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.
Keywords: Paeonia; essential oil; GC-MS; antioxidant; nitric oxide; DPPH; chromosome numbers
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 ([
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 ([
We have previously performed some isolation and bioactivity studies on some Paeonia species ([
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).
Table 1. Examined taxa with their voucher (ISTE) numbers, population numbers, locality and somatic chromosome numbers.
Species Voucher number Population number Locality 2n ISTE 84822 5801 Sivas; Zara 20 ISTE 84823 3301 Mersin; Tepeköy 1300 m 10 ISTE 84836 3101 Hatay; Yayladagi, 1100 m 20 ISTE 84824 1702 Çanakkale; Kalkim, 800-900 m 20 ISTE 84825 2901 Gümüşhane; Torul, 1750 m 10 ISTE 84826 4201 Konya; Doganhisar, 1650-1750 m 10 ISTE 84827 0302 Afyonkarahisar; Sultan mountain, 1400-1500 m 20 ISTE 84828 1708 Çanakkale; Kalkim 20 ISTE 84829 ISTE 84830 1601 1001 Bursa; M. Kemalpaşa, 500-600 m Balıkesir; Savaştepe -20 - ISTE 84831 3901 4501 Kırklareli; Dereköy, 350-400 m Manisa; Spil mountain - 20 ISTE 84832 2201 Edirne; Lalapaşa, Ortakça village 10 - ISTE 72288 0701 - Antalya; Elmali, 1800-1850 m Burdur; Altinyayla 20 ISTE 84837 5301 Rize; Ikizdere 20 ISTE 84833 1703 Çanakkale; Yenice 20
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.
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.
Table 2. Composition of the essential oils of 14 Anatolian Paeonia samples.
RRI Compound 1244 2-Pentyl furan 0.1 0.4 0.2 tr tr - 0.2 tr tr 0.3 0.2 - 0.5 0.1 1541 Benzaldehyde - - - - 0.1 - - - - tr 0.1 - tr - 1547 Dihydroachillene 0.1 tr tr tr - tr 0.2 tr 0.2 tr 0.2 - 0.1 0.1 1548 ( 0.1 0.2 tr - - tr tr - 0.1 0.1 - 0.4 - 1560 16.7 9.7 15.4 3.3 1.7 10.3 27.9 7.4 42.2 45.7 16.6 59.7 5.5 7.2 1570 2.2 1.2 1.7 tr 0.2 1.2 3.2 0.9 5.1 6.6 2.2 6.9 0.8 0.9 1601 Nopinone 2.2 1.0 1.8 10.2 0.5 1.3 5.0 1.7 3.5 1.1 2.1 0.5 4.7 0.8 1614 Carvacrol methyl ether tr tr tr - - - - tr - - 0.1 - tr - 1641 Methyl benzoate 0.1 tr - - - tr - tr - - 0.1 - 0.1 - 1648 Myrtenal 4.2 1.2 2.5 2.7 0.1 0.7 3.9 2.4 3.4 3.5 2.1 1.7 1.7 0.5 1685 Ethyl benzoate - - - - - - - - tr 0.1 - - - 1703 Salicylaldehyde 10.0 59.0 64.2 30.4 94.4 76.8 29.1 42.0 14.9 14.7 41.0 6.4 65.5 79.8 1729 α-Methyl cinnamaldehyde 2.3 1.8 3.2 3.4 tr 0.8 3.6 4.2 3.1 3.1 2.1 1.2 0.9 1.1 1744 Phellandral 0.5 tr tr - tr 0.1 0.8 0.4 tr 0.5 0.4 0.3 0.3 0.2 1798 Methyl salicylate 52.2 10.1 8.3 - 2.0 1.7 15.9 20.5 17.8 3.3 16.1 10.6 1.5 1.0 1804 Myrtenol 0.2 - - - tr 0.1 0.6 - 0.7 0.7 0.2 tr 0.3 0.3 1807 Perilla aldehyde 0.6 0.1 tr 1.8 tr 0.1 1.3 0.4 0.5 0.3 0.5 tr 0.6 0.3 1834 Ethyl salicylate 0.2 tr - tr - tr - tr - tr 0.1 - - - 1872 1.5 1.0 0.5 1.6 0.1 0.7 2.1 0.5 3.4 2.2 1.4 0.5 0.9 0.4 1879 0.7 0.5 0.8 1.3 0.1 0.3 4.2 1.0 3.7 1.7 1.8 0.4 1.0 0.2 1965 2-Ethyl hexanoic acid 0.1 - - - - tr 0.3 0.3 - 0.4 tr 0.1 - 2029 Perilla alcohol 0.3 0.3 - 1.3 tr 0.2 0.6 0.1 0.6 0.2 0.3 - 1.5 0.3 2186 Eugenol - - - - tr 0.2 - 0.3 - - 0.3 - 0.4 0.3 2198 Thymol 0.1 - - - 0.1 0.1 0.3 tr 0.2 - 0.9 - tr 0.9 2239 Carvacrol 0.2 tr 1.1 - 0.1 0.4 0.3 1.4 - tr 0.1 0.4 1.4 0.2 2242 Methyl hexadecanate - tr - tr tr 0.1 - 0.4 - tr 0.1 tr 0.2 tr 2250 Paenol - - - - - - - 0.3 - - 0.4 - - - 2262 Ethyl hexadecanate - - - - tr - - 0.4 - - - - - 2282 tr - - - tr 0.1 tr - 0.4 - 0.3 tr 0.2 1.4 2380 Hexyl cinnamic aldehyde - - - - - - - - - 0.1 - - - 2384 1-Hexadecanol 0.1 - - - tr tr tr 0.3 - 0.2 0.2 - 0.3 tr 2503 Dodecanoic acid - - - - - - - - - 0.7 - 0.6 - 2509 Methyl linoleate tr 0.7 - 1.6 tr 0.1 - 0.4 - 0.7 - tr 0.2 tr 2538 Ethyl linoleate - - - - tr - - tr - - - - - - 2583 Methyl linolenate - - - tr - - tr - - - - 0.1 - 2607 1-Octadecanol - - - - tr tr - - - - - - 0.3 - 2613 Ethyl linolenate - - - - - - - tr - - - - - 2670 Tetradecanoic acid - tr - tr - tr - tr - - 0.8 - tr - 2804 Benzyl salicylate tr - - tr - tr - tr - - 0.3 - 0.7 0.5 2822 Pentadecanoic acid tr tr - tr - - - tr - - 0.9 0.9 - 2931 Hexadecanoic acid 4.0 11.4 - 34.1 0.3 4.1 - 13.1 - 14.9 6.3 8.4 5.6 3.3 28 24 15 21 26 28 21 34 17 24 34 19 34 24 98.7 98.6 99.7 91.7 99.7 99.4 99.5 98.4 99.7 99.8 98.7 97.9 97.3 99.8 trace trace trace trace 0.22 0.03 0.01 trace trace trace 0.02 trace trace 0.04
3 RRI: Relative retention indices calculated against n-alkanes; %, calculated from FID data; tr, (< 0.1%); *the northwestern Anatolian sample; **the western Anatolian sample; # the eastern Anatolian sample;Ψthe western Anatolian sample;
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
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 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) ([
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).
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 ([
Graph
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%) ([
The concentration of total phenols in the extracts was determined by UV spectrophotometry using Folin-Ciocalteau's reagent ([
All cytological observations were made from the root tips of the materials shown in Table 1. 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.
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).
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.
Species TPC (mg GA/100 mg extract) %RSA of the extracts against DPPH* %RSA of the extracts against NO* 6 48.34 ± 0.049 51.3 ± 0.003 10.3 31.45 ± 0.049 55.9 ± 0.354 7.7 46.4 ± 0.006 55.6 ± 0.204 12.3 No activity 49.2 ± 0.009 14.9 58.21 ± 0.006 56.9 ± 0.12 5.3 46.11 ± 0.036 43.4 ± 0.28 14.1 29.81 ± 0.217 56.4 ± 0.047 14.3 No activity 56.3 ± 0.056 7.9 53.03 ± 0.051 54.5 ± 0.003 4.2 44.36 ± 0.006 56.4 ± 0.038 10.5 26.55 ± 0.026 54.4 ± 0.026 5.4 39.05 ± 0.045 49.7 ± 0.109 7.6 23.69 ± 0.338 55.4 ± 0.017 11 44.32 ± 0.055 56.3 ± 0.079 Standards Propyl gallate (DPPH) - 90 ± 0.002 Not tested Rutin (NO) - Not tested 79.9 ± 0.007
4 *n = 3 (Results of %RSA of the extracts and standards against DPPH and NO are expressed as average of three paralel experiments).
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% (Table 2). 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 (Table 2). 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 (Table 1). 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 ([
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 ([
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.
By Ilkay Orhan; Betül Demirci; Iman Omar; Huma Siddiqui; Erdal Kaya; M. Iqbal Choudhary; Gülay Ecevit-Genç; Neriman Özhatay; Bilge Şener and K. Hüsnü Can Başer
Reported by Author; Author; Author; Author; Author; Author; Author; Author; Author; Author