Investigation of Phenolic Constituents in Echinacea purpurea Grown in China

• Abstract
• Introduction
• Materials and Methods
• Growth regions of the herb in China
• Sample collection
• Extraction conditions
• HPLC apparatus and chromatographic conditions
• Determination of total phenolics
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Echinacea is a North American native medicinal herb. In 1990 s, it was introduced in China. Nowadays, Echinacea is growing successfully in a number of places in China, and has been used as a crude drug. However, the phytochemical variation in the plant grown in China has not been studied. In this study, the contents of total phenolics and caffeic acid derivatives in aerial parts and roots of Echinacea purpurea grown in China were investigated by high-performance liquid chromatography (HPLC) and colorimetric analysis. The effects of different drying methods on the components were also studied. The results show that the content of caffeic acid derivatives in E. purpurea reached its highest in the middle stage of full blossoming. The content of caffeic acid derivatives in fresh raw material was generally higher than that in dried raw material. There was no significant difference in the content of caffeic acid derivatives among three geographical populations of E. purpurea. Furthermore, the developmental pattern of total phenolics in E. purpurea was the same as that of caffeic acid derivatives. The stage of mid-bloom is an optimal harvesting period for both caffeic acid derivatives and total phenolics. In addition, the results show that the fresh raw material is the optimal material for pharmaceutical purposes, and that the optimal pharmaceutical parts are the roots, leaves and flowers.

Introduction

Echinacea purpurea is a popular herb in America and Europe. Native Americans were aware of the usefulness of the plant Echinacea along with other herbs in dealing with ailments. Of the several varieties of Echinacea with varying appearances, the most popular species are E. purpurea, E. angustifolia and E. pallida. Echinacea species are harvested for their roots, flower heads, seeds, or pressed as juice of the whole plant for pharmaceutical purposes [1], [2], [3], [4]. Echinacea is best known for its immunomodulatory function, and has also proven to offer other health benefits, such as for sore throats, toothaches, infections, wounds, snakebites, and skin problems. In addition, the species are mainly used for treatment of acute upper respiratory infections including common cold and influenza [5], [6], [7]. As reported in the literature, the major pharmaceutical components of Echinacea are caffeic acid derivatives, alkamides, polysaccharides, and glycoproteins, flavonoids and volatile oils [2], [8], [9], [10].

In 1990 s, Echinacea was introduced to China as a crude drug [11]. Now, it has been applied to commercial medical products widely. So, to utilize the herbal resource more efficiently, it is important to investigate the component background and optimize the harvesting conditions of the herb grown in China. In this paper, the seasonal variation of total phenolics and caffeic acid derivatives, the differences of contents of the components between the fresh and dried raw material, and the distribution of the components in different parts of E. purpurea grown in China were investigated in detail. Based on the results, the optimal harvesting time and the most suitable herbal parts as raw materials for commercial manufacture are suggested [12], [13], [14]. In addition, the contents of the components of herbs grown in 3 different regions of China were also compared. This is helpful to investigate any regional variability of the herb grown in China.

Materials and Methods

Growth regions of the herb in China

The herb samples were collected at the three following sites in China: 1) Changsha (28° 12′ N, 112° 55′ E): situated in the northeastern part of Hunan Province. Elevation is 100 m above sea level (asl). Annual precipitation is 1422.4 - 1556.4 mm. Mean annual temperature is 16.4 - 18.2 °C. 2) Changde (29° 03′ N, 111° 41′ E): situated in the northern part of Hunan Province. Elevation is 131.86 m asl. Annual precipitation is 1300 - 1600 mm. Mean annual temperature is 16.8 °C. 3) Guangyuan (32° 52′ N, 105° 59′ E): situated in the northeastern part of Sichuan Province. Elevation is 650 m asl. Annual precipitation is 1100 - 1500 mm. Mean annual temperature is 14.2 °C. All samples of E. purpurea were collected from the three sites.

Sample collection

The specimens were sampled four times every month and collected in the morning from March to November, 2006. Samples with similar height and growing conditions were randomly selected. The distance between two selected plants was more than 10 m. The samples of E. purpurea were washed and sucked dry with absorbent paper. Then, the roots, stems, leaves and flowers were separated quickly. The samples of Changde and Guangyuan were put in a bag, placed in pots filled with ice, and transported to the laboratory immediately. The plant materials were identified as Echinacea purpurea by Professor Jian-Zhong Li, Department of Botany, Hunan Normal University, China. Voucher specimens (no. RS0001 - RS0390) are deposited in the Institute of Chemistry, Hunan Normal University. 390 samples were collected during this study.

Extraction conditions

All samples were divided into four parts. Three parts were dried by different methods such as oven drying to constant weight at 60 °C, drying under sun light, and drying in the shade, respectively. The fourth part was saved as fresh plant material. After determination of the water content of the samples, the water content of fresh plant material was 73.5 - 90.8 %, and the water content of dried plant material was 4.7 - 10.5 %. Because the water contents of fresh and dried plant material were different, extraction solvents must be investigated. After optimizing the extraction solvents, it was found that the optimum extraction solvent for fresh plant material is 0.5 % phosphoric acid in methanol, and 70 % aqueous methanol solution containing 0.5 % phosphoric acid for dried plant material, respectively. The dried plant material was ground to powder with a tissue grinder and was passed through a 2-mm size mesh. One gram of the powder was extracted with 100 mL of 70 % (v/v) aqueous methanol solution containing 0.5 % (v/v) H₃PO₄ for 45 min in a calyptrate conical flask in an ultrasonic extractor. The fresh plant material was cut into pieces. Three grams of fresh chip sample were extracted with 100 mL of methanol with 0.5 % (v/v) H₃PO₄ for 45 min in a calyptrate conical flask in an ultrasonic extractor. The extraction solution was transferred to a 100-mL volumetric flask, and diluted to volume with methanol. The extraction was filtered through a 0.45 μm Millipore filter. The filtrate was analyzed by high-performance liquid chromatography (HPLC).

HPLC apparatus and chromatographic conditions

The content of caffeic acid derivatives was determined according to reported methods [15], [16], [17] by an HPLC apparatus consisting of a Waters 1525 Binary HPLC pump, a Waters 717 plus Autosampler, and a Waters 2996 Photodiode Array Detector, its detection wavelength was set at 330 nm. The separation was completed on a 4.6 × 200 mm Diamonsil 5 μm C₁₈ column. Acetonitrile (Solution A) and 0.1 % H₃PO₄ aqueous solution (Solution B) were used as mobile phases. The flow rate was 1.0 mL/min. The gradient program is shown in Table [1]. Chlorogenic acid (> 98 %, HPLC) purchased from Sigma (St. Louis, MO, USA) was used as an external calibration standard. A series of standard working solutions were analyzed to obtain a calibration curve. Injection volume was 10 μL. The correlation coefficient was 0.9993. The quantification of caffeic acid derivatives in samples was completed according to their relative response factor vs. chlorogenic acid at 330 nm. The identification of caffeic acid derivatives was completed by HPLC/ESI-MS [16]. The relative response factor (Fi) is 0.695 for cichoric acid, 0.881 for caftaric acid, 1.000 for chlorogenic acid, and 2.220 for echinacoside, respectively [15]. The percentage of caftaric acid, cichoric acid, chlorogenic acid and echinacoside in the samples was calculated according to the following equation:

% w/w (of plant material) individual phenolic compound = 10 000 × Fi × (Ai/As) × (C/W)

where Fi is the relative response factor; C is the concentration of chlorogenic acid in the standard solution (mg/mL); W is the sample weight (mg); Ai is the peak area of the relevant caffeic acid derivatives in the sample solution; As is the peak area of chlorogenic acid in the standard solution.

Determination of total phenolics

The content of total phenolics in E. purpurea was determined by the Folin-Denis method [12], [18]. The raw material was ground to a 20 mesh powder. One gram of the powder was extracted in an ultrasonic wave bath with 80 mL of aqueous ethanol solution (70 %) for 2 h. After cooling down, the volume of the solution was adjusted to 100 mL. The final solution was centrifuged prior to the colorimetric determination. Chlorogenic acid standards (10, 30, and 50 mg) were dissolved in 100 mL of aqueous ethanol solution (70 %), respectively. 10 mL of Folin-Denis reagent was added to either 1 mL of extract solution or 1 mL of standard solution. After reacting for 3 min, 10 mL of 35 % sodium carbonate solution was added and the test solution was diluted to 100 mL with H₂O and mixed. After 45 min, an aliquot was centrifuged for 5 min. Then the clear solution was transferred into a cuvette and the absorption coefficient measured at 745 nm. The standard chlorogenic acid solutions were taken as equivalent to 1, 3, and 5 % total phenolics calculated as chlorogenic acid. The total phenolic contents of the extracts were calculated using the linear regression coefficient from the standards.

Statistical analysis

ANOVA was performed to investigate the effect of season, region, different plant parts and different drying methods on the content of caffeic acid derivatives and total phenolics.

Results and Discussion

The schematic presentation of the seasonal variation of caffeic acid derivatives in the different plant parts E. purpurea grown in China is shown in Fig. [1]. From the results, we can see that the stems, leaves and flowers presented the same seasonal variation patterns of caffeic acid derivatives and total phenolics. The contents of caffeic acid derivatives and total phenolics decreased significantly (p < 0.001) during the whole period of growth. The contents of caffeic acid derivatives and total phenolics in the roots of E. purpurea decreased significantly (p < 0.001) during the vegetative growth of the plants, and reached its lowest value in the middle stage of blossoming, then increased (p < 0.001) and reached their highest value in winter. Fig. [1] E shows that the variation patterns of caffeic acid derivatives and total phenolics in flowers during the florescence. From the budding stage to the withering stage, the contents of the compounds decrease stage by stage. Fig. [2] shows the yield variation profile of the compounds in a whole plant in seedling stage and florescence. The results present that the yield of biosynthesis of the caffeic acid derivatives and total phenolics is maximal in the middle stage of blossoming. The results are in agreement with those reported in the literature [19]. So, the middle stage of blossoming would be the best harvesting time for the purpose of extracting caffeic acid derivatives and total phenolics. However, the best harvesting time for the roots would be the wintertime in November. In addition, the percentage of the stems is about 27 - 35 % in a whole plant weight in the mid-stage of blossoming, and the total content of the compounds in the stems is the lowest among that in the different parts of the plant. Thus, the stems should be abnegated in the manufacture of the extracts for saving solvents and energy.

As the assimilation is often higher than disassimilation during the vegetative growth, the secondary metabolic products of the plants accumulate continually during the course. When plants come into reproductive growth, a considerable amount of photosynthates flows into reproductive organs [20], [21]. So the contents of caffeic acid derivatives and total phenolics in the leaves decreases significantly after florescence. As temperature and light conditions change in fall, leaves turn to brown yellow and caffeic acid derivatives in the leaves decompose or transfer into storage organs, such as roots, rhizomes before the leaves fall, which is reflected by the fact that the content of caffeic acid derivatives and total phenolics in the aerial part of E. purpurea decreases to low levels in the fall as observed in other studies [22], [23].

The effect of regions on the contents of the compounds was also studied (see Table [2]). This shows that the contents of phenolic constituents in E. purpurea grown in Changsha were higher than those in Changde and Guangyuan. However, the differences of the contents of the compounds in the plants grown in the three regions are not appreciable because the three geographical sites have similar environmental conditions such as air temperature, sunlight, rainfall and soil, which affect biosynthesis and biodecomposition of phenolic compounds [24]. Thus, the similar contents of phenolics in the three populations are creditable. The contents of cichoric acid and total phenolics were 1.594 % and 5.310 % in the dried plant materials obtained from Changsha, respectively. This was higher than that in the plants grown in Beijing (cichoric acid, 1.108 %) [11], Australia (1.25 - 1.38 %) [25] and America (0.092 - 0.899 %) [22].

The contents of caffeic acid derivatives and total phenolics in the fresh samples were remarkably higher than that in the dried samples (see Table [3]). Therefore, most of the active constituents such as cichoric acid and caftaric acid were destroyed in the process of drying. Heat drying is the best way of drying among the three drying methods. The preservation rate of cichoric acid, caftaric acid and total phenolics was 26.7 - 73.4 %, 56.5 - 87.9 %, 63.9 - 89.7 %, respectively. The preservation rate under the heat drying is higher than that under the other drying methods. There were two possible reasons for this variation. First, fresh E. purpurea contain natural polyphenol oxidase enzymes. The polyphenol oxidase is responsible for the degradation of phenolic compounds [26], [27]. Heat drying can rapidly make it inactive. Besides, the enzyme reaction time was shortened remarkably during the heat drying. So, heat drying (at 60 °C) is better than drying in the shade and in the sun. A shorter drying time for E. purpurea is more desirable from an industrial efficiency perspective, and this can certainly be achieved by increasing the air temperature. Cichoric acid is sensitive to heat, the increase in temperature will result in enhanced loss of cichoric acid [26], [28] , [29]. So, the heat drying temperature is suggested to be not more than 60 °C. Alternatively, considering the loss of the compounds during the process of dryness, the fresh plant materials should be better raw materials for pharmaceutical purposes.

Acknowledgements

We are grateful to Ms. L. J. Luo and Ms. Q. Luo from Hunan Phytoway Inc. for their expert technical assistance and fruitful discussion during the study, to Mr. H. J. Yuan and Mr. J. Zhou for their help in sample collection. This work was financially supported by the National ”973” project & Key research project of Hunan Province (2006CB504701, 05SK2006).

References

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Prof. Dr. Bo Chen

Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research

Ministry of Education

Hunan Normal University

Changsha 410081

People’s Republic of China

Phone: +86-731-8865-515

Fax: +86-731-8865-515

Email: dr-chenpo@vip.sina.com

References

• 1 McGregor R L. The taxonomy of the genus Echinacea (Compositae).  Univ Kans Sci Bull. 1968;  48 113.
  PubMedSearch in Google Scholar
• 2 Perry N B, Wills R BH, Stuart D L. Factors affecting Echinacea quality: Agronomy and processing.  Boca Raton, FL: CRC. Press;  2004 111-26.
  PubMedSearch in Google Scholar
• 3 Schulthess B H, Ginger E, Baumann T W. Echinacea: anatomy, phytochemical pattern, and germination of the achene.  Planta Med. 1991;  57 384.
  PubMedSearch in Google Scholar
• 4 Bauer R. Standardization of Echinacea purpurea expressed juice with reference to cichoric acid and alkamides.  J Herbs Spices Med Plants. 1999;  6 51-62.
  PubMedSearch in Google Scholar
• 5 Barrett B. Medicinal properties of Echinacea .  Phytomedicine. 2003;  10 66-86.
  CrossrefPubMedSearch in Google Scholar
• 6 Bukovsky M, Vaverkova S, Kostalova D. Immunomodulating activity of Echinacea gloriosa L., Echinacea angustifolia DC., and Rudbeckia speciosa Wenderoth ethanol-water extracts.  Pol J Pharmacol. 1995;  4 175-7.
  PubMedSearch in Google Scholar
• 7 Bergeron C, Gafner S, Batcha L L, Angerhofer K. Stabilization of caffeic acid derivatives in Echinacea purpurea L. glycerin extract.  J Agric Food Chem. 2002;  50 3967-70.
  CrossrefPubMedSearch in Google Scholar
• 8 Hu C, Kitts D D, Zawistowski J. The chemistry of antioxidant constituents of Echinacea . Boca Raton, FL; CRC Press 2004: 73-89.
  Search in Google Scholar
• 9 Thygesen L, Thulin J, Mortensen A, Skibsted L H, Molgaard P. Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea, alone and in combination.  Food Chem. 2006;  101 74-81.
  PubMedSearch in Google Scholar
• 10 Percival S. Use of Echinacea in medicine.  Biochem Pharmacol. 2000;  60 155.
  CrossrefPubMedSearch in Google Scholar
• 11 Dou D M, Cui S Y, Cao Y J, Yan Y J, Shuai F. Assaying of cichoric acid in introducing plant of Echinacea purpurea .  J Chin Traditional Herb Drugs. 2001;  32 977-8.
  PubMedSearch in Google Scholar
• 12 Nigel B P, Elaine J B, Leanne G V. Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species.  J Agric Food Chem. 2001;  49 1702-6.
  CrossrefPubMedSearch in Google Scholar
• 13 Gary D E, Pallardy S G, Garrett H E, Rottinghaus G E. Acute drought stress and plant age effects on alkamide and phenolic acid content in purple coneflower roots.  Planta Med. 2003;  69 50-5.
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Perry N B, van Klink J W, Burgess E J, Parmenter G A. Alkamide levels in Echinacea purpurea: effects of processing, drying and storage.  Planta Med. 2000;  66 54-6.
  Thieme ConnectPubMedSearch in Google Scholar
• 15 USP29-NF24. Washington, D.C; The U. S. Pharmacopeial Convention, Inc. press 2006: 2318-21
  Search in Google Scholar
• 16 Luo X B, Chen B, Yao S Z, Zeng J G. Simultaneous analysis of caffeic acid derivatives and alkamides in roots and extracts of Echinacea purpurea by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry.  J Chromatogr A. 2003;  986 73-81.
  CrossrefPubMedSearch in Google Scholar
• 17 Federica P, Stefania B, Michele M, Todd L. Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC.  Phytochem Anal. 2005;  16 77-85.
  CrossrefPubMedSearch in Google Scholar
• 18 Shahidi F, Naczk M. Folin-Denis assay. Food phenolics: source, chemistry, effects, applications. Lancaster, PA; Technomic Publishing Co., Inc. Press 1995: 292.
  Search in Google Scholar
• 19 Miao Z L. The cultivation techniques of Echinacea .  Li Shizhen Med Material Med Res. 2006;  17 482.
  PubMedSearch in Google Scholar
• 20 Guan Y M, Wang J, You H L, Liu X. A study on the changing rule for total flavonoids in Ginkgo leaves in different climate season and age.  Chin Traditional Pat Med. 2000;  22 368-70.
  PubMedSearch in Google Scholar
• 21 Cheng S Y, Gu M R, Shu H R. Advance in the study of the formation and regulation of flavonoids Ginkgo biloba leaves.  Resour Sci. 2000;  22 46-8.
  PubMedSearch in Google Scholar
• 22 Binns S E, Livesey J F, Arnason J T, Baum B R. Phytochemical variation in Echinacea from roots and flowerheads of wild and cultivated populations.  J Agric Food Chem. 2002;  50 3673-87.
  CrossrefPubMedSearch in Google Scholar
• 23 Binns S E, Arnason J T, Baum B R. Phytochemical variation within populations of Echinacea angustifolia (Asteraceae).  Biochem Syst Ecol. 2002;  30 837-54.
  CrossrefPubMedSearch in Google Scholar
• 24 Wang S Y, Zhang W. Effect of plant growth temperature on antioxidant capacity in strawberry.  J Agric Food Chem. 2001;  49 4977-82.
  CrossrefPubMedSearch in Google Scholar
• 25 Wills R BH, Stuart D L. Alkylamide and cichoric acid levels in Echinacea purpurea grown in Australia.  Food Chem. 1999;  67 385-8.
  CrossrefPubMedSearch in Google Scholar
• 26 Nusslein B, Kurzmann M, Bauer R, Kreis W. Enzymatic degradation of cichoric acid in Echinacea purpurea preparations.  J Nat Prod. 2000;  63 1615-8.
  CrossrefPubMedSearch in Google Scholar
• 27 Nusslein B, Kurzmann M, Kreis W. Enzymatic degradation of caffeic acid derivatives in Echinacea purpurea herbs. 46th Annual Congress of the Society for Medicinal Plant Research Vienna; 1998.
  Search in Google Scholar
• 28 Douglas L S, Ron B HW. Effect of drying temperature on alkylamide and cichoric acid concentrations of Echinacea purpurea .  J Agric Food Chem. 2003;  51 1608-10.
  CrossrefPubMedSearch in Google Scholar
• 29 Kim H O, Durance T D, Scaman C H, Kitts D D. Retention of caffeic acid derivatives in dried Echinacea purpurea .  J Agric Food Chem. 2000;  48 4182-6.
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Bo Chen

Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research

Ministry of Education

Hunan Normal University

Changsha 410081

People’s Republic of China

Phone: +86-731-8865-515

Fax: +86-731-8865-515

Email: dr-chenpo@vip.sina.com