The Effects of Plant Age and Harvesting Time on Chicoric and Caftaric Acids Content of E. purpurea (L.) Moench

Document Type: Research Paper

Authors

1 Traditional Medicine and Materia Medica Research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Food and Drug Laboratory Research Center, and Food and Drug Control Laboratories, Ministry of Health and Medical Education, Tehran, Iran.

3 Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Abstract

      Plants of Asteraceae are used in traditional medicine and phytotherapy. The two main caffeic acid derivatives, chicoric and caftaric acid which are found in many genus of Asteraceae including Echinacea exhibit important biological activities. The level of these acids in E. purpurea is affected by many factors such as growing situations, extraction methods, storage conditions and plant age. In this investigation, chicoric acid and caftaric acid content in aerial parts and roots of E. purpurea harvested in spring and summer from 1-, 2- and 3-years old plants cultivated in Iran were determined by using HPLC method. The results revealed that maximum level of chicoric acid achieved in aerial parts of 1- and 2-years old plants beside 2-years old roots collected in spring. Aerial parts of 1- and 2-years old plants harvested in spring had maximum content of caftaric acid as well. It is concluded that total parts of 2-years old E. purpurea harvested in spring can be a good source of caffeic acid derivatives and used for preparation of the plant products.

Keywords


1. Introduction

 

 

    The genus of Echinacea (Asteraceae) includes limited species. One of the three medicinal species is E. purpurea which is native to central and eastern parts of United States [1, 2]. It is cultivated in many other countries, including Iran and numerous phar-macological and phytochemical studies have been performed on this species [3].

 

    In the past, Echinacea has been used against infectious diseases and snakebite [4, 5]. Nowadays, it is administered for prevention and treatment of colds and other respiratory tract infections to ameliorate the symptoms and shorten the duration of illness [2, 4, 6, 7]. The main constituents of Echinacea are alkamides, polysaccharides, caffeic acid derivatives, and glycoproteins [4]. These constituents are responsible for the plant effects such as immunomodulatory, antioxidant, anti-inflammatory, antifungal and antiviral activities [1, 3, 6, 8-18].

 

    Chicoric acid and caftaric acid (Figure 1) are the main caffeic acid derivatives in E. purpurea which are responsible for some biological effects of the plant such as hyaluronidase inhibitory activity, antioxidant property and enhancement of insulin secretion [5, 8, 17].

 

    The therapeutic effectiveness of different Echinacea products is depended on chemical composition of the plant. Many factors including species of Echinacea (E. purpu-rea, E. pallida or E. angustifolia), plant parts (leaves, flowers, stems or roots), plant age, the time of harvesting, grow¬ing, drying and storage conditions and method of extraction may influence the product quality [4, 10, 19-21]. So determination of the active ingredients level in different parts of the plant and best time of harvesting is very important for the industrial purposes. For this reason, we decided to measure chicoric acid and caftaric acid level in roots and aerial parts of E. purpurea harvested in spring and summer from 1- , 2- and 3-years old plants by using HPLC which is one of the most popular and precise method for analysis of herbal components [22-24].

 

2. Experimental

 

2.1. Plant material

 

    Aerial parts and roots of E. purpurea were collected from 1-, 2- and 3-years old plants from their cultivating area in Gorgan, province of Golestan, Iran, by Dr. M.H. Soleimani, Giyah Essence Company and identified by Mrs. M. Khatamsaz, Botanist, from Research Institute of Forests and Rangelands (Tehran).

 

2.2. Chemicals

 

   Acetonitrile (HPLC grade), ethanol (analytical grade), O-phosphoric acid (analytical grade) were purchased from Merck (Germany). The standard material of chlorogenic acid was prepared from Aldrich (Germany). The water used in HPLC and for sample preparation was produced with a Purelab UHQ (ELGA) with a resistivity over 18 MΩ-cm.

 

2.3. Instrumentation

 

    HPLC experiment was performed using Waters Alliance 2695 system equipped with a vacuum degasser, quaternary solvent mixing, auto sampler and a waters 2996 photodiode array detector. UV spectra were collected across the range of 200-900 nm extracting 330 nm for chromatograms. EmpowerTM chromatography data software was utilized for instrument control, data collection and processing. The column, an ACE C18 (4.6-mm 25cm, 5μm) was maintained at 35 ˚C. The mobile phase was a mixture of acetonitrile and phosphoric acid 0.085% in gradient mode (Table 1). The flow rate was 1.5 ml/minute. Injection volume for all test and standard solutions was 10 μl. Each test solution was injected 3 times.

 

Figure 1.Structures of chicoric acid and caftaric acid.

 

2.4. Preparation of solutions

 

2.4.1. Standard solutions preparation

 

     Stock standard solution was prepared accurately by weighing 10 mg of chlorogenic acid reference standard into 10 ml volumetric flask and dissolving in ethanol:water (7:3). Serial dilutions (10-500 µg/ml) were made from the stock solution.

 

2.4.2. Sample preparation

 

     For the extraction of phenolic compounds of aerial parts and roots of E. purpurea, 125 mg of dried and finely powdered material from each plant sample was accurately weighed and transferred to a round bottom flask, and 25 ml of solvent (ethanol:water, 7:3) was added and heated under reflux system for 15 min. The resulting mixture was filtered and diluted to 25 ml with the solvent [25]. Three replicate of each sample were prepared as described.

 

2.5. Determination of chicoric and caftaric acid content in the plant samples

 

     Quantitative determination of caftaric and chicoric acid was performed by using chlorogenic acid standard calibration curve. Since different compounds absorb various amounts of radiation at a fixed wavelength, it is necessary to use a correction factor to calculate caftaric and chicoric acid percentage by this method. This factor is determined by BP for each of the compounds, and they are equal to 0.695 and 0.881 for chicoric acid and caftaric acid, respectively [25]. At first, areas under the caftaric and chicoric acid curves were multiplied in the related correction factor to obtain area under the curve according to chlorogenic acid. Then concentration of the compounds in the test solution was calculated using the calibration curve and following equation:

%w/w=(C×V)/10M

 

C: concentration of phenolic compound (ppm) from linear regression analysis;

 

V: Extract solution volume (25 ml); M: Sample weight (125 mg).

 

2.6. Statistical analysis

 Comparisons between groups were made by SPSS software and ANOVA test.

 

 

Differences with p < 0.05 between groups were considered statistically significant

Figure 2. HPLC chromatogram of a sample from spring/2y E. purpurea aerial parts; the 5.2 min peak is related to caftaric acid, and 13.8 min peak is related to chicoric acid.

 


3. Results and discussion

 

     Main peaks were observed in retention times about 5.2 and 13.8 min in the HPLC chromatograms of the test solutions (Figure 2). Chlorogenic acid ST peaks appeared about 5.7 min in reference standard solutions chromatograms. Caftaric and chicoric acid were identified by comparison with authentic chromatogram in British Pharmacopeia [25]. Relative retention times with reference to chlorogenic acid ST were about 0.9 and 2.4 for two main peaks in test chromatograms and had good compatibility with BP relative retention times for caftaric and chicoric acid, respectively. So, main peaks have been recognized as caftaric (5.2 min) and chicoric acid (13.8 min).

 

     According to chlorogenic acid standard calibration curve (y=2.0708x-10.361, r2=0.9993), the content of both phenolic compounds in the plant samples were determined. Caftaric and chicoric acid percentages in the aerial parts and roots of E. purpurea are shown in Figures 3 and 4, respectively.

 

    The results of quantitative determination of chicoric acid and caftaric acid by HPLC demonstrated that 1- and 2-years old aerial parts, in addition 2-years old roots harvested in spring had maximum amount of chicoric acid and there was no statistical difference between these 3 sample groups (p>0.05). Maximum level of caftaric acid found in aerial parts of 1- and 2-years old plants harvested in spring. The minimum amounts of caftaric and chicoric acid were observed in the roots collected in summer from 2-years old plants.

 

    The results also showed that in all samples, caftaric acid content of the roots was less than the aerial parts, but no distinct manner was found in chicoric acid content of roots and aerial parts. Chicoric acid amount was higher in the aerial parts than the roots in the plants harvested in spring (1 and 3 years) and summer (2 and 3 years), and it was higher in roots of summer (1 year) samples. Spring (2 years) roots and aerial parts had the same content of chicoric acid.

 

    Chicoric acid is subsequently used as the indicator of caffeoyl phenols in the commercial products [26]. As it is obvious in Figures 2 and 3, chicoric acid content in the plant samples is higher than caftaric acid, so in general, it can be concluded that spring (2 years) plant has the best quality according to phenolic compounds and it is better to use total parts of the plant for preparation of the Echinacea products.

 

      In a study, the effects of plantation age of E. purpurea on the flavonoids and phenolics content has been investigated and no relationship was found between age of plants and flavonoid and phenolic content in flower heads. This study has proved that the phenolic acids content was slightly higher in the flower heads than in the rhizomes [27]. Another investigation indicated that E. purpurea flower heads from 2-years old cultivated plants contain about the same amount of chicoric acid as the young roots of that species. They demonstrated that caffeic acid derivatives in Echinacea contain compounds with closely structure that vary throughout the growth of the plants. In that study, the content of phenolic compounds of E. purpurea roots has been decreased with age [28]. According to references [25, 29] aerial parts of E. purpurea and roots of E. angustifolia are used as medicinal parts but this study showed that the cultivated plant in Iran is rich in phenolics in both aerial parts and roots, therefore, in order to produce a qualified product from E. purpurea it is necessary to consider the age and harvesting time of the plant.

 

Figure 3. Caftaric acid content in aerial parts and roots of E. purpurea, harvested from 1-, 2- and 3-years old plants in spring and summer.

 

Figure 4. Chicoric acid content in aerial parts and roots of E. purpurea, harvested from 1-, 2- and 3-years old plants in spring and summer.

4. Conclusion

 

     Some of the most important factors which cause inconsistency in industrial products are plant parts and plant age in the time of harvesting. This study showed that 2- years old E. purpurea aerial parts and roots harvested in spring had the most content of chicoric and caftaric acids and it is better to use total plant for preparation of E. purpurea products.

 

Acknowledgment

 

The authors wish to thanks Dr. M.H. Soleimani from Giyah Essence Company for plants collection. The results have been obtained from thesis of Pharm.D. student.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[1]     Percival  SS.  Use  of  Echinacea  in  medicine. Biochem Pharmacol 2000; 60: 155-8.

 

[2]    Blumenthal  M,  Goldberg A,  Brinckmann  J. Herbal Medicine. Expanded Commission E monographs: Austin: Integrative Medicine Communications, 2000.

 

[3]     Pellati F, Benvenuti S, Magro L, Melegari M, Soragni F. Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. J Pharm Biomed Anal 2004; 35: 289-301.

[4]    Miller SC, Yu HC. Echinacea: the genus Echinacea: London: CRC Press, 2004.

 

[5]     Barrett B. Medicinal properties of Echinacea: a critical review. Phytomedicine 2003; 10: 66-86.

[6]    Cepae BA. WHO monographs on selected medicinal plants. Geneva: World Health Organization, 1999.

 

[7]     Shah SA, Sander S, White CM, Rinaldi M, Coleman CI. Evaluation of Echinacea for the prevention and treatment of the common cold: a meta-analysis. Lancet Infect Dis 2007; 7: 473-80.

 

[8]    Thygesen L, Thulin J, Mortensen A, Skibsted LH, Molgaard P. Antioxidant activity of chicoric acid and alkamides from Echinacea purpurea, alone and in combination. Food Chem 2007; 101: 74-81.

 

[9]    Burger RA, Torres AR, Warren RP, Caldwell VD, Hughes BG. Echinacea-induced cytokine production by human macrophages. Intern J Immunopharmacol 1997; 19: 371-9.

 

[10]   Rininger JA, Kickner S, Chigurupati P, McLean A, Franck Z. Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells. J Leukocyte Biol 2000; 68: 503-10.

 

[11]   Raso GM, Pacilio M, Carlo G, Esposito E, Pinto L, Meli R. In vivo and in vitro anti- inflammatory effect of Echinacea purpurea and Hypericum perforatum. J Pharm Pharmacol 2002; 54: 1379-83.

 

[12]   Clifford L, Nair M, Rana J, Dewitt D. Bioactivity of alkamides isolated from Echinacea purpurea (L.) Moench. Phytomedicine 2002; 9: 249-53.

[13]  Merali S, Binns S, Paulin-Levasseur M, Ficker C, Smith M, Baum B, et al. Antifungal and anti-inflammatory activity of the genus Echinacea. Pharm Biol 2003; 41: 412-20.

 

[14]  Binns S, Purgina B, Bergeron C, Smith M, Ball L, Baum B, et al. Light-mediated antifungal activity of Echinacea extracts. Planta Med 2000; 66: 241-4.

 

[15]  Binns S, Hudson J, Merali S, Arnason J. Antiviral activity of characterized extracts from Echinacea spp.(Heliantheae: Asteraceae) against Herpes simplex virus (HSV-1). Planta Med 2002; 68: 780-3.

 

[16]   Lee JY, Yoon KJ, Lee YS. Catechol-Substituted-Chicoric acid analogues as HIV integrase inhibitors. Bioorg Med Chem Lett 2003; 13: 4331-4.

 

[17]   Tousch D, Lajoix AD, Hosy E, Azay-Milhau J, Ferrare K, Jahannault C, et al. Chicoric acid, a new compound able to enhance insulin release and glucose uptake. Biochem Biophys Res Commun

2008; 377: 131-5.

 

[18]  Bauer R, Wagner H. Echinacea species as potential immunostimulatory drugs. Econ Med Plant Res 1991; 5: 5.

 

[19]  Iranshahi M, Amanzadeh Y. Rapid isocratic HPLC analysis of caffeic acid derivatives from Echinacea purpurea cultivated in Iran. Chem Nat Comp 2008; 44: 190-3.

 

[20]  Gray DE, Pallardy SG, Garrett H, Rottinghaus GE. Acute drought stress and plant age effects on alkamide and phenolic acid content in purple coneflower roots. Planta Med 2003; 69: 50-5.

[21]  Hajimehdipoor H, Khanavi M, Shekarchi, Abedi Z, Pirali Hamedani M . Investigation of the best method for extraction of phenolic compounds from Echinaceae purpurea L. (Moench). J Med Plants 2010; 8: 145-52.

 

[22]  Shekarchi M, Hajimehdipoor H, Khanavi M, Adib N, Bozorgi M, Akbari-adergani B, Pirali Hamedani M. A validated method for analysis of swerchirin in Swertia longifolia Boiss. by high performance liquid chromatography. Phcog Mag 2010; 6: 13-8.

 

[23]  Hajimehdipoor H, Shekarchi M, Khanavi M, Adib N, Amiri M. A validated HPLC method for analysis of thymol and carvacrol in Thymus vulgaris L. Volatile Oil. Phcog Mag 2010; 6: 154-8.

 

[24]   Shekarchi M, Khalili F, Mostofi Y, Hajimehdipoor H. High performance liquid chromatographic method determination of ascorbic acid in Brassica oleracea L. var. italica Plenck. Biosci Biotech Res Asia 2011; 8: 95-100.

 

[25]  BP. Vol. 4, London: The Stationary Office. 2011; pp. 3468-72.

 

[26]  Wills RBH, Stuart DL. Alkylamide and chicoric acid levels in Echinacea purpurea grown in Austria. Food Chem 1999; 67: 385-8.

[27]  Seemannova Z, Mistrikova I, Vaverkova S. Effects of growing methods and plant age on the yield, and on the content of flavonoids and phenolic acids in Echinacea purpurea (L.) Moench. Plant Soil Environ 2006; 52: 449-53.

 

[28]   Binns SE, Livesey JF, Arnason JT, Baum BR. Phytochemical variation in Echinacea from roots and flower heads of wild and cultivated populations. J Agr Food Chem 2002; 50: 3673-87.

 

[29]  USP31, NF26. Vol 1, Washington DC: The Board of Trustees; 2008. p 933.