Method validation
Xu et al.[28] determined diterpenoids in A. paniculata leaves by silver ion complexation in the mobile phase to separate AP3 and AP6 with a 25-min analysis time. The analysis time in the present study was less than 18 min. The chromatograms in Figure 2 showed good separation of these four active diterpenoids; a chromatogram of the standard solution is shown, compared with samples containing the highest contents of AP6 and AP1 at the transfer and mature-seed stages, respectively. The calibration curves showed good linearity in the range of 0.5 to 1000 μg/mL, and the correlation coefficients were 0.9999 for AP1, AP3, AP4, and AP6. Accuracy was determined (expressed as percentage recovery) by adding three known amounts of standard solutions to the plant samples, 10 replicates per concentration, and then extracting the samples and analyzing by the HPLC method described above. The mean recoveries of the four active diterpenoids, AP1, AP3, AP4, and AP6, were 99.66 ± 2.22%, 98.91 ± 2.29%, 100.20 ± 1.85%, and 97.98 ± 1.90%, respectively, indicating good accuracy of the method. To access the precision of the method, we analyzed the extracted sample solutions 10 times on the same day and 10 times on 3 consecutive days. The results showed acceptable intra- and inter-day precision, as represented by the percent relative standard deviation (%RSD) ranging from 0.80% to 1.02% (content) and from 0.41% to 0.66% (retention time) for intra-day precision, and from 3.42% to 4.92% (content) and from 0.39% to 0.74% (retention time) for inter-day precision. The limit of detection (LOD) and limit of quantitation (LOQ) were measured based on the signal to noise ratio of 3:1 and 10:1, respectively. The LOD of AP1, AP3, AP4, and AP6 were 0.10, 0.25, 0.25, and 0.25 μg/mL, respectively, and the LOQ were 0.25, 0.5, 0.5, and 0.5 μg/mL, respectively. These results showed that our analytical method met acceptable criteria for all analytes, and could be used for routine analysis for the four active diterpenoids in A. paniculata.
Greenhouse experiment
There were large variations in contents of the four active diterpenoids in different plant organs at different growth stages. We studied the patterns of accumulation of these four active diterpenoids in two cultivation periods: September 2006 (Figure 3) and January 2007 (Figure 4). The contents of four active diterpenoids in seeds and roots of A. paniculata were lower than limit of detection (LOD). From the seedling to the first true leaf stages, the highest content of AP6 was in the cotyledons (Figure 3A and B). At the transfer stage, the highest content of AP6 was in leaves both in 2006 and 2007 (28.02 ± 0.15 and 36.05 ± 0.69 mg/g, respectively; Figures 3C and 4C). In 2006, the highest content of AP1 was in leaves at the mature-seed stage (24.02 ± 1.18 mg/g) (Figure 3G), whereas in 2007, the highest content of AP1 was at the seed-forming stage (24.72 ± 1.89 mg/g) (Figure 4F). Flowers at the 50% flowering stage also contained high contents of AP1 in both 2006 and 2007 (21.82 ± 1.61 and 21.42 ± 3.74 mg/g, respectively) (Figures 3E and 4E). In contrast, flowers contained low contents of the other three active diterpenoids (≤ 0.9 mg/g). There were low levels of AP3 in all plant organs at all growth stages (≤ 1.4 mg/g). The highest AP3 content was at the mature-seed stage in 2006 (1.4 ± 0.12 mg/g) and at the vegetative stage in 2007 (0.77 ± 0.09 mg/g) (Figures 3G and 4D). The highest content of AP4 was in cotyledons at the transfer stage in 2006 and 2007 (3.28 ± 0.23 and 16.65 ± 4.48 mg/g, respectively) (Figures 3C and 4C). The range of AP4 in leaves was from 0.22 mg/g to 3.72 mg/g and the content increased as plants matured. The highest content of AP4 in leaves was at the mature-seed stage in 2006 and 2007 (3.72 ± 1.31 and 1.88 ± 0.15 mg/g, respectively) (Figures 3G and 4G). The AP6 content in leaves was lower in 2006 (28.02 ± 0.15 mg/g) than in 2007 (36.05 ± 0.69 mg/g), while the contents of the other three active diterpenoids were similar in both cultivation times. The present results also indicated that the young plants at seedling and first true leaf stages (harvesting time ≤ 14 days) contained high contents of these four active diterpenoids.
Field experiment
In greenhouse and field experiments, the changes in the contents of the four active diterpenoids at different growth stages were similar in leaves and the aerial part (Figures 5 and 6). However, leaves contained higher contents of four active diterpenoids than the aerial part. In the greenhouse experiment, the highest AP1 content in leaves (24.02 ± 1.18 mg/g) was about three times higher than that in the aerial part (8.64 ± 0.25 mg/g) at the mature-seed stage in 2006 (Figures 5A and 6A). Similarly, in 2007, the highest AP1 content in leaves was four times higher than that in the aerial part at the seed-forming stage (24.72 ± 1.89 mg/g in leaves, 5.71 ± 0.65 mg/g in aerial part) (Figures 5B and 6B). In the field experiment, the highest AP1 content in leaves was only twice that in the aerial part at the vegetative stage (43.16 ± 0.92 mg/g in leaves, 24.31 ± 1.68 mg/g in aerial part; Figures 5C and 6C). In greenhouse (2006 and 2007) and field experiments, there was a drastic increase of AP1 in leaves from the transfer stage (7.78 ± 0.03, 9.09 ± 0.47, and 18.43 ± 0.54 mg/g, respectively) to the vegetative stage (19.61 ± 0.28, 15.39 ± 0.06, and 43.16 ± 0.92 mg/g, respectively; Figure 5). In the field experiment, the highest content of AP1 was at the early growth stage (vegetative stage) whereas it was at the mature-seed and seed-forming stages in the greenhouse experiment in 2006 and 2007, respectively. The highest content of AP6 in leaves was at the transfer stage in the field experiment (30.59 ± 1.39 mg/g) (Figure 5C) and in the greenhouse experiment (28.02 ± 0.15 and 36.05 ± 0.69 mg/g in 2006 and 2007, respectively) (Figure 5A, 5B). In the greenhouse experiment, the AP6 content in leaves and the aerial part increased markedly from the first true leaf to the transfer stages and then decreased at maturity. The pattern of AP6 accumulation in plants was similar in the field and greenhouse experiments (Figures 5 and 6). Moreover, there were low levels of AP3 and AP4 in leaves and the aerial part at all growth stages in both the greenhouse and field experiments. A. paniculata grown in field conditions contained higher levels of the four active diterpenoids than plants grown in greenhouse conditions. For example, AP1 contents in leaves at all growth stages (transfer to mature-seed stages) were higher in field-grown plants than in greenhouse-grown plants. Because A. paniculata was grown under natural conditions in the field experiment, it is difficult to control cultivation conditions that may affect the levels of these active diterpenoids and quality of this medicinal plant. However, these results showed that the pattern of accumulation of the four active diterpenoids was similar in the field and greenhouse experiments.
To our knowledge, this is the first report to evaluate the contents of these four active diterpenoids in different plant organs during plant development in greenhouse and field experiments. A. paniculata is normally harvested at the 50-60% flowering stage, since it was proposed that plants at this stage contain the highest levels of AP1[22, 23]. Among the active compounds extracted from A. paniculata, AP1 is generally reported as the major active compound [2]. Matsuda et al.[26] analyzed whole A. paniculata plants and reported the following diterpenoids contents on a dry weight basis: AP1 (0.6%), AP3 (0.06%), AP4 (0.005%), and AP6 (0.02%). Sharma et al.[29] reported that the leaves of A. paniculata contained the highest content of AP1 (2.39%), similar to our results (2.4%) in 2006 and 2007 in the greenhouse experiment. However, the highest content of AP1 in our field experiment (4.3%) was higher than that reported previously. A new finding in our study was that the most abundant diterpenoid was not AP1 but AP6 (in 2006 and 2007 at the seedling to vegetative stages in greenhouse experiments, and at transfer stage in the field experiment). The highest content of AP6 was found in leaves at the transfer stage; it was four times higher than AP1 in 2006 (28.02 ± 0.15 and 7.78 ± 0.03 mg/g, respectively) and 2007 (36.05 ± 0.69 and 9.09 ± 0.47 mg/g, respectively). A. paniculata at the transfer stage with short harvesting time (39–45 days) contained the highest level of AP6, which showed bioactivity as an effective vasorelaxant [16].
Since the four active diterpenoids of A. paniculata have different pharmacological properties, knowledge of their different patterns of accumulation among various plant organs and growth stages will be helpful to select plant materials for particular purposes or disease treatments. The patterns of accumulation of AP1 and AP6 were similar to those reported by Bhan et al.[22]. AP1 was continuously produced during plant growth while AP6 content in leaves peaked at the transfer stage and then decreased as the plants matured. In a previous report, harvesting at 100 days after transplantation was recommended to obtain plants with the highest AP1 content [22]. Prathanturarug et al.[23] reported levels of AP1 and AP3 similar to those detected at 50% flowering stage in the present study, although the A. paniculata plants were harvested at the 60% flowering stage in their study. Recently, Parasher et al.[24] analyzed the AP1 content in leaves at different growth stages, and found the highest levels of AP1 in leaves at 120 days of maturity, similar to our results (highest AP1 content was in leaves at mature-seed and seed-forming stages). The content of AP3 was lower than that of other diterpenoids in these experiments. However, in our previous study, we observed that the AP3 increased in plants and plant products stored for a period of time [25]. Furthermore, the stability of the amorphous form of AP1 was temperature-dependent, and it could be converted to AP3[30]. Fresh mature plants with low AP3 content and high AP1 content should be used to treat the common cold, rather than products that have been stored for a period of time, which may exert cardiovascular side effects [31]. The highest content of AP4 was at the transfer stage, similar to AP6, but in cotyledons rather than leaves. The AP4 content in leaves reported in this study was low and slightly increased with maturity. Bhan et al.[22] reported that the simultaneous dehydration and glycosylation of AP1 formed andrographoside and AP4. However, the AP4 content in our study was lower than that reported in their study, which may be because of genetic variations and/or cultivation conditions.