EXTRACTION AND DETERMINATION OF MAJOR HYPOTENSIVE COMPOUNDS IN BARK OF EUCOMMIA ULMOIDES OLIV

A reversed-phase liquid chromatographic method was developed for the quantitative determination of three major hypotensive compounds, namely geniposidic acid, chlorogenic acid, and geniposide in the bark of Eucommia ulmoides. Soxhlet extraction of GPA, GPS, and CA from E. ulmoides was optimized according to the Taguchi experimental design. Maximum global yields were obtained using the following conditions: extraction temperature, 80°C; extraction time, 1 h; number of extractions, three; solvent volume, 16 ml/g of sample; and 50% ethanol concentration in water. Optimal conditions of separation and detection were achieved on a Diamonsil ODS C18 column (150 mm × 4.6 mm, 5 μm) with a linear gradient of methanol and 0.04% aqueous phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detection wavelength of 240 nm. All calibration curves showed good linearity (r2 > 0.999) within test ranges. The relative deviation of this method was less than 3% for intraand inter-day assays, and the recovery percentage of the method was 95-104%, with a relative standard deviation (R.S.D.) of less than 5%. The current assay method was used for quantitative determination of geniposidic acid, chlorogenic acid, and geniposide in five samples of E. ulmoides with different age. The results indicate that the developed method could be readily utilized as a quality control method in working with E. ulmoides.


INTRODUCTION
The tree Eucommia ulmoides, called Du-zhong in Chinese, is the source of a traditional medicine used as a tonic for reducing blood pressure (Huang et al., 2002).The medicine obtained from E. ulmoides is sweet in taste, naturally warm, and active in nourishing the liver and kidney, strengthening bone and muscle, and preventing abortion (Deyama et al., 2001).Previous studies have found that many bioactive components are present in E. ulmoides, including derivatives of iridoids, flavonoids, and phenolic compounds (Deyama, 1983;Deyama et al., 1985Deyama et al., , 1986aDeyama et al., , 1986bDeyama et al., , 1987aDeyama et al., , 1987b;;Cheung et al., 2003).Geniposidic acid (GPA), chlorogenic acid (CA), and geniposide (GPS) (Fig. 1) are three important active compounds in E. ulmoides.The first of these may promote collagen synthesis (Li et al., 1998), while the last two have anti-inflammatory activity (inhibition of 5-lipoxygenase) (Nishizawa et al., 1988), are active against tumor-promoting 12-Otetradecanoyl-phorbol-3-acetate (Ueda et al., 1991;Lee et al., 1995), and have been used for treatment of a liver disorder (inhibition of P450-3A monooxygenase) (Kang et al., 1997).Du-zhong leaves, which contain the same components as Eucommia cortex, have recently been the focus of medical research (Nakamura et al., 1997;Yen andHsieh, 2000, 2002;Lee et al., 2004;Lee et al., 2005).Yen and Hsieh (2000) reported that extracts of Du-zhong leaves exhibit antioxidant activity toward various lipid peroxidation models, with a good correlation between polyphenol content in the extracts and their antioxidant activity.Leaves of E. ulmoides improve hyperglycemia in streptozotocin (STZ)-induced diabetes of rats (Lee et al., 2005).The plant shows inhibitory activity toward adipose conversion, which is a major cause of obesity (Lee et al., 2004).Aqueous extracts from leaves, raw cortex, and roasted cortex of E. ulmoides had effects on the oxidative modification Abstract -A reversed-phase liquid chromatographic method was developed for the quantitative determination of three major hypotensive compounds, namely geniposidic acid, chlorogenic acid, and geniposide in the bark of Eucommia ulmoides.Soxhlet extraction of GPA, GPS, and CA from E. ulmoides was optimized according to the Taguchi experimental design.Maximum global yields were obtained using the following conditions: extraction temperature, 80°C; extraction time, 1 h; number of extractions, three; solvent volume, 16 ml/g of sample; and 50% ethanol concentration in water.Optimal conditions of separation and detection were achieved on a Diamonsil ODS C18 column (150 mm × 4.6 mm, 5 μm) with a linear gradient of methanol and 0.04% aqueous phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detection wavelength of 240 nm.All calibration curves showed good linearity (r2 > 0.999) within test ranges.The relative deviation of this method was less than 3% for intra-and inter-day assays, and the recovery percentage of the method was 95-104%, with a relative standard deviation (R.S.D.) of less than 5%.The current assay method was used for quantitative determination of geniposidic acid, chlorogenic acid, and geniposide in five samples of E. ulmoides with different age.The results indicate that the developed method could be readily utilized as a quality control method in working with E. ulmoides.
of human low-density lipoprotein (LDL) induced by the copper ion (Cu 2+ ) (Yen and Hsieh, 2002).Geniposidic acid, geniposide, and chlorogenic acid were found to be the main hypotensive compounds in E. ulmoides.
This work was therefore designed to develop an HPLC system to separate and quantify the aforementioned three hypotensive compounds in ethanol and aqueous extracts.The main validation parameters of the method are also determined for these complex matrices.

Material and chemicals
The acetic acid and methanol used were chromatographic grade.The solvents were filtered through a 0.45-mm nylon membrane filter (Schleicher and Schnell, Keene, NH, USA) prior to utilization.Ethanol was analytical grade.Standards of geniposidic acid (>98%), chlorogenic acid (>98%), and geniposide (>98%) were generously provided by Dr. Deyama, a Japanese professor.Dried barks of E. ulmoides were obtained from the Traditional Chinese Medicine Co., Ltd.(Hunan, China).The specimen (No. 20051364) was identified by Zhou Zijing at the Biology Department of Guangxi Chinese Medical University.

Preparation of standards
Stock solutions of GPA (1.36 mg/ml), GPS (1.32 mg/ml), and CA (1.42 mg/ml) were prepared with methanol.An aliquot of 1 ml of each stock solution was transferred to a 10-ml flask and diluted to line with methanol to obtain the standard mixture solution.This mixed standard solution was then diluted stepwise to obtain a series of working solutions.All stock and working solutions were stored in a refrigerator and brought to room temperature before use.

Instrumentation
We used the Agilent 1100 HPLC system equipped with a G1314A isocratic pump, a thermostatted column compartment, and a variable-wavelength UV detector (VWD) in conjunction with Agilent Chemstation software.

Chromatographic conditions
The analysis was performed on a Diamonsil ODS C 18 column (150 mm × 4.6 mm, 5 μm, Hanbon Science and Technology Co., Ltd.) connected with a Zorbax SB-C18 guard column (20 mm × 4 mm, 5 μm).The optimal separation by HPLC was achieved with a mobile phase composed of methanol and a 0.04% aqueous phosphoric acid solution (12: 88, v/v) at a flow-rate of 1.0 ml/min, sensitivity of the procedure being 0.05 AUFS.The volume of sample injected was 20 μl.The monitoring wavelength was 240 nm.The column temperature was set at 35°C.

Soxhlet extraction
For Soxhlet extraction, the sample (10 g) was housed in a 200-ml Soxhlet thimble.The apparatus was fitted with a 250-ml round-bottomed flask.The extraction temperature was controlled between 50 and 80°C with a regulator.Because these two components are easily dissolved in water and ethanol, an ethanol:water mixture was used as the extraction solvent in preliminary tests.The flask was heated for 0.5-2.0h and the solvent was refluxed until a given time was up.

Optimization of extraction conditions
In order to obtain optimal extraction efficiency, variables involved in the procedure (such as the solvent and the extraction method) were optimized.
Chloroform, methanol, and chloroform-methanol solutions were tried as the extraction solvent.
Chloroform was chosen at last as the extraction solvent because the three compounds of interest to us can be efficiently extracted and are well resolved from the background.Ultrasonic extraction was compared with refluxing.It was found that the ultrasonic extraction method has lower extraction efficiency.Soxhlet extraction was therefore chosen as the preferred method.An L 16 (4 5 ) orthogonal-design graph was used to reduce the number of experiments (Table 2), with the result that only 16 were needed.The yields of GPA, GPS, and CA obtained under orthogonal conditions are also shown in Table 2.As indicated by the results of our preliminary test, the best results for extraction of GPA, GPS, and CA were: extraction temperature, 80°C; extraction time, 1 h; number of extractions, three; solvent volume, 16 ml/g of sample; and 50% ethanol concentration in water (No. 14).

Optimization of chromatographic conditions
In view of the absorption maxima of the three compounds on UV spectra with three-dimensional chromatograms of HPLC-DAD, the detection was performed at 240 nm.It was also found that the separation was improved when column temperature was increased to 35ºC and the mobile phase was delivered at a flow rate of 1.0 ml/min.Composition of the mobile phase and the ratio of phosphoric acid in water were also optimized.A mixture of methanol and water containing 0.04% phosphoric acid was chosen as the eluting solvent system since it gave not only the desired separation, but also an acceptable tailing factor were acquired.
Chromatograms of the standard mixture and E. ulmoides extract are shown in Figs. 2 and 3, respectively.The chromatographic peaks of E. ulmoides extract were identified by comparing them with the retention time of each reference compound and the UV spectrum recorded using a diode array detector.Suitability of the system was determined using the standard mixture solution and evaluated on the basis of five replicate injections.The system was deemed to be suitable for use because the tailing factor was less than 1.3, the resolution was greater than 1.5, and the theoretical column plate number was more than 10,000 for each compound analyzed.

Method of validation
The method was validated for parameters such as linearity, precision, accuracy, and stability following the International Conference on Harmonization (ICH) guidelines (ICH, 1995).

Calibration curves
The calibration curves for GPA in the range of 4.0-16.0μg/ml had excellent correlation coefficients (r = 0.998 9).By regression analysis of peak area X against concentration Y (μg/ml), the regression equation was obtained.The linear equation for GPA was Y GPA = 23114 + 27273.775X.The calibration curves for GPS in the range of 5.1-20.4µg/ml also had excellent correlation coefficients (r = 0.999 1).By regression analysis of peak area X against concentration Y (μg/ml), the regression equation was calculated.The linear equation for GPS was Y GPS = 33314.5+ 13026.13725X.Finally, the calibration curves for CA in the range of 1.42-16.2µg/ml had excellent correlation coefficients (r = 0.999 3) too.By regression analysis of peak area X against concentration Y (μg/ml), the regression equation was computed.The linear equation for GPS was Y CA = -58837.93013+ 1633614X.The test solution was subjected to HPLC analysis five times.The retention times of these three compounds were 6.712, 10.498, and 21.113 min, respectively.

Precision
Measurement of intra-and inter-day variability was performed to determine repeatability of the developed assay method.The intra-day repeatability was examined on six individual sample solutions prepared from Eucommia ulmoides on the same day, while inter-day repeatability was determined on three independent days.The relative standard deviation (R.S.D.) was taken as a measure of repeatability.The repeatability and reproducibility were < 2.94% and < 1.37%, respectively.These results indicate that the present method can be used for quantitative analyses of GPA, CA, and GPS in Eucommia ulmoides.

Limits of detection (LOD) and quantification (LOQ)
The standard stock solutions were diluted with methanol to provide a series of solutions with the appropriate concentrations.The limit of detection and quantification under chromatographic conditions were determined by measuring the signal-tonoise ratio for each compound.This was done by injecting a series of solutions until a S/N ratio of 3 was obtained for LOD and 10 for LOQ.Table 3 shows detection and quantification limits for the GPA, CA, and GPS analyzed.

Accuracy
Known quantities of the mixed standard solution were added to known amounts of E. ulmoides samples, after which the resultant samples were extracted and analyzed by the established HPLC method.The added standard solution was prepared in the concentration range of a calibration curve with three different concentration levels (high, medium, and low) and triplicate experiments were performed at each level.The recovery percentages were evaluated by calculating the ratio of the detected amount versus the added amount.As shown in Table 4, the developed analytical method was reproducible with good accuracy for all compounds analyzed.The recovery percentages were in the range of 95-104%, with R.S.D of less than 5% for GPA, CA, and GPS.

Analysis of three compounds in E. ulmoides
As shown in Table 5, the developed RP-HPLC assay method was successfully applied for determination of GPA, GPS, and CA in different E. ulmoides samples.On the basis of these experiments, we conclude that the content and composition of E. ulmoides differed significantly in different samples because of differences in age, cultivating conditions, or the manufacturing process.These results indicate that samples from older trees had higher content of these three compounds than younger trees.CONCLUSION This is the first report on the simultaneous determination of three major hypotensive compounds in E. ulmoides.The described method proved to be simple, rapid, accurate, and reliable.Under the employed multiple optimized HPLC conditions, these three hypotensive compounds were totally separated and eluted individually within 30 min.The validation procedure confirmed that this method is suitable not only for analysis of the given three hypotensive compounds, but also for evaluation of the quality of E. ulmoides products.

EXTRACTION AND DETERMINATION OF MAJOR HYPOTENSIVE COMPOUNDS IN BARK OF EUCOMMIA ULMOIDES OLIV.
JIANBO XIAO, XINLIN WEI, and YUANFENG WANG Institute of Food Engineering, College of Life and Environment Science, Shanghai Normal University, 200234 Shanghai, PR China

Table 1 .
Since various parameters potentially affect the extraction process, optimization of the experimental conditions represents a critical step in the development of Soxhlet extraction.Extraction temperature Factors and levels for the orthogonal design.
a T 3 represents the total concentration of three compounds in E. ulmoides (mg/g)

Table 3 .
Detection and quantification limits for the GPA, CA, and GPS analyzed.