Abstract
Essential oil compositions and antioxidant potentials of fourteen ethanol (75%) root extracts prepared from twelve taxa of the genus Paeonia (Paeoniaceae), including P. arietina Anders., P. daurica Andrews, P. xkayae N. Özhatay, P. kesrouanensis Thiéb., P. mascula (L.) Miller subsp. arasicola G. Kaynak, ö. Yilmaz & R. Daşkin, P. mascula (L.) Miller subsp. bodurii N. Özhatay, P. cf. mascula L. (Mill.) subsp. mascula (two samples from central and northeastern Anatolia), P. cf. officinalis Retz., P. peregrina Miller (two samples from western and northwestern Anatolia), P. tenuifolia L., P. turcica Davis & Cullen, and P. wittmanniana Hartwiss ex Lindl. were assessed. The chromosome numbers of the root tips of the species were examined using chromosome staining technique with Shiff’s reagent under Leitz microscope. The essential oils of the roots of the Paeonia species were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and the major components were identified as salicylaldehyde (10%–94.4%), cis-myrtanal (5.5%–59.7%), and methyl salicylate (2%–52.2%). Antioxidant potentials were tested against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radicals using propyl gallate and rutin as the references. Total phenolic contents of the ethanol extracts were determined using Folin-Ciocalteau’s method. The extracts exerted moderate NO scavenger effect and displayed insignificant DPPH radical scavenger activity at 500 μg mL−1. On the other hand, P. daurica, P. tenuifolia and P. cf. mascula subsp. mascula are diploids with 2n = 10, while other nine taxa are tetraploids with 2n = 20.
Introduction
The genus Paeonia L. (Paeoniaceae), known as “şakayik, ayi gülü, bocur, etc.” in Turkey, is only found in the northern hemisphere and comprises about 35 species. The genus has historically been subdivided into three sections, Moutan DC, Onaepia Lindley, and Paeonia DC (CitationDavis & Cullen, 1965; CitationDavis et al., 1988; CitationÖzhatay, 2000) and the section Paeonia is distributed in eastern and central Asia, the western Himalayas as well as the Mediterranean region. This section is composed of approximately 25 herbaceous species of both diploids and tetraploids (CitationHong et al., 2001), among them twelve taxa of Paeonia were recorded in Turkey, which is the most important gene center worldwide for this genus. Dependence on crucial income obtained by export of the plant means different species are thoughtlessly collected from the wild by digging out the roots in its natural habitat, which causes a slow extinction of the genus. Therefore, it has been decided to rigorously preserve the genus in Turkey by Atatürk Central Horticultural Research Institute in Yalova (Turkey) (personal communication).
Apart from its importance as an attractive ornamental plant, Paeonia (peony) species have also been utilized as medicinal plants. For instance, the Ottomans used Paeonia ssp. to treat internal diseases, pains, and epilepsy (CitationLew, 2002). Some of the peony species in Anatolia have been consumed as tea, which it has been recommended to drink two or three glasses per day against constipation and epilepsy as well as for antitussive purposes (CitationBaytop, 1999). The genus Paeonia is also one of the most important crude drugs in Chinese traditional medicine used against atopic eczema as well as for anticoagulant, anti-inflammatory, analgesic, and sedative purposes (CitationKirby & Schmidt, 1997; CitationMutlugil, 1989).
We have previously performed some isolation and bioactivity studies on some Paeonia species (CitationYeşilada et al., 1992), and we also thought it worthwhile to ascertain essential oil composition and antioxidant potential of Turkish Paeonia species. We have herein examined the ethanol extracts prepared from the roots of P. arietina, P. daurica daurica (three samples from western and northeastern and southern Anatolia), P. x kayae, P. kesrouanensis, P. mascula subsp. arasicola, P. mascula subsp. bodurii, P. cf. mascula subsp. mascula (two samples from central and northeastern Anatolia), P. cf. officinalis, P. peregrina (two samples from western and northwestern Anatolia), P. tenuifolia, P. turcica and P. wittmanniana by 1,1-diphenylpicrylhydrazyl (DPPH) radical and nitric oxide (NO) scavenging activities at 500 μg mL−1 using an ELISA microplate reader. Total phenolic content (TPC) of the extracts was established by Folin-Ciocalteau’s method.
Materials and methods
Plant materials
The roots of P. arietina, P. daurica, P. xkayae, P. kesrouanensis, P. mascula subsp. arasicola, P. mascula subsp. bodurii, P. cf. mascula subsp. mascula (two samples from central and northeastern Anatolia), P. cf. officinalis, P. peregrina, P. tenuifolia, P. turcica, and P. wittmanniana were obtained in 2007 at the experimental garden of Atatürk Central Horticultural Research Institute in Yalova (Turkey). All examined materials had been collected from the wild population and were planted in the experimental garden in Yalova. Population numbers of the species were given by Erdal Kaya, while the voucher specimens are housed in the Herbarium of the Faculty of Pharmacy, Istanbul University (ISTE) ().
Table 1. Examined taxa with their voucher (ISTE) numbers, population numbers, locality and somatic chromosome numbers.
Preparation of the extracts
The dried and powdered root materials of twelve above-mentioned Paeonia taxa were extracted with 300 mL (x 2) of ethanol (75%). All twelve pooled separately which were evaporated in vacuo until dryness to obtain the crude ethanol extracts which were later subjected to the antioxidant assays.
Isolation of the essential oils
Air-dried roots were crushed using a mortar and immediately hydrodistilled for 3 h using a Clevenger apparatus to provide essential oils. The oil yields are given in .
Table 2. Composition of the essential oils of 14 Anatolian Paeonia samples.
Gas chromatography-mass spectrometry analysis
The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas (0.8 mL min−1). GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min, and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.
Gas chromatographic analysis
The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300°C. In order to obtain the same elution order with GC-MS, simultaneous autoinjection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from the FID chromatograms.
Identification of components
Identification of the essential oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library; Adams Library; MassFinder 3 Library) (CitationJennings & Shibamoto, 1980; CitationAdams, 2001; CitationKönig et al., 2004) and in-house Başer Library of Essential Oil Constituents built up by genuine compounds and components of the known oils, as well as MS literature data (CitationJoulain & König, 1988; CitationMcLafferty & Stauffer, 1989; CitationESO, 2000; CitationShaheen et al., 2005) were employed in identification of essential oil composition.
Antioxidant assays
Nitroprusside natrium was obtained from Merck 2549480 (Merck Co, Darmstadt, Germany), while sulfanilic acid from MP-Biomedicals (102990) (MP Biomedicals, CA, USA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and N-(1-naphthyl)ethylenediamine dihydrochloride and dimethyl sulfoxide (DMSO) were purchased from Sigma (St Louis, MO).
DPPH radical scavenging activity
The ethanol extracts were allowed to react with stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) for 1.5 h at 37°C as described in our previous publication (CitationBadami et al., 2003). After incubation, decrease in absorption was measured at 515 nm using an ELISA multiplate reader (Spectra MAX-340). Percentage radical scavenging activity (RSA) by samples was determined in comparison with a DMSO-treated control group, and was calculated by using the following formula where “A” is referred to “Absorbance at 515 nm”:
NO scavenging activity
In the present experiment, modified Griess-Illosvoy reaction was employed by using N-(1-naphthyl)ethylenediamine dihydrochloride (0.1% w/v) instead of 1-naphthylamine (5%) (CitationSkerget et al., 2005). The reaction mixture (200 μL) contained 10 μL of each sample, 20 μL of potassium phosphate buffer (0.1 mM, pH 7.4), and 70 μL of sodium nitroprusside (10 mM), then incubated at 25°C for 90 min. Following addition of 50 μL of N-(1-naphthyl) ethylenediamine dihydrochloride (0.1%, w/v) into the reaction mixture given above, 50 μL of sulfanilic acid reagent was then added and shaken carefully. Absorbance at 540 nm against the blank solution containing all reagents except for the test sample was determined in a microtiter plate by ELISA reader. RSA was calculated by using the above formula for DPPH assay.
Total phenolic contents (TPC) in the extracts
The concentration of total phenols in the extracts was determined by UV spectrophotometry using Folin-Ciocalteau’s reagent (CitationSingleton & Rossi, 1965). The absorbance was measured at 760 nm and the results obtained were expressed in mg of gallic acid (GA) per 100 mg of each extract (mg GA/100 mg extract).
Karyological study
All cytological observations were made from the root tips of the materials shown in . The seeds were germinated on soil in pots. Fresh root tips were cut about 1 cm long, pretreated in 1-bromonaphthalene at 4°C for 24 h, then fixed with 1:3 (glacial acetic acid:absolute alcohol) at 4°C for 24 h. The root tips were hydrolyzed in 1 N HCl at 60°C for 10 min and stained in Shiff’s reagent. Stained root tips were squashed with a drop of 2% aceto orcein. Permanent slides were made by mounting with Sandeural. Preparations were examined using a Leitz microscope.
Results and discussion
Antioxidant potentials of the ethanol extracts of the root samples of P. arietina, P. daurica P. xkayae, P. kesrouanensis, P. mascula subsp. arasicola, P. mascula subsp. bodurii, P. cf. mascula subsp. mascula (two samples from central and northeastern Anatolia), P. cf. officinalis, P. peregrina (two samples from the western and northwestern Anatolia), P. tenuifolia, P. turcica, and P. wittmanniana were determined by their radical scavenger activities against DPPH and NO along with their total phenolic contents ().
Table 3. Total phenolic contents (TPC) and percentage of DPPH and NO radical scavenging activity (%RSA) of the ethanolic root extracts from the Paeonia species.
Our results demonstrate the Paeonia species screened here showed a moderate anti-radical activity against DPPH causing inhibition between 23.9% and 58.2% as compared to the propyl gallate (90%), and they displayed similar activity against NO ranging between 43.4% and 56.9% (). TPCs were the highest in P. cf. mascula subsp. mascula of the central Anatolia (14.9 mg), followed by P. mascula subsp. bodurii (14.3 mg), and P. mascula subsp. arasicola (14.1 mg). Interestingly, P. cf. mascula subsp. mascula of the northeastern Anatolia was found to contain approximately one third the amount (5.3 mg) of the central sample, which may depend on various factors such as climate conditions, soil composition, etc. The same difference was also observed between two P. peregrina samples collected from the western and northwestern regions of Turkey, which may well be due to the above-mentioned factors. The sample of P. cf. mascula subsp. mascula obtained from central Anatolia that has the highest TPC among the others also exerted the highest antioxidant capacity against DPPH and NO radicals.
The results obtained by essential oil analyses have highlighted great compositional variation in all fourteen oils in which salicylaldehyde, cis-myrtanal, and methyl saliciylate were the major components (). Salicylaldehyde was dominant in P. cf. mascula subsp. mascula samples of central and northeastern Anatolia, P. xkayae, P. daurica, P. mascula subsp. bodurii, P. peregrina of northwestern Anatolia, P. turcica, and P. wittmanniana, whereas methylsalicylate was the leading component only in P. arietina. Cis-myrtanal was the major compound in P. tenuifolia (59.7%), followed by P. peregrina of west Anatolia (45.7%), and P. cf. officinalis (42.2%).
Chromosome numbers are known to be important to improve agriculturally important plants and to determine relationships between plants. Since Paeonia species are also considered one of the ornamentally important plants and no karyological study has been so far performed on the genus, we determined their chromosome numbers. Our karyological results indicated that the somatic chromosome numbers of the species of Paeonia is either 2n = 10 or 2n = 20. P. daurica P. tenuifolia and P. cf. mascula subsp. mascula are diploids with 2n = 2x = 10. The other nine taxa are tetraploids with 2n = 4x = 20 (). Considering their essential oil compositions, there has been no correlation observed between diploid and tetraploid taxa.
There have been several reports on the antioxidative effect of Paeonia species of Chinese origin. For instance; in a study (CitationKirby & Schmidt, 1997), an herbal mixture used as a tea in China consisting of many Chinese plants including P. lactiflora, was tested for each ingredient plant separately against DPPH radical and superoxide (SO) anion for their antioxidant effects as well as the tea itself. Among them, the aqueous extract of P. lactiflora was found to be the most active against DPPH, whereas it was one of the inactive plants towards SO. Conversely, seventy Chinese medicinal plants were screened for their SO and hydroxyl radical scavenging activities and P. lactiflora potently scavenged SO radical (CitationLiu & Ng, 2000). In another study, twelve Chinese medicinal plants including P. suffruticosa were screened for their antioxidant activity through lipid peroxidation in rat kidney and brain homogenates, SO and hydroxyl radical scavenging effects (CitationLee et al., 2003) and P. suffruticosa strongly inhibited generation of hydroxyl and SO radicals at 100 μg mL−1. It was also one of the most potent plants in lipid peroxidation, where it had a very low pro-oxidant effect. P. suffruticosa was reported to have a very high level of DPPH radical scavenging activity causing over 90% of inhibition (Li et al., 2008). Also, this plant induced 43%, 28%, and 45% increases in superoxide dismutase, catalase, and gluthation peroxidase levels, respectively. On the other hand, P. lactiflora and P. suffruticosa were found to contain 26.75 and 24.51 mg TPC expressed as GA equivalent/g extract, and these two plants were concluded to possess high antioxidant capacity in scavenging free radicals and reducing (CitationMiyazawa et al., 1983).
However, there have been few reports on volatile constituents of Paeonia species. Although it is a well-reputed Chinese traditional plant, only two papers mention the volatile oils of P. lactiflora and P. suffruticosa of Chinese origin, in which benzoic acid was the dominant component (CitationMiyazawa et al., 1984; CitationPapandreou et al., 2002). The essential oils of three taxa including P. clusii subsp. clusii, P. mascula subsp. hellenica, and P. parnassica growing in Greece were analyzed by GC-MS (CitationIvanova et al., 2002), and among the twelve components identified, salicylaldehyde was found to be the major one all in three species. Methyl salicylate was the second major constituent in P. parnassica (24.7%), while the second one was paeonol in P. clusii subsp. clusii (32.6%). Benzoic acid, the major compound found in Chinese P. lactiflora and P. suffruticosa, was only found in P. clusii subsp. clusii constituting 7.7% of the oil. Interestingly, salicylaldehyde, the main compound in the essential oils of the Greek and some of the Turkish Paeonia species mentioned herein, has been never reported from Chinese peony roots. Also, benzoic acid and its several derivatives were observed to dominate the essential oils from P. peregrina and P. tenuifolia growing in Bulgaria, which are consistent with their Chinese counterparts (CitationIvanova et al., 2002). However, benzoic acid was not detected at all in the essential oils of our Paeonia species.
Considering the literature on Paeonia, except for two Chinese Paeonia species (P. lactiflora and P. suffruticosa), there has been so far no information about the antioxidant potential of other members of the genus Paeonia of non-Chinese origin. Accordingly, the essential oils from Paeonia taxa of Turkish origin seem quite similar to the Greek taxa of Paeonia since salicylaldehyde is the main component in most of their oils. To the best of our knowledge, this is the first report on antioxidant properties of these twelve Paeonia taxa and their total phenolic contents along with the chromosome numbers and essential oil compositions of the Turkish Paeonia taxa.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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