Anthraquinone profile, antioxidant and enzyme inhibitory effect of root extracts of eight Asphodeline taxa from Turkey: can Asphodeline roots be considered as a new source of natural compounds?

Abstract

Plant-based foods have become attractive for scientists and food producers. Beneficial effects related to their consumption as dietary supplements are due to the presence of natural occurring secondary metabolites. In this context, studies on these products are important for natural and safely food ingredients evaluation. The aim of this study was to evaluate root extract of eight Asphodeline species as antioxidants, enzyme inhibitors and phytochemical content. Spectrophotometric antioxidant and enzyme inhibitory assays were performed. Total phenolic and flavonoids contents as well as the chemical free-anthraquinones profiles were determined using routinely procedure (HPLC-PDA). Data show that Asphodeline roots can be considered as a new source of natural compounds and can be used as a valuable dietary supplement. Some differences related to biological activities can be inferred to other phytochemicals that can be considered in the future for their synergic or competitive activities.

• Anthraquinones
• antioxidant
• Asphodeline
• enzyme inhibitory effects
• Turkey

Introduction

Functional foods or plant-based foods have recently become an attractive subject for many scientists and food producers. Several epidemiological studies have shown an association between their intake and some diseases such as Alzheimer, diabetes, cancer, cardiovascular disease and cataracts1. Beneficial effects are mainly due to the presence of plants secondary metabolites. Among plant secondary metabolites, phenolics are of special interest related to their activities, such as antioxidants2^,3, antimutagenic4^,5, anti-inflammatory6^,7 and inhibitions of enzymes associated with global diseases including Alzheimer, diabetes mellitus8–11. In this context, new studies performed on plants or plant-derived products are very important for the search of natural and safely functional food ingredients.

Anthraquinones are a group of phenolics and are widely distributed in many plant families such as Fabaceae, Liliaceae, Rubiaceae and Rhamnaceae12. Anthraquinones derivatives such as emodine, physcione, rhein and chrysophanol have been used as colorants in food, drugs and cosmetics. Nowadays, anthraquinones attracted attention especially related to their interesting biological properties. They are known for their different biological activities including antimicrobial13, anticancer14, antioxidant15 and anti-inflammatory16. In this direction, it is suggested that consumption of anthraquinones-rich plants such as Rhamnus and Frangula species17–19 can represent a valid intake way in order to benefit of their biological activities. From this point, the presence of anthraquinones is valuable as important criterion in the plants quality used for medicinal purposes.

Asphodeline (Xanthorrhoeaceae, the genus was recently classified under the family Liliaceae) is a genus of about 14 species, widespread in Mediterranean Region, mainly the Middle East-countries. The genus is presented in Europe by only three species, which are A. lutea, A. liburnica and A. taurica. In Turkey, this genus contains 20 taxa, 12 of which are endemic to this country20^,21. Plants of the genus Asphodeline have traditionally used as medicinal plants in various parts of the world. For example, some members of Asphodeline (A. damascena subsp. damascena and A. tenuior subsp. tenuiflora var. tenuiflora) are used to alleviate warts and heal wounds in Turkey folk medicine. Likewise, A. cilicica and A. globifera are used as folk medicine for the treatment of earaches and hemorrhoids, respectively. Again, several Asphodeline species (A. cilicica, A. damascena, A. globifera, A. lutea and A. taurica) are consumed as salad vegetables in different Turkey regions22. Recent studies show that Asphodeline leaves had good nutritional quality with high anthraquinones, essential amino acids and polyphenols levels23–26. However, to the best of our knowledge, the information on the anthraquinone profiles and biological effects of Asphodeline is scarce. Thus, the aim of this study was to evaluate root extract of eight Asphodeline species as antioxidants and enzyme inhibitors. In addition, total phenolic and flavonoids contents as well as the chemical free-anthraquinones profile were determined.

Methods

Plant material and methanol extracts

Asphodeline species were collected at flowering stage (May–July) in Turkey regions, and their information and localities are supplied in Supplementary material Section S.1. Voucher specimens were deposited in KNYA Herbarium (Department of Biology, Selcuk University, Konya, Turkey). The roots, dried at ambient conditions in the dark, were finely triturated (5–10 g) and macerated overnight with 250 mL of methanol at room temperature (25 °C). The concentrated extracts (under vacuum; 40 °C) were stored at +4 °C in dark until analyses (extraction yields are reported in Table 1).

Table 1. Extraction yields, total phenolics, flavonoids and free anthraquinones content for reported taxa.

	Extraction yield (%)	Phenolic content (mgGAEs/g extract)	Flavonoid content (mgREs/g extract)	Anthraquinones content (mg/g)
A. anatolica	12.2	24.2 ± 0.7*	11.4 ± 0.6	211.8
A. baytopae	24.9	18.3 ± 0.4	17.3 ± 0.1	1136
A. brevicaulis	25.7	13.5 ± 0.3	10.6 ± 0.5	193.6
A. cilicica	6.53	49.2 ± 1.5	30.9 ± 1.4	314.9
A. sertachea	16.2	25.6 ± 0.2	23.7 ± 0.4	617.8
A. globifera	11.6	23.9 ± 0.9	24.2 ± 0.7	603.7
A. peshmeniana	16.8	24.6 ± 0.3	30.3 ± .0.6	603.4
A. rigidifolia	11.9	27.8 ± 0.2	25.3 ± 2.6	689.5

GAE, gallic acid equivalents; RE, rutin equivalents.

*Expressed are means ± SD of three measurements.

HPLC chemicals and reagents

Anthraquinones chemical standards (all >99%) were purchased from Extrasynthese (Genay, France). Methanol (HPLC-grade) and formic acid (99%) were obtained from Carlo Erba Reagenti (Milan, Italy). Double-distilled water was obtained using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA).

Determination of total bioactive components

Total phenolics, flavonoids and free anthraquinones contents

The total phenolics content was determined by a reported method27 with slight modification and expressed as gallic acid equivalents (GAEs/g extract), while total flavonoids content was determined by a reported method28 with slightly modification and expressed as rutin equivalents (REs/g extract). HPLC analyses were performed following validated method6 using a Waters model 600 solvent pump, a C18 column (GraceSmart RP18, 4.6 mm × 150 mm, 5 μm; Grace, Deerfield, IL) at 28 ± 1 °C, a 2996 photodiode array detector and Empower v.2 Software (Waters Spa, Milford, MA) was used for data acquisition in the range of 200–500 nm.

Biological activities evaluation

The antioxidant activity was evaluated by phosphomolybdenum and β-carotene bleaching methods28. Radical scavenging activities, measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2 azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS)28, were expressed as trolox equivalents (TEs/g extract). The reducing power measured using cupric ion reducing (CUPRAC) and ferric ion reducing antioxidant power (FRAP). Metal chelating activity on ferrous ions, determined by the method described by Zengin et al.28, was expressed as EDTA equivalents (EDTAEs/g extract). Acetylcholinesterase (AChE) or butyrylcholinesterase (BChE), α-amylase, α-glucosidase and tyrosinase inhibitory activities were carried out by the method described by Zengin et al.28

Results and discussion

Total phenolic and flavonoids content

Total phenolics and flavonoids are presented in Table 1. The highest and lowest levels were found in A. cilica and A. brevicaulis, respectively, with values comparable with A. lutea (22.45 mgGAEs/g for Bulgarian origin and 17.26 mgGAEs/g for Turkish origin26). Total phenolics content in A. anatolica29 is higher than reported in the present study, but the differences can be associated to the different extraction method (soxhlet and maceration) used, in agreement with earlier studies30–32.

Total free anthraquinones content and HPLC analyses

Total free-anthraquinones fractions are presented in Table 1 (expressed as mg/g of dry raw material, see also Supplementary material, Section S.2). Figure 1 are reported the chromatographic profiles for the analyzed root plant samples.

Figure 1. Chromatographic profiles for the analyzed root plant samples.

Free radical scavenging activity (DPPH and ABTS assays), reducing power (FRAP and CUPRAC assays) and chelating activity

DPPH and ABTS free radicals used to evaluate radical scavenging activity. Antioxidant compounds interact with free radicals (DPPH and ABTS) by electron or hydrogen atom transfer, thus converting them into stable and non-radical molecules. A. cilicica exhibited better performance against DPPH, followed by A. sertachae, and A. peshmeniana (Table 2). As to ABTS radical scavenging activity, A. cilicica and A. rigidifolia extracts possessed the highest antioxidant capacity. ABTS activities can be explained with the flavonoids level, in accordance with a positive correlation33^,34. A. brevicaulis had the lowest radical scavenging activity. According to the trolox equivalent, A. brevicaulis had less effective antioxidant capacity than both A. lutea and A. anatolica26^,29. These differences may be explained by different phenolics level in extracts. FRAP and CUPRAC assays were employed to evaluate reducing abilities (Table 2). High reducing power variation was observed among the samples. A. cilicica exhibited higher value than other extracts, and could be related to high polyphenols, flavonoids or anthraquinones contents. Emodine and rhein show the highest levels in A. cilicica, A. anatolica and A. peshmeniana, and the strongest reducing capacity for these anthraquinones was reported in the literature18^,35, which reported that high emodine and rhein levels make a positive contribution to antioxidant properties, including reducing power.

Table 2. Free radical scavenging, reducing power and metal chelating activity.

	Radical scavenging (mgTEs/g extract)		Reducing power (mgTEs/g extract)		Metal chelating (mgEDTAEs/g extract)
Samples	DPPH	ABTS	FRAP	CUPRAC	Ferrous ion chelating
A. anatolica	27.52 ± 1.08*	137.60 ± 2.11	58.11 ± 1.25	72.61 ± 0.99	15.09 ± 0.52
A. baytopae	17.42 ± 1.31	50.88 ± 8.20	38.55 ± 2.46	54.85 ± 1.38	9.81 ± 0.20
A. brevicaulis	6.08 ± 2.35	31.57 ± 0.96	30.61 ± 1.14	41.95 ± 0.61	9.84 ± 0.28
A. cilicica	39.67 ± 1.12	213.71 ± 3.64	77.92 ± 2.03	123.59 ± 1.41	11.11 ± 0.48
A. globifera	30.01 ± 1.33	125.10 ± 7.32	54.80 ± 2.35	77.72 ± 2.32	16.01 ± 0.32
A. peshmeniana	27.71 ± 0.40	111.73 ± 3.31	56.93 ± 3.16	76.57 ± 0.76	15.72 ± 0.13
A. rigidifolia	26.70 ± 1.62	140.00 ± 2.10	55.78 ± 1.75	85.27 ± 1.55	13.70 ± 1.03
A. sertachae	29.63 ± 1.61	122.49 ± 3.91	55.44 ± 1.79	88.19 ± 0.90	13.03 ± 0.52

TE, Trolox equivalents; EDTAE, EDTA equivalents.

*Expressed are means ± SD of three measurements.

Chelating ferrous ion (Fe²⁺) may prevent reactive oxygen species (ROS) production36, and all extracts demonstrated this ability (Table 2), with higher values in A. globifera, followed by A. peshmeniana, and A. anatolica. Chelating results are inconsistent with total phenolics content in Asphodeline extracts, and there are contradictory reports regarding this capacity37^,38. In this direction, extracts chelating abilities could be ascribed to anthraquinones (emodine, rhein, chrysophanol, etc.) that show this ability, as previously reported35.

Total antioxidant capacity by phosphomolybdenum and β-carotene/linoleic acid assays

Results for phosphomolybdenum assay are given in Table 3. A. cilicica and A. anatolica extracts showed the highest total antioxidant capacity. Other Asphodeline extracts have similar antioxidant capacity. Zengin and Aktumsek29 also reported that the trolox equivalent values of different parts of A. anatolica ranged between 258.34 and 438.32 mgTEs/g, and Lazarova et al.26 found to be 236.80 and 232.44 mg ascorbic acid equivalents (AEs/g) for phosphomolybdenum reduction activity in A. lutea roots from Bulgaria and Turkey, respectively.

Table 3. Antioxidant activity by phosphomolybdenum and β-carotene/linoleic acid assays.

	Total antioxidant capacity
Samples	Phosphomolybdenum (mmolTEs/g extract)	β-Carotene/linoleic acid assay (%)
A. anatolica	1.76 ± 0.15*	85.82 ± 0.28
A. baytopae	1.44 ± 0.01	81.14 ± 1.38
A. brevicaulis	1.43 ± 0.08	70.77 ± 1.36
A. cilicica	2.94 ± 0.11	97.65 ± 0.42
A. globifera	1.45 ± 0.03	92.73 ± 0.79
A. peshmeniana	1.40 ± 0.03	89.54 ± 0.06
A. rigidifolia	1.43 ± 0.04	92.74 ± 0.80
A. sertachae	1.35 ± 0.03	94.98 ± 0.73
BHT	n.t.	97.32 ± 0.17
Trolox	n.t.	93.34 ± 0.27

TE, Trolox equivalents; n.t., no tested.

*Expressed are means ± SD of three parallel measurements.

The discoloration rate of β-carotene depends on the antioxidant capacity39. All extracts were able to inhibit linoleic acid oxidation. A. cilicica, A. sertachae and A. rigidifolia inhibited the linoleic acid oxidation (Table 3). This trend was similar to that observed in total flavonoids content, where these extracts have the higher amount. The high inhibition ability could be explained by the presence of flavonoids level, accordingly to the literature38^,39. A. cilicica showed higher inhibition compared to trolox, although close to that of BHA. Results showed also that the least activity was found in A. brevicaulis with the lowest inhibition value. However, the Asphodeline extracts can be considered as natural inhibitors of lipid oxidation in food industry due to concerns on the use of synthetic antioxidants, in agreement with those obtained by Zengin and Aktumsek29 for A. anatolica extracts.

Enzyme inhibitory activities

Recently, key enzymes inhibition involved in the pathogenesis of several severe diseases is considered as one of the most effective strategies in these diseases treatment. Many synthetic inhibitors have been developed (galanthamine, acarbose or kojic acid), even if some of these are suspected of being responsible for secondary adverse effects such as gastrointestinal disturbances and liver damage40^,41. There is an increasing interest in finding natural enzyme inhibitors from plant materials in order to replace synthetic ones. The Asphodeline enzyme inhibitor activities are reported here for the first time. The extracts were tested for their in vitro cholinesterase inhibitory activities (AChE and BChE) by the spectrophotometric Ellman’s method. Results are provided in Table 4. A. peshmeniana exhibited an activity against AChE, while the highest inhibitory effect on BChE was obtained by A. cilicica. Interestingly, A. peshmeniana showed the lowest inhibitory activity against BChE. This variation may be explained by differences in phytochemicals of the Asphodeline species. Extracts show amylase and glucosidase inhibitory activity. A. cilicica, A. sertachae, A. rigidifolia and A. anatolica have higher amylase and glucosidase inhibitory activities than other Asphodeline species. A. cilica, which had the highest level of phenolics, shows the best activity against both amylase and glucosidase. In this sense, phenolics may be a valuable responsible for the inhibitory capacity, in accordance with the literature42^,43. A. baytopae and A. brevicaulis displayed the lowest inhibitory activity with lower phenolics levels. A. sertachae showed potent antityrosinase activity compared to other extracts. A. sertachae showed tyrosinase inhibitory activity approximately twofold higher than that of A. cilicica extract. A. cilicia has the lowest inhibitory activities on tyrosinase despite the high content of total phenolics, and strong antioxidant properties compared to other tested extracts. These findings were in contrast with the literature44^,45 where was found a correlation between phenolics content and antityrosinase activity. The lack of correlation can result from the difference in mechanism involved. From these data, it can be inferred that other phytochemicals, not investigated in this project, besides phenolics in Asphodeline extract contributed to the greater levels of tyrosinase inhibitory activity.

Table 4. Enzyme inhibitory activity of Asphodeline taxa.

	Cholinesterase inhibition		Antidiabetic assay		Skin care effects
Samples	AChE inhibition*	BChE inhibition*	α-Amylase inhibition†	α-Glucosidase inhibition†	Tyrosinase inhibition‡
A. anatolica	1.73 ± 0.03¶	1.76 ± 0.07	0.50 ± 0.02	2.42 ± 0.08	22.27 ± 2.64
A. baytopae	1.67 ± 0.11	1.94 ± 0.37	0.34 ± 0.02	1.63 ± 0.13	25.04 ± 1.45
A. brevicaulis	1.61 ± 0.04	1.73 ± 0.28	0.37 ± 0.02	1.10 ± 0.21	25.50 ± 0.41
A. cilicica	1.62 ± 0.12	1.98 ± 0.40	1.24 ± 0.03	4.99 ± 0.11	18.57 ± 0.64
A. globifera	1.76 ± 0.09	1.75 ± 0.15	0.60 ± 0.03	2.12 ± 0.28	23.44 ± 0.63
A. peshmeniana	1.85 ± 0.05	1.67 ± 0.14	0.48 ± 0.01	2.23 ± 0.20	23.56 ± 2.17
A. rigidifolia	1.67 ± 0.04	1.88 ± 0.12	0.65 ± 0.02	3.15 ± 0.16	28.38 ± 1.03
A. sertachae	1.66 ± 0.14	1.93 ± 0.20	0.77 ± 0.02	3.07 ± 0.16	33.84 ± 1.92

*Expressed as mg GALAEs/g extract; GALAE, galanthamine equivalents.

†Expressed as mmol ACEs/g extract; ACE, acarbose equivalents.

‡Expressed as mg KAE/g extract; KAE, kojic acid equivalents.

¶Values expressed are means ± SD of three parallel measurements.

Conclusion

The aim of this study was to evaluate root extract activities of eight Asphodeline species, in order to test their use as functional or plant-based foods. Total phenolics and flavonoids contents and free anthraquinones profiles were reported for each species. The enzyme inhibitory activities of the Asphodeline species are reported for the first time as well as anthraquinone profiles. Generally, results showed a strong relationship between biological activities (antioxidant and enzyme inhibitory activities) and bioactive components in Asphodeline species. However, other phytochemicals, besides phenolics and anthraquinones, could contribute to tyrosinase inhibitory activity reported. Indeed, there is a current need for availability of new plant derived bioactive molecules, thus Asphodeline species may be a great natural source for preparing of new functional foods and drug formulations.

Supplementary materials online only – For review only at proofing stage.

Acknowledgements

Authors thank Dr. Luciano Malatesta for his helpful, and friendly collaboration during analyses.

Declaration of interest

Authors declare no conflict of interest. Financial support for this research is from the Selcuk University, Science Faculty, Department of Biology and from University “G. d’Annunzio” of Chieti-Pescara.