Acta Chromatographica 20(2008)2, 259–267 DOI: 10.1556/AChrom.20.2008.2.10 Screening for Phenolic Acids in Five Species of Iris Collected in Mongolia A. MACHALSKA1,*, K. SKALICKA-WOŹNIAK1, J. WIDELSKI1, K. GŁOWNIAK1, G. PUREVSUREN2, Z. OYUN2, D. KHISHGEE2, AND B. URJIN2 1Department of Pharmacognosy with Medicinal Plant Laboratory, Medical University of Lublin, 1 Chodźki St., 20-093 Lublin, Poland 2Traditional Medical Science, Technology and Production Corporation of Mongolia, Ulaanbaatar-20, 209, Mongolia E-mail: [email protected] Summary. Five species of Iris commonly used in Mongolian traditional medicine (Iris di- chotoma Pall., Iris flavissima Pall., Iris bungei Maxim., Iris lactea Pall., and Iris tenuifolia Pall.) were analyzed for the presence of phenolic acids. This was the first study of the phenolic acid content of these species. Samples containing the phenolic acids were pre- pared by the method of Świątek and then analysed by HPLC with UV–visible diode- array detection (DAD). Identification was performed by comparing retention times with those of standards. Quantitative determination was performed at the absorbance maxi- mum of each phenolic acid (320 nm for ferulic, p-coumaric, and caffeic acids, 280 nm for trans-cinnamic, syringic, and gallic acids, and 254 nm for vanillic, m-hydroxybenzoic, p- hydroxybenzoic, and protocatechuic acids). As the result of our study ten phenolic acids, both free and liberated by alkaline and acid hydrolysis, were identified by HPLC. Chromatographic investigation revealed the presence of vanillic acid, protocatechuic acid, trans-cinnamic acid, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, gallic acid, syringic acid, m-hydroxybenzoic acid, and caffeic acid. Quantitative analysis of these acids was also performed. Finally, the presence or absence of some phenolic acids after alkaline or acid hydrolysis was also observed. Introduction Iris species (Family Iridaceae) are used in Mongolian traditional medicine, especially for treatment of cancer, inflammation, and bacterial infection [1, 2]. Iris dichotoma Pall. is known for its antipyretic and antioedemic activity; it can also improve blood circulation. Chemical analysis of plant from the Iris genus has revealed the presence of numerous biologically active sub- stances, for example flavones and isoflavones, peltogynoids (iridoids A, B, 0231–2522 © 2008 Akadémiai Kiadó, Budapest Unauthenticated | Downloaded 09/24/21 06:34 PM UTC 260 A. Machalska et al. C, D, and E), saponins, coumarins, and benzoquinones [1–4]. The last of these have been used as anticancer agents in modern Chinese medicine [1, 2]. Five species of Iris (Iris bungei Maxim., Iris dichotoma Pall., Iris flavissima Pall., Iris lactea Pall., Iris tenuifolia Pall.) collected in Mongolia have been analyzed for the presence of phenolic acids. Studies on the phenolic acid content of these species, which is very important because of their varied physiological activity, has not previously been performed. Experimental Plant Material The plant material was collected in 2006, in Mongolia. It was identified by a specialist from the Herbarium of the Botanical Institute of the Mongolian Academy of Science, Ulaanbaatar, Mongolia, where voucher specimens of the plants have been deposited. The species examined are listed in Table I. Table I. The species examined No. Plant Symbol 1 Iris bungei Maxim.a Ib 2 Iris lactea Pall.a Il 3 Iris tenuifolia Pall.a It 4 Iris dichotoma Pall.b Id 5 Iris flavissima Pall.b If aAerial parts bRhizomes Examination of different plant tissue in this study (aerial parts of Ib, Il, It and underground parts of Id and If) was motivated by the widespread use of these tissues from these species in traditional medicine. Extraction The plant materials (40 g Iris lactea Pall., Iris flavissima Pall. and Iris dichotoma Pall., 25 g Iris tenuifolia Pall., and 27 g Iris bungei Maxim.) were pre-purified by extraction in a Soxhlet apparatus with petroleum ether (250 mL for Ib and It, and 450 mL for Id, If, and Il). They were then extracted exhaustively Unauthenticated | Downloaded 09/24/21 06:34 PM UTC Phenolic Acids in Five Species of Iris from Mongolia 261 with 100% methanol in a Soxhlet apparatus. The extracts were concentrated under reduced pressure and purified from ballast with 200 mL hot water. After 24 h, ballast substances were removed by filtration through quantita- tive disc filters (Filtrak) and the filtrates were extracted 10 times with 20 mL diethyl ether. Two fractions were obtained, the diethyl ether and aqueous fractions. Both were left for further study of phenolic acids by the method of Świątek [5]: • analysis of free phenolic acids (FPhAs), • analysis of bonded phenolic acids liberated by acid hydrolysis (AcPhAs), • analysis of bonded phenolic acids liberated by alkaline hydrolysis (AlPhAs). Both qualitative and quantitative analysis were performed on fractions ob- tained as described above. Before HPLC analysis the extracts were passed through PTFE membrane filters. RP-HPLC Analysis Samples were analysed using an Agilent 1100 liquid chromatograph with UV–visible diode-array detection (DAD). Compounds were separated on a 250 mm × 4.6 mm stainless-steel column packed with 5-μm Hypersil BDS C18 (Shandon, UK). The mobile phase was a gradient prepared from water containing 1% acetic acid and acetonitrile (Table II) [6]. The mobile phase Table II. The mobile phase gradient [6] Time (min) Acetonitrile (%) Water containing 1% acetic acid (%) 0 10 90 10 10 90 15 20 80 30 30 70 35 100 0 40 100 0 flow rate was 1 mL min−1, the sample injection volume was 10 μL, and elu- tion was performed at 25°C. Identification was performed by comparing re- tention times with those of standards (p-hydroxybenzoic, m-hydroxy- benzoic, protocatechuic, gallic, vanillic, syringic acid, trans-cinnamic, p- coumaric, caffeic, ferulic acid, rosmarinic acid, chlorogenic acid, m- Unauthenticated | Downloaded 09/24/21 06:34 PM UTC 262 A. Machalska et al. coumaric acid, o-coumaric acid, isovanillic acid, and gentisic acid) [6]. Quantitative determination was performed at the wavelength of maximum absorption of the acids – 320 nm for ferulic, p-coumaric, and caffeic acids, 280 nm for trans-cinnamic, syringic, and gallic acids, and 254 nm for vanil- lic, m-hydroxybenzoic, p-hydroxybenzoic, and protocatechuic acids. Calibration plots for the phenolic acids were highly linear (R2 > 0.999) in the concentration range 0.01–2.00 mg per 10 mL (n = 3). Each extract was injected in triplicate on the same day. RSD values (%) ranged from 0.00 to 8.91 (0.01–6.34 for ferulic acid, 0.27–0.51 for gallic acid, 2.28–4.81 for caffeic acid, 0.18–1.95 for m-hydroxybenzoic acid, 0.04–4.74 for p-coumaric acid, 0.00–3.67 for p-hydroxybenzoic acid, 0.10–8.91 for protocatechuic acid, 0.13– 2.97 for syringic acid, 0.70–2.49 for trans-cinnamic acid, and 0.03–4.40 for vanillic acid). Results and Discussion HPLC analysis of fifteen isolated fractions enabled identification of ten phenolic acids, both hydroxybenzoic acid derivatives (p-hydroxybenzoic, m-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids) and hydroxycinnamic acid derivatives (trans-cinnamic, p-coumaric, caffeic, and ferulic acid). Typical chromatograms obtained from the extracts are shown in Figs 1–5. 5 10 15 20 25 30 35 40 min Fig. 1. HPLC chromatogram obtained from analysis of Iris dichotoma Pall. FPhAs (280 nm). Phenolic acids: 1, protocatechuic acid; 2, p-hydroxybenzoic acid; 3, vanillic acid; 6, ferulic acid Unauthenticated | Downloaded 09/24/21 06:34 PM UTC Phenolic Acids in Five Species of Iris from Mongolia 263 5 10 15 20 25 30 35 40 min Fig. 2. HPLC chromatogram obtained from analysis of Iris flavissima Pall. FPhAs (280 nm). Phenolic acids: 1, protocatechuic acid; 2, p-hydroxybenzoic acid; 3, vanillic acid; 4, syringic acid; 5, p-coumaric acid; 6, ferulic acid 5 10 15 20 25 30 35 40 min Fig. 3. HPLC chromatogram obtained from analysis of Iris flavissima Pall. FPhAs (254 nm). Phenolic acids: 1, protocatechuic acid; 2, p-hydroxybenzoic acid; 3, vanillic acid; 4, syringic acid Unauthenticated | Downloaded 09/24/21 06:34 PM UTC 264 A. Machalska et al. 5 10 15 20 25 30 35 min Fig. 4. HPLC chromatogram obtained from analysis of Iris lactea Pall. FPhAs (280 nm). Phenolic acids: 1, protocatechuic acid; 3, vanillic acid; 4, syringic acid; 5, p-coumaric acid; 6, ferulic acid 5 10 15 20 25 30 35 min Fig. 5. HPLC chromatogram obtained from analysis of Iris lactea Pall. AlPhAs (280 nm). Phenolic acids: 1, protocatechuic acid; 2, p-hydroxybenzoic acid; 3, vanillic acid; 4, syringic acid; 5, p-coumaric acid; 6, ferulic acid; 7, gallic acid Unauthenticated | Downloaded 09/24/21 06:34 PM UTC Phenolic Acids in Five Species of Iris from Mongolia 265 Total amounts of phenolic acids (free or bonded in the glycoside or ester- like forms and liberated after acid and alkaline hydrolysis) were: 10.411 mg per 100 g dry wt in Iris bungei Maxim., 9.922 mg per 100 g dry wt in Iris lac- tea Pall., 7.067 mg per 100 g dry wt in Iris flavissima Pall., 5.704 mg per 100 g dry wt in Iris dichotoma Pall., and 2.249 mg per 100 g dry wt in Iris tenuifolia Pall. (Tables III and IV). Table III. Results from qualitative and quantitative analysis of phenolic acids identified in the underground parts of Iris flavissima Pall. (If) and Iris dichotoma Pall. (Id) Fraction Phenolic acid content (mg per 100 g dry wt) exam- Hydroxybenzoic Hydroxycinnamic ined Total V pHB PC S F pC C FPhA 1.170 0.630 0.196 0.078 0.206 0.200 – 2.48 AcPhA 1.031 – – 0.354 0.258 – – 1.643 If AlPhA 0.800 0.152 0.128 0.234 1.438 0.192 – 2.944 Total 3.001 0.782 0.324 0.666 1.902 0.392 – 7.067 FPhA 0.179 0.239 0.031 – 0.269 – – 0.718 AcPhA 0.209 0.086 – 0.544 0.267 – – 1.106 Id AlPhA 0.471 0.181 0.029 0.622 1.763 0.72 0.094 3.88 Total 0.859 0.506 0.06 1.166 2.299 0.72 0.094 5.704 V, vanillic acid; pHB, p-hydroxybenzoic acid; PC, protocatechuic acid; S, syringic acid; F, ferulic acid; pC, p-coumaric acid; C, caffeic acid Amounts of every phenolic acid were estimated by HPLC.