Assessment of Phenolic Acid Content and In Vitro Antiradical Characteristics of Hawthorn

Abstract

The infusions and extracts obtained from leaves with flowers, fruit peel, and seed from hawthorn (Crataegus monogyna Jacq., Family Rosaceae) were subjected to evaluation as potential sources of antioxidant phytochemicals on the basis of their total content of phenolics, levels of phenolic acids, and in vitro antiradical activity. Total phenolic content of extracts was determined using the modified Folin–Ciocalteau method. Antioxidant activity was determined for phenolic extracts by a method involving the use of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Phenolic acids containing extracts and infusions from hawthorn leaves, fruit peel, and seeds were obtained using different polarity solvents and separated by reverse-phase high-performance liquid chromatography, which enabled improved separation by the use of a C₁₈ column, an acidic mobile phase, and gradient elusion. The highest total phenolic content (343.54 mg of gallic acid equivalents/g) and the highest DPPH radical scavenging activity as the inhibition percentage (60.36%) were obtained in ethyl acetate extract from hawthorn leaves with flower. Also, the highest phenolic acid content was measured in the extracts of hawthorn leaves with flowers: protocathechuic (108–128 mg/100 g), p-hydroxy benzoic (141–468 mg/100 g), caffeic (137–3,580 mg/100 g), chlorogenic (925–4,637 mg/100 g), ferulic (3,363–3,462 mg/100 g), vanillic (214 mg/100 g), and syringic (126 mg/100 g) acids. The results indicate that hawthorn is a promising plant because of its high antioxidant activity.

Introduction

H awthorn is the common name of all plant species in the genus Crataegus [especially Crataegus monogyna Jacq. and Crataegus laevigata (Poiret) DC. (syn. Crataegus oxyacantha L.)] of the Rosaceae family, and there are approximately 280 known species. It is known as a traditional medicinal plant in some countries. The fruits of native hawthorns are also edible.¹

C. monogyna fruits, leaves, and flowers are known to contain numerous chemical constituents, such as 1–2% flavonoids (hyperoside, apigenin, luteolin-7-glucoside, rutin, quercetin, vitexin, vitexin rhamnosides, and spirein),^2,3 2–3% proanthocyanidins,⁴ sterols, trace amounts of cardioactive amines,⁵ triterpenes,⁶ phenolic acids (chlorogenic and caffeic acids),^2,7 and essential oil.⁴

Extracts of hawthorn (C. monogyna Jacq.) have become a popular herbal drug for their well-recognized cardiotonic effects in addition to other activities, including anti-arrhythmic,⁶ hypotensive,⁸ hypolipidemic,⁴ and antioxidant^7,9,10 effects.

It is reported that reactive oxygen species cause the development of many chronic disorders, such as cancer, arteriosclerosis, nephritis, diabetes mellitus, rheumatism, and cardiovascular diseases, as well as being active in gastrointestinal tract disorders and inflammatory injury.¹¹ Phenolic compounds (such as flavonoids, anthocyanins, proanthocyanins, and phenolic acids), naturally occurring substances essentially in all plant materials, are also potent antioxidants that play an important role in human nutrition as preventative agents against several diseases, protecting the body tissues against oxidative stress.¹²

Phenolic acids are secondary metabolites that are commonly found in plant-derived foods. Substituted derivatives of hydroxybenzoic and hydroxycinnamic acids are the predominant phenolic acids in plants, with hydroxycinnamic acids being the more common. These derivatives differ in the patterns of the hydroxylations and methoxylations of their aromatic rings.¹³ They can be analyzed by thin-layer chromatography,^14,15 gas chromatography,¹⁵ high-performance liquid chromatography (HPLC)–diode array detection,^2,15 and capillary electrophoresis.^16
–18

According to our literature survey, there has been no study yet on the chemical and physical properties of hawthorn fruit peel and seeds. Thus, it was aimed to investigate the content of phenolic acids in seeds, fruit peel, and leaves with flowers of hawthorn (C. monogyna) using certain extraction methods with solvents of different polarities. In addition, the total phenolic content (TPC) values were determined, and each extract was evaluated for antiradical activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In assay-guided screening of plant samples, simple, sensitive, and rapid methods that are applicable to different sample types and give reproducible results are preferable. The DPPH method, which is very rapid, simple, sensitive, and reproducible, does not require special instrumentation. In this study, it was very convenient for the screening of large numbers of samples of different polarity. The scavenging abilities of the extracts were also compared with that of tert-butylhydroxytoluene (BHT) as a synthetic antioxidant.

Materials and Methods

Reagents

Chemicals used for HPLC (methanol and formic acid) were of chromatographic grade (Sigma-Aldrich, St. Louis, MO, USA). The phenolic acid standards gallic acid (GA), protocathechuic acid (protoCA), p-hydroxybenzoic acid (p-hydBA), vanillic acid (VA), caffeic acid (CA), chlorogenic acid (ChA), syringic acid (SA), and ferulic acid (FA), propylparaben as the internal standard, DPPH, and Folin–Ciocalteau reagent were purchased from Sigma-Aldrich and Merck GmbH (Darmstadt, Germany).

Samples

Plant materials, purchased from a local herbalist store in Eskişehir, Turkey, had been prepared in different times of the seasons. It was stated that leaves with flowers were harvested wilting and fruits were collected and prepared after the fruits were grown to some extent to be eaten.

Extraction

Extraction procedures were applied as described elsewhere.² Dried leaves with flowers, fruit peel, and seeds of hawthorn were ground and extracted with petroleum ether in a Soxhlet apparatus. Fat-free air-dried material was extracted with methanol:water (70:30 vol/vol) in a 40°C water bath for 30 minutes four times. The extract was concentrated to dryness in vacuo, and aqueous solution was lyophilized (methanol extract [ME extract]). Second extracts were prepared as follows: fat-free air-dried material was extracted with methanol:water (70:30 vol/vol) in a 40°C water bath for 30 minutes four times and concentrated in vacuo, and the aqueous phase was extracted with ethyl acetate at room temperature. Then it was concentrated to dryness in vacuo (ethyl acetate extract [EA extract]), and the aqueous solution was lyophilized (water extract [W extract]). The infusions from dried plant material were prepared as 2% solutions, and then they were lyophilized (infusion). The phenolic acids were then recovered with solvents of different polarities. All extracts obtained were weighed to determine the yields of soluble constituents.

Analytical HPLC

An HPLC system consisting of a model 600 E HPLC pump, model 717 Plus Autosampler, model 996 photodiode array detector, and the data processor of a Millenium 32 chromatograph was used for the analysis (all from Waters Corp., Milford, MA, USA). Standards or hawthorn extracts were analyzed on a reverse-phase C₁₈ Ultrasphere column (Teknokroma, Barcelona, Spain) (100×4.6 mm i.d.; particle size, 3 μm), and a Shimadzu (Kyoto, Japan) model 160A spectrophotometer was used.

Ultra pure deionized water was purified by Synergy Water Purification System (Millipore, Rotterdam, Netherlands) to a specific resistance of 18 mΩ cm.

Determination of TPC

The amounts of total phenols were determined spectrophotometrically at 750 nm, based on a colorimetric measurement for extracts as described previously.^19,20 This method gives a general measurement of phenolic content, as it is not completely specific for phenolic compounds and not all phenolic compounds exhibit the same level of activity in the assay.

Separation and analysis of phenolic acids by HPLC

Chromatographic analysis of the extracts was carried out by a gradient elution (solution A, methanol:water:formic acid [10:88:2 by volume]; solution B, methanol:water:formic acid [90:8:2 by volume]) as reported elsewhere.²¹ Chromatography was performed by using a linear gradient program. The flow-rate was 1 mL/minute, and the injection volume was 10 μL. Signals were detected at 280 nm. The internal standard technique was applied to increase the repeatability.

The relevant extracts were dissolved in a mixture of methanol and water (1:1 vol/vol), and the mixture was injected into the HPLC apparatus.

Radical scavenging activity using the DPPH method

The DPPH assay was used as a rapid spectroscopic method to provide an evaluation of antioxidant activity due to scavenging free radicals. Being a stable free radical colored purple, DPPH^· is reduced to the yellow diphenylpicryl hydrazine.

Free radical scavenging effects of the extracts on DPPH^· were estimated according to the method of Sanchez-Moreno et al. ²² applying some modifications. The reaction mixture was left at ambient temperature for 30 minutes in dark, and the absorbance of the resulting solution was then measured spectrophotometrically at 517 nm.

Results and Discussion

Analysis of phenolic acids by HPLC

Chromatographic analysis was used to identify and quantify phenolic acids present in hawthorn. Screening of the studies indicates us that little information is available in the literature on the content and types of phenolic acids in hawthorn. In addition to that lack of knowledge, isolation and quantifications are difficult because of the great variety of species present and the wide variations in their levels. Thus, it is essential to devise a sample preparation stage that will ensure reliable identification and quantification.

The phenolic acids in different parts of hawthorn were analyzed by gradient elution using a validated HPLC method that has a good repeatability using the internal standard technique in the range of 0.35%–1.65% relative standard deviation (RSD); as percent they have limits of detection (LOD) values in the range of 2.49×10⁻⁶–9.69×10⁻⁶ M and limits of quantification (LOQ) values of 1.27×10⁻⁶–2.93×10⁻⁵ M as reported elsewhere.²¹

Most of the phenolic acids have absorption maxima in the ultraviolet absorption spectra at a wavelength of 280 nm, and they were identified by matching their retention times (peak normalization) and ultraviolet spectra of samples with those of authentic standards, using the HPLC–diode array detection system. The original chromatograms of hawthorn EA extracts from fruit peel, seeds, and leaves with flowers and methanol containing only internal standard (blank) are shown in Figure 1.

FIG. 1.

Representative chromatograms of ethyl acetate extracts of hawthorn fruit peel (HPEA), seeds (HSEA), and leaves with flowers (HLFEA) and methanol containing only internal standard (MeOH+IS) (blank). The phenolic acids marked are gallic acid (peak 1), protocathechuic acid (peak 2), p-hydroxybenzoic acid (peak 3), vanillic acid (peak 4), caffeic acid (peak 5), chlorogenic acid (peak 6), syringic acid (peak 7), ferulic acid (peak 8), and propylparaben internal standard (peak 9) on the chromatograms. AU, arbitrary units.

The amounts of the relevant phenolic acids were calculated by using their calibration equations (Table 1). As shown in Table 1, by HPLC analysis ChA was most abundant (43–4,637 mg/100 g of extract), followed by FA (3,363–3,462 mg/100 g of extract), CA (12–3,580 mg/100 g of extract), p-hydBA (41–2,651 mg/100 g of extract), protoCA (19–588 mg/100 g of extract), VA (34–214 mg/100 g of extract), SA (21–126 mg/100 g of extract), and GA (19.8–86 mg/100 g of extract).

Table 1.

Content of Phenolic Acids in Hawthorn Fruit Peel, Seeds, and Leaves with Flowers Extracted into Different Solvents

	Phenolic acid (mg/100 g of extract)
Extract	GA	ProtoCA	p-hydBA	VA	CA	ChA	SA	FA
HLF
ME	—	128.0	350.0	—	137.0	4,145.0	—	3,384.0
EA	—	128.0	141.0	—	206.30	4,637.0	—	3,462.0
W	—	108.0	468.0	—	762.0	4,145.0	—	3,363.0
Inf	—	211.0	347.0	214.0	3,580.0	925.0	126.0	3,363.0
HP
ME	26.0	—	54.3	—	31.0	58.0	26.4	—
EA	38.0	286.0	474.0	142.0	79.0	1,353.0	21.0	—
W	—	—	ND	—	ND	ND	—	—
Inf	19.8	32.0	76.5	—	20.0	—	—	—
HS
ME	24.7	23.7	76.8	34.0	26.0	43.0	—
EA	86.0	588.0	2,651.0	—	296.0	594.0	—
W	—	19.0	—	—	12.0	46.0	—
Inf	33.5	73.5	41.7	42.0	30.2	—	24.7

CA, caffeic acid; ChA, chlorogenic acid; EA, ethyl acetate extract; FA, ferulic acid; GA, gallic acid; HLF, hawthorn leaves with flowers; HP, hawthorn fruit peel; HS, hawthorn fruit seed; p-hydBA, p-hydroxybenzoic acid; Inf, infusion; ME, methanol extract; ND, not detectable; protoCA, protocathechuic acid; SA, syringic acid; VA, vanillic acid; W, water extract.

There are five phenolic acids (ChA, CA, protoCA, p-hydBA, and FA) in the extracts of C. monogyna leaves with flowers. These extracts contain high levels of ChA, with the concentration in the ME extract being 4,145 mg/100 g, in the EA extract 4,637 mg/100 g, and in the W extract 4,145 mg/100 g.

Although the presence of ChA and CA in dried flowers and leaves of hawthorn has already been shown in previous studies using HPLC methods with reference compounds,^2,3 protoCA, p-hydBA, and FA have not been reported previously. In the present study, we found these three compounds in addition to ChA and CA for the first time.

In the fruit peel of C. monogyna, according to our HPLC findings, the most abundant compound is ChA in ME and EA extracts; however, the infusion form has mostly p-hydBA. The ME fraction has also five phenolic acids (GA, p-hydBA, CA, ChA, and SA), whereas the EA fraction has seven phenolic acids (GA, protoCA, p-hydBA, VA, CA, ChA, and SA). The amounts of phenolic acids detected in the W extract are very small.

The main component in the seed of the hawthorn fruit is p-hydBA. FA is not detectable in the seeds as well as in the leaves with flowers. The seeds contain GA, protoCA, ChA, and CA. GA, protoCA, p-hydBA, VA, CA, and SA are found in the infusion of hawthorn fruit seeds. In general agreement with our findings, the presence of ChA in dried hawthorn fruits has been demonstrated in a previous study.²³

Based on the quantitative results, it is evident that phenolic acids are much more abundant in leaves with flowers than in fruit peel and seeds of hawthorn.

TPC and antiradical activity

The major shortcoming in the qualitative or quantitative evaluation of plant phenolics lies in the current lack of an appropriate methodology. At present, the most widely used method for evaluating the polyphenolic content of plant materials is the Folin–Ciocalteau colorimetric assay. This method is not an absolute measurement of the amount of phenolics because some other substances such as organic acids, residual sugars, amino acids, proteins, and other hydrophilic compounds interfere with this assay.²⁴

In our study, the Folin–Ciocalteau assay was very convenient for the screening of large numbers of samples of different polarity. This method is not discriminative with respect to the radical species but gives a general idea about the radical quenching ability.²⁵

The results of TPC and free radical scavenging potentials tested by the DPPH method of different extracts from hawthorn leaves with flowers, fruit peel, and seed and BHT at different concentrations are given in Table 2.

Table 2.

Total Phenolic Content and 2,2-Diphenyl-1-Picrylhydrazyl Inhibition in Various Hawthorn Extracts Obtained with Different Solvents

		Inhibition (%) with added amount (μg/mL)
Extract	Total phenolic content (mg of GAE/g of extract)	9.6×10⁻⁴	1.8×10⁻³	3.6×10⁻³
HLF
ME	245.78	19.49	34.34	47.23
EA	343.54	25.57	49.94	60.36
W	108.65	6.17	15.79	21.01
Inf	148.33	14.51	28.14	38.12
HP
ME	93.43	4.01	9.99	17.01
EA	216.96	17.73	32.25	41.18
W	35.89	4.98	9.49	15.82
Inf	70.23	11.67	24.43	36.35
HS
ME	44.30	3.17	9.79	15.01
EA	95.25	10.86	14.51	28.14
W	33.56	6.52	10.98	16.73
Inf	54.25	9.24	21.97	28.41
BHT		27.98	48.95	72.69

BHT, tert-butylhydroxytoluene; GAE, GA equivalents.

The TPC for the extracts studied falls within the range of 33.56 (hawthorn fruit seed W extract) to 343.54 (hawthorn leaves with flowers EA extract) mg of GA equivalents/g. The TPC for EA extracts, however, is over double that for infusions.

The hawthorn extracts investigated showed dose-dependent radical scavenging activities. The percentage inhibition value is a parameter widely used to measure the free radical scavenging activity. That is, a higher percentage inhibition value corresponds to a higher antioxidant activity.²⁶

Antiradical activity ranges from very active (hawthorn leaves with flowers EA extract, 60.36 μg/mL) to inactive (hawthorn fruit seed W extract, 16.73 μg/mL). Hawthorn leaves with flowers EA extract, with higher than average TPC (343.54 mg of GA equivalents/g), is the most active extract in the DPPH assay. Its antioxidant activity was concentration-dependent. These data indicate that extract obtained from C. monogyna leaves with flowers was as effective as BHT in radical scavenging ability. The high radical scavenging activity observed in C. monogyna leaves with flowers is due, most probably, to the presence of phenolic glycosides and phenolic acids (especially CA and ChA). CA and ChA have also been studied, and these compounds were found to have effective antioxidant capacity using ferric reducing antioxidant power and 2,2′-azinobis-3-ethylbenzthiazoline-6-sulfonic acid assays²⁷ or β-carotene bleaching and DPPH methods.²⁸

It was found that the average antioxidant activity of hawthorn fruit peel extracts was higher than the activity of hawthorn seed at those same concentrations. The ranking of antioxidant activity of the extracts is as follows: EA > ME > infusion > W extracts.

Conclusions

In general, antioxidant activity of plant materials significantly increases with the presence of high levels of TPC. In our study, TPC was more closely correlated with antiradical activity, suggesting that the antiradical activity of these extracts is a complicated synergy that may also involve compounds not yet identified or quantified. A potent antioxidant activity of extracts obtained from C. monogyna leaves with flowers was shown, considering the scavenging abilities displayed against DPPH. This scavenging activity can be mainly attributed to the phenolic derivatives present in the extracts.

Phenolic acids are responsible for the pharmacological activity attributed to medicinal plant extracts. Therefore, the profile of their content represents a useful fingerprint to find out the identity and the quality of these crude drug and phytopreparations.¹³ The results mentioned above showed the proposed liquid chromatography method was very suitable for rapid determination of phenolic acids in extracts of hawthorn. This work also confirmed that HPLC is a powerful technique for the investigation of phenolic acids in the complex extracts obtained from medicinal plants.

Footnotes

Acknowledgments

The authors acknowledge the Research Foundation of University of Anadolu (project number 030353) and the Plant, Drug and Scientific Research Centre of Anadolu University for their kind support of this study.

Author Disclosure Statement

No competing financial interests exist.

References

Blumenthal

, Goldberg

, Brinckmann

. Hawthorn. Herbal Medicine Expanded: Commission E Monographs. American Botanical Council, Integrative Medicine Communications: Newton, MA, 2000; 182–189.

Svedstrom

, Vuorela

, Kostiainen

, Laakso

, Hiltunen

. Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to HPLC analysis. J Chromatogr A, 2006; 1112:103–111.

Rehwald

, Meier

, Sticher

. Qualitative and quantitative reversed-phase high-performance liquid chromatography of flavonoids in Crataegus leaves and flowers. J Chromatogr A, 1994; 677:25–33.

Chang

, Zuo

, Harrison

, Chow

MSS

. Hawthorn. J Clin Pharmacol, 2002; 42:605–612.

Wagner

, Grevel

. Cardioactive drugs: IV. Cardiotonic amines from Crataegus oxyacantha. Planta Med, 1982; 45:98–101.

Garcia

, Saenz

, Ahumada

, Cert

. Isolation of three triterpenes and several aliphatic alcohols from Crataegus monogyna. J Chromatogr A, 1997; 767:340–342.

Bahorun

, Trotin

, Pommery

, Vasseur

, Pinkas

. Antioxidant activities of Crataegus monogyna extracts. Planta Med, 1994; 60:323–328.

Walker

, Marakis

, Morris

, Robinson

. Promising hypotensive effect of hawthorn extract: a randomized double-blind pilot study of mild, essential hypertension. Phytother Res, 2002; 16:48–54.

Bahorun

, Aumjaud

, Ramphul

, Rycha

, Luximon-Ramma

, Trotin

, Aruoma

. Phenolic constituents and antioxidant capacities of Crataegus monogyna (hawthorn) callus extracts. Nahrung/Food, 2003; 47:191–198.

10.

Ljubuncic

, Portnaya

, Cogan

, Azaizeh

, Bomzon

. Antioxidant activity of Crataegus aronia aqueous extract used in traditional Arab medicine in Israel. J Ethnopharmacol, 2005; 101:153–161.

11.

Frei

. Natural Antioxidants in Human Health and Disease. Academic Press: San Diego, CA, 1994; 303–352.

12.

Pratt

. Natural antoxidants from natural material. Phenolic Compounds in Food and Their Effects on Health, 2 ^nd. Huang

, Ho

, Lee

. American Chemical Society: Washington, 1992; 54–71.

13.

Simonovska

, Vovk

, Andrensek

, Valentova

, Ulrichova

. Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers. J Chromatogr A, 2003; 1016:89–98.

14.

Males

, Medic-Saric

. Optimization of TLC analysis of flavonoids and phenolic acids of Helleborus atrorubens Waldst. et Kit. J Pharm Biomed Anal, 2001; 24:353–359.

15.

Waksmundzka-Hajnos

. Chromatographic separations of aromatic carboxylic acids. J Chromatogr B, 1998; 717:93–118.

16.

Urbanek

, Pospisilová

, Polásek

. On-line coupling of capillary isotachophoresis and zone electrophoresis for the assay of phenolic compounds in plant extracts. Electrophoresis, 2002; 23:1045–1052.

17.

Suntornsuk

. Capillary electrophoresis of phytochemical substances. J Pharm Biomed Anal, 2002; 27:679–698.

18.

Pomponio

, Gotti

, Hudaib

, Cavrini

. Analysis of phenolic acids by micellar electrokinetic chromatography: application to Echinacea purpurea plant extracts. J Chromatogr A, 2002; 945:239–247.

19.

Folin

, Ciocalteu

. On tyrosine and tryptophane determinations in proteins. J Biol Chem, 1928; 73:627–650.

20.

Singleton

, Rossi

. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am J Enol Vitic, 1965; 16:144–158.

21.

öztürk

, Tunçel

. Determination of phenolic acids by a modified HPLC: its application to various plant materials. J Liquid Chromatogr Rel Technol, 2007; 30:587–596.

22.

Sanchez-Moreno

, Larrauri

, Saura-Calixto

. A procedure to measure the antiradical efficiency of polyphenols. J Sci Food Agric, 1998; 76:270–276.

23.

Chang

, Zuo

, Ho

WKK

, Chow

MSS

. Comparison of the pharmacokinetics of hawthorn phenolics in extract versus individual pure compound. J Clin Pharmacol, 2005; 45:106–112.

24.

Zhang

, Chang

, Zhu

, Huang

, Ho

WKK

, Chen

Z-Y

. Characterization of antioxidants present in hawthorn fruits. J Nutr Biochem, 2001; 12:144–152.

25.

Cuvelier

, Richard

, Berset

. Comparison of the antioxidative activity of some acid-phenols: structure-activity relationship. Biosci Biotechnol Biochem, 1992; 56:324–325.

26.

Cetkovic

, Canadanovic-Brunet

, Djilas

, Savatovic

, Mandic

, Tumbas

. Assessment of polyphenolic content and in vitro antiradical characteristics of apple pomace. Food Chem, 2008; 10:340–347.

27.

Nilsson

, Pillai

, Onning

, Persson

, Nilsson

, Akesson

. Comparison of the 2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid (ABTS) and ferric reducing anti-oxidant power (FRAP) methods to asses the total antioxidant capacity in extracts of fruit and vegetables. Mol Nutr Food Res, 2005; 49:239–246.

28.

Fukumoto

, Mazza

. Assessing antioxidant and prooxidant activities of phenolic compounds. J Agric Food Chem, 2000; 48:3597–3604.