Analysis of Yield of Eleutherosides B and E in Acanthopanax divaricatus and A. koreanum Grown with Varying Cultivation Methods

An analysis of the yield of eleutherosides B and E in Acanthopanax divaricatus and A. koreanum was performed using high performance liquid chromatography to evaluate production by different cultivation methods. In A. divaricatus and A. koreanum, the total content of eleutherosides B and E was 2.466–7.360 mg/g varying by plant section, 3.886–11.506 mg/g by pinching site, 3.655–10.083 mg/g by planting time, and 3.652–10.108 mg/g by fertilizer ratio. Thus the total content of eleutherosides B and E in A. divaricatus and A. koreanum differed depending on cultivation methods. These results present useful information for high eleutheroside content applications in A. divaricatus and A. koreanum. This information can affect selection of plant section and cultivation methods for nutraceutical, pharmaceutical, and cosmeceutical material development.

There have been many studies on the activities of eleutherosides B and E. Eleutherosides B and E have been reported to have protective effects against amyloid (25-35)-induced neuritic atrophy in cultured rat cortical neurons [7], neuroprotective effects against transient global cerebral ischemia in rats [8], protective effects in dopaminergic neurons in Parkinson's disease mice [9], and antioxidant properties [10]. There have been many studies determining the effects of eleutherosides B and E. The quantitative analysis of eleutherosides B and E from Acanthopanax species has been reported from various parts of members of Acanthopanax species [11,12], including A. sessiliflorus fruits and fermented wine made from them [13] and in the roots of A. senticosus [14].
Acanthopanax species are cultivated and grow wild in various areas in Korea, and the variety of their pharmacological effects has attracted consumers' interests. However, there are many difficulties in producing high quality Acanthopanax species, depending upon the location and method of culture, which affect different pharmacological ingredients [15].
This study, therefore, analyzed eleutherosides B and E in A. divaricatus and A. koreanum, depending on plant section and cultivation method, using high performance liquid chromatography (HPLC) with the goal of optimizing the content of eleutherosides B and E by cultivation method, to suggest efficient cultivation methods for A. divaricatus and A. koreanum.

Cultivation Methods.
Tilling was carried out on March 20, during which fully fermented compost (1,500 kg/10a) was added to the soil, and A. divaricatus and A. koreanum seedlings were planted on March 30. The compound fertilizer (2N-2P-2K, 21-17-17) fertilized the soil once in the June 10. The harvest time of all samples was February 13 of the following year. The cultivation conditions (2007) were 13.1 ∘ C average temperature and 1,142 mm average precipitation. The soil conditions were mature soil, pH 7.0, 3.4% soil organic content, 435 ppm available phosphate, and 0.36, 3.0, and 1.2 cmol + /kg of K, Ca, and Mg, respectively [16]. All samples were cultivated under the same above conditions.

Sample Preparation.
To analyze the eleutherosides B (1) and E (2) content in A. divaricatus and A. koreanum, 5 g of each of A. divaricatus and A. koreanum was extracted with 50% MeOH (3 × 100 mL) by reflux and evaporated in vacuo. The residue was dissolved in 1 mL of MeOH and filtered with a 0.45 m filter. The resulting solution was used for HPLC analysis.

HPLC Conditions. HPLC separation of eleutherosides B
(1) and E (2) for qualitative and quantitative analysis was performed using a reverse phase system. A Discovery C18 (4.6 × 250 mm, 5 m) column was used with a mobile phase that consisted of water and acetonitrile. A gradient solvent system of water and acetonitrile (90 : 10 to 70 : 30 for 20 min) was used for the elution program. UV detection was conducted at 350 nm. The injection volume was 10 L and the flow rate was 1 mL/min. The temperature was maintained at 25 ∘ C. All injections were performed in triplicate.

Calibration Curve.
A stock solution (1 mg/mL) of each eleutherosides B (1) and E (2) isolate was prepared in MeOH, and then the solution content was successively reduced to 50% to create different concentrations. The analyte contents were determined from the corresponding calibration curves. The calibration functions of the eleutherosides B (1) and E (2) isolate were calculated using the peak area ( ), concentration ( , g/10 L), and mean values ( = 3) ± standard deviation (SD).

Statistical
Analysis. Data for each sample was expressed as mean ± SD. ANOVA using the SAS Enterprise Guide software was calculated and the significance between the means of each group was determined using Duncan's multiple test.

Results and Discussion
Content analysis of eleutherosides B (1) and E (2) in A. divaricatus and A. koreanum by plant section and cultivation method was conducted by HPLC. Eleutherosides B (1) and E (2) (Figure 2) have previously been isolated from A. senticosus, A. divaricatus, and A. koreanum [12,17,18].
HPLC was used to separate eleutherosides B (1) and E (2) for qualitative and quantitative analysis using a reverse phase system. HPLC conditions were selected to analyze eleutherosides B (1) and E (2) with good linearity (eleutheroside B, 2 = 0.9999; eleutheroside E, 2 = 0.9997). The contents of eleutherosides B (1) and E (2) in A. divaricatus and A. koreanum were determined, examining variations in plant section, pinching site, planting time, and fertilizer ratio, as described in Section 2. The HPLC chromatograms of 4 The Scientific World Journal 3.801 ± 0.013 a 3.559 ± 0.020 b 7.360 ± 0.033 * Plant sections are as shown in Figure 1(a).
Data are presented as the mean ± SD ( = 3) in mg/g of the dried samples. Means followed by the different superscript letters (a-e) are significantly different by Duncan's multiple range tests.  Figure 1(b).
Data are presented as the mean ± SD ( = 3) in mg/g of the dried samples.
eleutherosides B (1) and E (2) standards and of A. divaricatus and A. koreanum 50% MeOH extracts are shown in Figure 3. The total eleutherosides B (1) and E (2) content in different plant sections (Ps-1 to -5) ranged from 2.466 to 5.841 mg/g in A. divaricatus and from 4.339 to 7.360 mg/g in A. koreanum ( Table 1). The total eleutherosides B (1) and E (2) content in the upper part (Ps-1) was greater than that in the lower part (Ps-5) of A. divaricatus, butthe result in A. koreanum was the opposite. The total eleutherosides B (1) and E (2) content was the highest in Ps-1 of A. divaricatus and Ps-5 of A. koreanum.
The total eleutherosides B (1) and E (2) content was found to be similar in the upper and lower parts of A. divaricatus and A. koreanum when different cultivation methods were employed. Pinching (30 and 60 cm) yielded total eleutherosides B (1) and E (2) contents of 6.301-6.827 and 7.858-11.506 mg/g in the upper and lower parts of A. divaricatus, respectively, while in A. koreanum, the total eleutherosides B (1) and E (2) contents were 4.439-7.847 and 3.886-4.161 mg/g in the upper and lower parts, respectively (Table 2). When planting time was varied (March 30, April 15, and April 30), the total eleutherosides B (1) and E (2) contents were found to be 4.290-6.526 and 3.769-10.083 mg/g in the upper and lower parts of A. divaricatus, respectively, while the total eleutherosides B (1) and E (2) contents were found to be 4.546-5.693 and 3.655-6.529 mg/g in the upper and lower parts of A. koreanum, respectively (Table 3). When fertilizer ratio was varied (N-P-K, 2N-P-K, N-2P-K, N-P-2K, and 2N-2P-2K), the total eleutherosides B (1) and E (2) contents were found to range from 4.417 to 6.905 and from 3.652 to 7.227 mg/g in the upper and lower parts of A. divaricatus, respectively, and from 4.591 to 10.108 and from 3.834 to 9.079 mg/g in the upper and lower parts of A. koreanum, respectively (Table 4).
A previous paper reported that eleutherosides B (1) and E (2) contents were 0 and 1.804 mg/g in the stem of A. divaricatus, 0 and 1.016 mg/g in the root of A. divaricatus, 0.621 and 0.885 mg/g in the stem of A. koreanum, and 0.478 The Scientific World Journal 5   The Scientific World Journal and 0.538 mg/g in the root of A. koreanum, respectively [11]. Our results showed a similar trend. The best conditions to increase the eleutherosides B (1) and E (2) content in A. divaricatus were determined to be with 30 cm pinching, April 30 planting time, and a N-2P-K fertilizer ratio. In comparison, for A. koreanum, the eleutherosides B (1) and E (2) content was obtained with 30 cm pinching, March 30 planting time, and a 2N-2P-2 K fertilizer ratio. Moreover, the total eleutherosides B (1) and E (2) contents in the upper part of the plant were greater than in the lower part of A. divaricatus by pinching and fertilizer ratio and in the lower part of the plant they were greater than in the upper part of A. divaricatus by planting time. However, the result of the A. koreanum was the opposite. Therefore, the cultivation methods of Acanthopanax species should be established on an individual basis for each species considered. In conclusion, these results can be applied to optimize eleutherosides B (1) and E (2) production for harvesting from A. divaricatus and A. koreanum in nutraceutical, pharmaceutical, and cosmeceutical development.