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Crop Science 41:1159-1161 (2001)
© 2001 Crop Science Society of America

CROP ECOLOGY, MANAGEMENT & QUALITY

Quantification of Root Chicoric Acid in Purple Coneflower by Near Infrared Reflectance Spectroscopy

D. E. Gray*,a, C. A. Robertsc, G. E. Rottinghausd, H. E. Garrettb and S. G. Pallardyb

a Herb Pharm, Williams, OR, 97544
b School of Natural Resources
c Dep. of Agronomy
d College of Veterinary Medicine, Univ. of Missouri, Columbia, MO 65211

* Corresponding author (dgray{at}herb-pharm.com)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Purple coneflower [Echinacea purpurea (L.) Moench] is an increasingly popular crop because of its value in the U.S. botanical industry. At present, there is no rapid method of analyzing it for chicoric acid, the predominant phenolic acid associated with immunostimulation in humans. The objective of this study was to quantify chicoric acid in purple coneflower roots by near infrared (NIR) reflectance spectroscopy. One hundred sixty-nine plants were harvested and their root tissues analyzed for chicoric acid by high performance liquid chromatography (HPLC). Root samples were scanned between 1100 and 2498 nm and reflectance data recorded. The HPLC data were regressed against infrared spectra to develop empirical prediction equations. The optimum prediction equation produced coefficients of determination for calibration and cross validation of 0.90 and 0.86, respectively. The mean chicoric acid concentration was 8.29 g kg DM-1, and the standard errors of calibration and cross validation were 0.89 and 1.06 g kg DM-1, respectively. We conclude that NIR reflectance spectroscopy can accurately quantitate chicoric acid in purple coneflower.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
PURPLE CONEFLOWER is a crop gaining popularity for its value as a dietary supplement. This species is grown, extracted, and made into dietary supplements that currently rank among the highest selling in the USA (Brevoort, 1998). Supplements containing purple coneflower are used for the treatment and prevention of colds and upper respiratory infections in humans (Rehman et al., 1999; Barrett et al., 1999; Melchart et al., 1998; Brinkeborn et al., 1998).

The beneficial effect of purple coneflower extract is reportedly due to its immunostimulatory activity (Rehman et al., 1999; Barrett et al., 1999; Melchart et al., 1998; Brinkeborn et al., 1998; Alschuler et al., 1997). Although the extract contains many natural products that may act synergistically to produce its putative therapeutic efficacy, one of its constituents, chicoric acid, also known as cichoric acid, is believed to be the predominant phenolic acid responsible for this immunostimulatory activity (Bauer and Wagner, 1991). L-Chicoric acid has additionally been shown to be a selective inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (King et al., 1999; King and Robinson, 1998; Robinson, 1998).

As purple coneflower production increases, the industry is developing a need for crop quality analysis. At present, standardized purple coneflower products generally contain 40 mg g-1 total phenolics. Current quality control methods for this crop do not specifically quantify individual phenolics that have proven therapeutic value. Nor do current methods provide rapid, accurate procedures. If a method could be developed for efficient quantification of an important phenolic acid, such as chicoric acid, purple coneflower could be partially evaluated on the basis of a biologically active ingredient.

A method for efficient quantification of chicoric acid may be developed by means of NIR spectroscopy, an empirical technology that offers a rapid, nondestructive, and accurate analysis of a wide range of plant products. The NIR method involves obtaining infrared spectra from a set of samples, analyzing the samples in the laboratory to obtain known values, then regressing the spectral data against the laboratory-derived data (Roberts et al., 1997). Successful regression results in a standard calibration equation that permits the prediction of laboratory values from infrared spectra, thereby eliminating the cost and time required for routine chemical analysis.

Recent investigators have used NIR reflectance spectroscopy to determine levels of ergovaline in tall fescue (Festuca arundinacea Schreber; Roberts et al., 1997), alkaloids and phenolics in green tea [Camellia sinensis (L.) O. Kuntze; Schulz et al., 1999], ginsenosides in American ginseng (Panax quinquefolium L.; Ren and Chen, 1999), tannins in birdsfoot trefoil (Lotus corniculatus L.; Roberts et al., 1993), and total dietary fiber in cereal grains (Kays et al., 1996; Windham et al., 1997; Kays et al., 1998; Kays et al., 1997). The objective of this study was to quantify chicoric acid in purple coneflower by NIR reflectance spectroscopy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Sample Selection and Preparation
One hundred sixty-nine root samples of purple coneflower were collected from ongoing container and field experiments at the Horticulture and Agroforestry Research Center, New Franklin, MO. Samples represented a wide range of age, propagation, and harvest season, and were collected from container-grown plants throughout 1997 and 1998, as well as from field-cultivated plants from the autumn of 1998. Plants ranged from 1 to 3 yr old.

Following harvest, roots were separated from top growth, rinsed with water, and dried at 60°C. After drying, samples were ground to pass a 1-mm screen with a cyclone-type grinder. Ground samples were stored at -20°C until spectral and chemical analyses.

NIR Spectra Collection
All 169 root samples were analyzed spectrally with a NIRSystems scanning monochromator, model 5000 (Silver Spring, MD) with software developed by Infrasoft Inernational (Port Matilda, PA). Samples were scanned with near infrared radiation from 1110 to 2490 nm, and log 1/reflectance (log 1/R) was recorded at 2-nm intervals.

Chemical Analysis
After spectral analysis, samples were analyzed in triplicate for chicoric acid by the following procedure. A 0.5-g subsample was weighed into a 20-mL screw cap bottle and 20 mL methanol were added. Each sample was extracted for 24 h on a rotating shaker. Contents were allowed to settle, and a 100-µL aliquot was combined with 400-µL distilled water and immediately analyzed by HPLC.

A modified method described by Bauer and Wagner (1991) was used for HPLC quantification of root chicoric acid. Twenty microliters of sample were injected onto a Hitachi L-7100 gradient HPLC pump equipped with a Hitachi L-7400 UV detector (284 nm) and a Luna 5-µm C18 450- by 4.60-mm reversed-phase column (Phenomenex, Torrance, CA). The mobile phase was filtered and degassed under vacuum and pumped at 1 mL/min. The mobile phase was 7:3 (H2O:acetonitrile) + 1% acetic acid, with isocratic determination of chicoric acid within 4 min.

NIR Equation Development
Spectral prediction equations for chicoric acid concentration were developed by regressing HPLC data against first and second derivative transformations of log 1/R. The regression procedure was modified partial least squares regression. Subsequent equations were validated with four validation groups, and outliers were eliminated in two outlier passes (Shenk and Westerhaus, 1991). Except for wavelength selection, an optimum equation was chosen according to criteria outlined by Windham et al. (1989). The optimum equation would have high coefficients of determination for calibration (R2) and 1-variance ratio (1-VR), a low standard error of calibration (SEC), and a low standard error of cross validation (SECV).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The detection of chicoric acid was attained isocratically within 4 min by UV detection (Fig. 1). A previous report demonstrated equally effective detection, although elution time was two to three times longer (Bauer and Wagner, 1991). Concentrations of chicoric acid ranged from 1.8 to 19.1 g kg DM-1.



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  		Fig. 1. Liquid chromatogram from coneflower root extract showing chicoric acid (1). Isocratic determination using 7:3 H2O:acetonitrile + 1% acetic acid with detection at UV 284 nm.

 
The HPLC data provided the precision required for development of an NIR spectroscopy prediction equation. The optimum equation empolyed first derivative spectra (Table 1).


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  		Table 1. Calibration and validation statistics for quantification of chicoric acid in purple coneflower using near infrared reflectance spectroscopy and modified partial least squares regres-sion.

 
The R2 of 0.90 calculated in equation development was typical of NIR equations based on constituents in low concentrations (Roberts et al., 1991; 1997). The same could be said of the 1-VR calculated during equation validation (Table 1). Such correlation statistics might appear to be low when compared to the high correlations reported in other studies. However, many of these other studies investigated compounds up to 10 times higher in concentration, thereby expediting detection that could be otherwise masked by spectral interference from background matrix.

We concluded that chicoric acid in purple coneflower root could be detected accurately by NIR reflectance spectroscopy. Although the equation reported in this study accurately predicted chicoric acid in a wide range of plant ages and propagation, it only demonstrated potential. A more robust equation must be developed before NIR spectroscopy can become a practical tool in quality control of purple coneflower. Such an equation should include samples from a wider genetic and geographical base.


   ACKNOWLEDGMENTS
 
The authors express their gratitude to Ms. Heather Benedict for excellent laboratory assistance in this research effort. This work was funded under cooperative agreement C R 826704-01-0 with the U.S. EPA. The results presented are the sole responsibility of the P.I. and/or the University of Missouri and may not represent the policies or positions of the EPA.

Received for publication April 19, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 


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Crop Science 2001 41: 991. [Full Text]  




This Article
Right arrow Abstract Freely available
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Right arrow Citing Articles via ISI Web of Science (1)
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