reversed-phase column (250 mm × 4.6 mm in length, 5 μm particle size). The phenolic compositions of the extracts were determined according to the method of Sarikurkcu et al.³⁷ (−)-Epicatechin, (+)-catechin, apigenin, benzoic acid, caffeic acid, chlorogenic acid, eriodictyol, ferulic acid, gallic acid, hesperidin, kaempferol, luteolin, naringin, naringenin, o-coumaric acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, quercetin, rosmarinic acid, rutin, sinapic acid, syringic acid, trans-cinnamic acid, and vanillin were used as standard compounds.

The analytical characteristics of the phenolics and flavonoids were presented in ESI file (S1) Appendix S1.† Identification of the compounds was carried out by separate injections of each standard solution. Thus, the resolution peak and run time data were determined for each compound. To verify the identification of the compounds, a chromatographic run was performed with the extracts spiked with the standard solution. Phenolic and flavonoid compounds in the extracts were identified by comparing their retention times and spectrums with those obtained by the injection of standard solution under the same conditions. For the quantitation of compounds, peak areas were measured by using an internal standard. Chromatographic profiles of the compounds and the extracts were presented in ESI files S2–S4 in Appendix S1.†

2.8. Statistical analysis

All of the assays were carried out in triplicate. The results were expressed as mean and standard deviation values (mean ± SD). Statistical differences between the extracts were analyzed using a Student's t-test (α = 0.01). All of the analyses were carried out by using Statistical Package for the Social Sciences (SPSS) v22.0 software.

3. Results and discussion

3.1. Phytochemical compositions of the extracts

It is not logical to evaluate the biological activities of the extracts without considering their chemistry. Biological activities of the plant species are strictly depending on the bioactive compounds occur naturally in the plant body. In order to make a healthy assessment on the biological activity potential of any plant species, it is necessary to reveal the chemical composition as well. As presented in the experimental section of this paper, the ethanol and water extracts of C. botrys were evaluated for their total phenolics, flavonoids, saponins, and condensed tannins (Table 1).

Table 1 Total bioactive components of C. botrys extracts (mean ± SD)^a

Assays	Ethanol	Water
a Different superscript letters in the same row indicate significant difference (p < 0.01).b GAE, gallic acid equivalent.c RE, rutin equivalent.d QAE, quillaja equivalent.e CE, catechin equivalent.
Total phenolics (mg of GAE per g of dry plant)^b	2.02 ± 0.07^a	3.85 ± 0.13^b
Total flavonoids (mg of RE per g of dry plant)^c	4.54 ± 0.04^a	4.18 ± 0.06^a
Total saponins (mg of QAE per g of dry plant)^d	25.45 ± 0.03^b	15.52 ± 0.34^a
Total condensed tannins (mg of CE per g of dry plant)^e	59.20 ± 0.57^b	43.28 ± 3.55^a

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As can be seen from the Table 1, condensed tannins were found to be the most abundant compounds in the extracts. It was followed by saponins, flavonoids, and phenolics, respectively. In the ethanol extract, total quantities of the flavonoids, saponins, and condensed tannins were found to be higher than that of the water extract. Total quantities of these compounds were determined as 4.54 mg of RE per g of dry plant, 25.45 mg of QAE per g of dry plant, and 59.20 mg of CE per g of dry plant in the ethanol extract, respectively. On the other hand, total phenolic assay was resulted in the superiority of the water extract. This extract contained 3.85 mg of GAE per g of dry plant of phenolic compounds. This value was found to be almost two folds greater than that of the total phenolic content of the ethanol extract. Total flavonoid contents of the ethanol and water extracts were found to be similar from the statistical point of view (p > 0.01).

In addition to the qualitative chromatographic analyses given above, the extracts were also screened for the quantification of twenty-five selected compounds as following: gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, (−)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapic acid, benzoic acid, o-coumaric acid, rutin, naringin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, naringenin, luteolin, kaempferol, and apigenin.

According to data presented in the Table 2, the RP-HPLC analyses were resulted in the superiority of the water extract. Benzoic acid was found to be the most abundant compound in the extracts. The quantities of this compound were measured as 59.93 and 2974.24 μg per g of dry plant in the ethanol and water extracts, respectively. In the water extract, benzoic acid was followed by ferulic (219.65 μg per g of dry plant) and chlorogenic acids (118.83 μg per g of dry plant), respectively. The amounts of syringic and trans-cinnamic acids were found to be equal in the water extract (21.60 μg per g of dry plant).

Table 2 Quantities of selected phytochemicals in C. botrys extracts (mean ± SD)^a

No	Phenolics and flavonoids	Concentration (μg per g of dry plant)
		Ethanol	Water
a Different superscript letters in the same row indicate significant difference (p < 0.01).b nd, not detected.
1	Gallic acid	nd^b	nd
2	Protocatechuic acid	nd	nd
3	(+)-Catechin	nd	nd
4	p-Hydroxybenzoic acid	13.40 ± 0.49	nd
5	Chlorogenic acid	nd	118.83 ± 14.40
6	Caffeic acid	4.23 ± 0.14	nd
7	(−)-Epicatechin	nd	90.02 ± 3.60
8	Syringic acid	nd	21.60 ± 0.72
9	Vanillin	1.41 ± 0.07	nd
10	p-Coumaric acid	3.53 ± 0.21	nd
11	Ferulic acid	7.05 ± 0.49^a	219.65 ± 7.20^b
12	Sinapic acid	nd	nd
13	Benzoic acid	59.93 ± 1.41^a	2974.24 ± 28.81^b
14	o-Coumaric acid	nd	nd
15	Rutin	nd	nd
16	Naringin	nd	nd
17	Hesperidin	nd	nd
18	Rosmarinic acid	nd	nd
19	Eriodictyol	nd	32.41 ± 1.80
20	trans-Cinnamic acid	nd	21.60 ± 0.36
21	Quercetin	nd	nd
22	Naringenin	nd	nd
23	Luteolin	11.28 ± 0.71	nd
24	Kaempferol	nd	nd
25	Apigenin	nd	nd

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In the case of the water extract, benzoic acid was followed by p-hydroxybenzoic acid (13.40 μg per g of dry plant) and luteolin (11.28 μg per g of dry plant), respectively. The quantities of p-hydroxybenzoic acid, luteolin, caffeic acid, p-coumaric acid, and vanillin in the ethanol extract were found to be higher than that of the water extract. According to the results of the RP-HPLC analyses, the extracts were found to contain no gallic acid, protocatechuic acid, (+)-catechin, sinapic acid, o-coumaric acid, rutin, naringin, hesperidin, rosmarinic acid, quercetin, naringenin, kaempferol, and apigenin.

As far as our literature survey could ascertain, studies on the phytochemical composition C. botrys led to the isolation of several classes of compounds such as flavonols [chrysoeriol, quercetin, quercetin-3-O-β-D-glucopyranoside, and quercetin-3-O-β-(D-glucopyranosyl-6-β-D-glucopyranoside)], flavones (hispidulin, salvigenin, 5-methylsalvigenin, 7-methyleupatulin, sinensetin, and jaceosidin),^38–42 alkaloids (betaine),^43–45 and phytoecdysteroids (ecdysteroids).⁴⁶ But, the compounds listed in the Table 2 were firstly reported by this study.

3.2. Antioxidant activity

Antioxidant activities of the ethanol and water extracts of C. botrys were evaluated by employing radical scavenging (on DPPH, ABTS˙⁺, O₂˙⁻, NO, and HO˙) (Table 3), reducing power [CUPRAC, FRAP, and iron(III) to iron(II) reduction], phosphomolybdenum, and chelating effect assays (Table 4).

Table 3 Radical scavenging activities of C. botrys extracts (mean ± SD)^a

Assays	Ethanol	Water
a Different superscript letters in the same row indicate significant difference (p < 0.01).b TE, trolox equivalent.c BHAE, Error! Hyperlink reference not valid.butylated hydroxyanisole equivalent.d ME, mannitol equivalent.
DPPH (mg of TE per g of dry plant)^b	4.38 ± 0.10^a	21.77 ± 0.16^b
ABTS cation (mg of TE per g of dry plant)^b	1.43 ± 0.01^a	12.88 ± 0.14^b
Superoxide anion (mg of BHAE per g of dry plant)^c	54.15 ± 0.45^a	191.70 ± 2.39^b
Nitric oxide (mg of BHAE per g of dry plant)^c	158.78 ± 7.49^a	289.01 ± 17.36^b
Hydroxyl (mg of ME per g of dry plant)^d	17.07 ± 0.56^a	73.01 ± 3.76^b

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Table 4 Reducing power, total antioxidant (by phosphomolybdenum method), and metal chelating activities of C. botrys extracts (mean ± SD)^a

Assays	Ethanol	Water
a Different superscript letters in the same row indicate significant difference (p < 0.01).b TE, trolox equivalent.c AAE, ascorbic acid equivalent.d EDTAE, ethylenediaminetetraacetic acid (disodium salt) equivalent.
CUPRAC (mg of TE per g of dry plant)^b	10.57 ± 0.07^a	21.10 ± 0.33^b
FRAP (mg of TE per g of dry plant)^b	3.63 ± 0.04^a	14.41 ± 0.32^b
Iron(III) to iron(II) reduction (mg of TE per g of dry plant)^b	3.27 ± 0.13^a	13.74 ± 0.79^b
Phosphomolybdenum (mg of AAE per g of dry plant)^c	76.19 ± 1.38^b	49.53 ± 0.52^a
Chelating effect (mg of EDTAE per g of dry plant)^d	2.28 ± 0.03^a	39.48 ± 2.19^b

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Radical scavenging assays were resulted in the superiority of the water extract. As can be seen from the data presented in the Table 3, the water extract showed remarkable scavenging activity on NO (289.01 mg of BHAEs per g of dry plant). O₂˙⁻ scavenging effect of this extract was determined as 191.70 mg of BHAE per g of dry plant. The ethanol extract also exhibited a promising activity on these radicals (158.78 and 54.15 mg of BHAE per g of dry plant, respectively). The water extract exhibited the weakest activity on ABTS˙⁺ (12.88 mg of TE per g of dry plant). As can be seen from the table, this value was found to be nine folds greater than that of the ethanol extract (1.43 mg of TE per g of dry plant). Scavenging activity of the water extract on HO˙ and DPPH radicals were determined as 73.01 mg of ME per g of dry plant and 21.77 mg of TE per g of dry plant, respectively.

As mentioned above, the reducing power of the ethanol and water extracts of C. botrys were determined by employing CUPRAC, FRAP, and iron(III) to iron(II) reduction assays (Table 4). As happened in the radical scavenging part of this study, all of the assays were resulted in the superiority of the water extract. Reducing power potential of the water extract was measured as 21.10, 14.41, and 13.74 mg of TE per g of dry plant in these test systems, respectively. The ethanol extract also exhibited a promising activity in CUPRAC assay (10.57 mg of TE per g of dry plant). Reducing power potential of the ethanol extract in FRAP and iron(III) to iron(II) reduction assays were found to be almost equal to the each other (3.63 and 3.27 mg of TE per g of dry plant, respectively).

Antioxidant activities of the extracts were also evaluated by using phosphomolybdenum assay (Table 4). Unlike the results obtained from the other test systems, phosphomolybdenum assay was resulted in the superiority of the ethanol extract (76.19 mg of AAE per g of dry plant). In this assay, the antioxidant activity of the water extracts was determined as 49.53 mg of AAE per g of dry plant. In the case of chelating effect assay, the activity of the water extract (39.48 mg of EDTAE per g of dry plant) was found to be higher than that of the ethanol extract (2.28 mg of EDTAE per g of dry plant).

As far as our literature survey could ascertain, we could reach no report concerning the antioxidant activity of C. botrys. Therefore, data presented here could be assumed as the first report on this topic. However, whole and milled fractions of C. quinoa, which is another member of Chenopodiaceae, were evaluated for their antioxidant properties.²² According to the results of this study, ferulic and vanillic acids were found to be the principal phenolic acids. Additionally, rutin and quercetin were found to be as the predominant flavonoids detected in whole grain and milled fractions. Hemalatha et al.²² claimed that despite having relatively lower phenolic contents dehulled and milled grain fractions showed significantly higher metal chelating activity than those of the other fractions.

As can be seen from the Table 2, the water extract contained considerable amounts of benzoic, ferulic, and chlorogenic acids (2974.24, 21.9.65, and 118.83 μg per g of dry plant, respectively). Our research team has previously reported the total antioxidant activities of the selected phenolic acids.⁴⁷ According to this report, total antioxidant activity of p-hydroxy benzoic acid was found to be 95.26%. Sevgi et al.⁴⁷ also reported the total antioxidant activities of ferulic, chlorogenic, syringic, and cinnamic acids as 94.06, 85.73, 91.94, and 93.14%, respectively. In this report, ferulic, cinnamic, and p-hydroxybenzoic acids were also found to have remarkable DPPH free radical scavenging activities at 0.5 mg mL⁻¹ concentration (88.69, 87.30, and 84.73%, respectively). At 0.3 mg mL⁻¹ concentration, p-hydroxybenzoic acid showed higher activity than that of the butylated hydroxyl anisole (BHA).⁴⁷

3.3. Enzyme inhibitory activity

C. botrys extracts were tested for their inhibitory activities on AChE, BChE, tyrosinase, α-amylase, and α-glucosidase (Table 5). As can be seen from the data presented in the table, inhibitory activity of the water extract (113.69 μg of GALAE per g of dry plant) was found to be superior to the ethanol extract (50.87 μg of GALAE per g of dry plant). On the other hand, the extracts exhibited a different activity profile in BChE inhibitory assay. According to the results of this assay, the ethanol extract showed higher inhibitory activity (60.90 μg of GALAE per g of dry plant) than the water extract (23.40 μg of GALAE per g of dry plant).

Table 5 Enzyme inhibitory activity of C. botrys extracts (mean ± SD)^a

Assays	Ethanol	Water
a Different superscript letters in the same row indicate significant difference (p < 0.01).b GALAE, galantine equivalent.c KAE, kojic acid equivalent.d ACE, acarbose equivalent.
Acetyl cholinesterase (μg GALAE per g of dry plant)^b	50.87 ± 0.47^a	113.69 ± 5.01^b
Butyryl cholinesterase (μg GALAE per g of dry plant)^b	60.90 ± 0.13^b	23.40 ± 1.00^a
Tyrosinase (μg KAE per g of dry plant)^c	39.45 ± 11.29^a	165.56 ± 7.33^b
α-Amylase (mg of ACE per g of dry plant)^d	6.80 ± 0.01^a	7.16 ± 0.05^b
α-Glucosidase (mg of ACE per g of dry plant)^d	4.41 ± 0.01^a	6.47 ± 0.12^b

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In tyrosinase, α-amylase, and α-glucosidase inhibitory assays, the water extract showed obviously the highest activity when compared with the ethanol extract. Inhibitory activity of the water extract on these enzymes was determined as 165.56 μg of KAE per g of dry plant, 7.16, and 6.47 mg of ACE per g of dry plant, respectively. It is extremely important to point out that α-amylase and α-glucosidase inhibitory activities of the water and methanol extracts were found to be too close to the each other.

As far as our literature survey could ascertain, enzyme inhibitory activity of C. botrys has not previously been reported elsewhere. However, whole and milled fractions of C. quinoa, another member of Chenopodiaceae, were evaluated for their phenolic compositions and their inhibitory effects on α-amylase and α-glucosidase were studied.²² According to the results of this study, the extracts of bran and hull fractions displayed strong inhibition on α-amylase [IC₅₀, 108.68 μg mL⁻¹ and 148.23 μg mL⁻¹, respectively] and α-glucosidase [IC₅₀, 62.1 μg mL⁻¹ and 68.14 μg mL⁻¹, respectively] activities.²²

4. Conclusions

The water extract of C. botrys showed a remarkable antioxidant activity in almost all test systems. Additionally, the water extract was found to be as the potent inhibitor of AChE, tyrosinase, α-amylase, and α-glucosidase. According to the results of the antioxidant and enzyme inhibitory activity assays, C. botrys may have the potential to prevent diseases associated with oxidative stress and to prevent AD, pigmentation diseases, hyperglycaemia and its associated complications. The water extract was found to contain considerable amounts of benzoic, ferulic, and chlorogenic acids. In order to establish a clear connection between the phytochemical composition and biological activity potential of the plant samples, biological activity guided chromatographic fractionation techniques should be applied as the next step of this study. By this way, phytochemicals responsible for these activities could be documented clearly.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors would like to thank to the Scientific Research Council of Celal Bayar University, Manisa-Turkey for the financial support (Project Number: 2015-088). The authors also would like to thank to Dr Olcay Ceylan for his kind help in identifying the plant material used in this study.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13229d

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