Comparison of growth characteristics of the three Evodia species

Table 1 summarizes the growth characteristics of plants of the three Evodia species, including the tree height (m), leaf length (cm), number of branches, inflorescences, and fruits per inflorescence; fruit diameter (mm), dry weight of 100 fruits (g), and dry weight of fruits per tree (g). Table 2 shows the diameter of fresh fruits collected from plants of the three Evodia species after 1, 2, and 3 weeks of flowering and during the fruit ripening period. Figure 1a–d shows images of the whole plants and leaves of the three Evodia species.

Table 1 Growth characteristics of plants of the three Evodia speciesTable 2 Fresh fruit diameter according to harvest time in the olnats of the three Evodia speciesFig. 1

Whole plant and leaves of the three species of Goshuyu plants. (a) Evodia rutaecarpa, (b) E. officinalis, (c) E. hupehensis, and (d) (left to right) leaves of E. rutaecarpa, E. officinalis, and E. hupehensis

The tree height and leaf length values increased in the order E. officinalis < E. hupehensis < E. rutaecarpa. A significant difference was observed between E. rutaecarpa and E. officinalis (p < 0.01). The number of branches, inflorescences, and fruit sets per inflorescence increased in the order E. rutaecarpa < E. officinalis < E. hupehensis, with a significant difference observed between E. rutaecarpa and E. hupehensis (p < 0.01). Notably, the fruit diameter and dry weight of fruit yield per tree increased in the following order: E. hupehensis < E. rutaecarpa < E. officinalis, with a significant difference between E. hupehensis and E. officinalis (p < 0.01). Furthermore, the values for 100 fruit dry weight increased in the order E. hupehensis < E. officinalis < E. rutaecarpa, with E. hupehensis and E. rutaecarpa significantly differing (p < 0.01). Fresh fruit diameter increased in the order of E. hupehensis < E. officinalis < E. rutaecarpa until 1 week post-flowering and then in the order of E. hupehensis < E. rutaecarpa < E. officinalis from 2 weeks post-flowering to the fully ripe stage. Significant differences were observed between E. rutaecarpa and E. hupehensis 1 week post-flowering, between E. officinalis and E. hupehensis 2 and 3 weeks post-flowering, and between E. rutaecarpa and E. officinalis during the fruit ripening period (p < 0.01).

Comparison of flowering start date and peak flowering period of the three Evodia species

Table 3 shows the flowering start dates and peak flowering periods of E. rutaecarpa, E. officinalis, and E. hupehensis plants. Figure 2a–d shows images of flowers of these species. Table 2 shows that both flowering parameters were in the following order (earlier to later): E. officinalis < E. hupehensis < E. rutaecarpa. Thus, the three Evodia species have different flowering periods, likely due to variations in growth characteristics, allowing them to avoid crossbreeding.

Table 3 Flowering start date and peak flowering period of the three Evodia speciesFig. 2

Flowers of the plants of the three Evodia species. (a) E. rutaecarpa, (b) E. officinalis, (c) E. hupehensis in the male stage, and (d) E. hupehensis in the female stage

Comparison of dry fruit weight per inflorescence among the three Evodia species

Table 4 shows the dry fruit weight per inflorescence and harvest date of the plants of the three Evodia species after 1, 2, and 3 weeks of the flowering and ripening periods. Figure 3a–c shows representative images of dry fruits of the three Evodia species, sorted by collection date. During all periods, the dry weight of fruit per inflorescence was in the order E. hupehensis < E. officinalis < E. rutaecarpa. After the ripe stage, the dry weight of E. hupehensis inflorescences was not measurable. The dried fruit weight of the three species did not significantly differ 1 week post-flowering. However, dried fruit weight significantly differed between E. rutaecarpa and E. officinalis 2 and 3 weeks post-flowering (p < 0.05). No significant differences in dried fruit weight were observed between E. rutaecarpa and E. officinalis at the fully ripe stage.

Table 4 Dry fruit weight per inflorescence and harvest date of the three Evodia speciesFig. 3

Dried fruits of the three Evodia species sorted by time of collection. (a) E. rutaecarpa, (b) E. officinalis, and (c) E. hupehensis

Considering the changes in dry fruit weight by collection time, the dry fruit weight of E. rutaecarpa and E. officinalis tended to increase as the number of days after flowering increased. However, the fruit remained immature until 3 weeks post-flowering in E. rutaecarpa and up to 2 weeks post-flowering in E. officinalis. The dry weight of E. hupehensis fruits could not be measured at the ripe stage because they did not fully ripen or fall.

Comparison of evodiamine and rutaecarpine content in immature fruits of the three Evodia species

Figure 4a–c shows the evodiamine and rutaecarpine content of the immature fruits of the three Evodia species at different collection times (1, 2, and 3 weeks post-flowering and the ripe stage). One week post-flowering, fruit evodiamine content was 0–0.01% in E. rutaecarpa, 0.07–0.17% in E. officinalis, and 0% in E. hupehensis. Two weeks post-flowering, fruit evodiamine contents were 0.03–0.04%, 0.14–0.27%, and 0–0.03% in E. rutaecarpa, E. officinalis, and E. hupehensis, respectively. Three weeks post-flowering, the contents were 0.16–0.18%, 0.21–0.36%, and 0.02–0.03% in E. rutaecarpa, E. officinalis, and E. hupehensis, respectively. At the fully ripe stage, fruit evodiamine contents were 1.82–2.28% for E. rutaecarpa, 0.59–0.86% for E. officinalis, and not measurable in E. hupehensis. Compared with the evodiamine content of commercially available E. rutaecarpa (0.19–0.37%) [16], our observed content was slightly lower 1 week post-flowering, roughly the same 2 and 2 weeks post-flowering, and approximately sevenfold higher at full ripeness. The evodiamine content of E. officinalis in the present study was slightly lower 1 week post-flowering, roughly the same 2 and 3 weeks post-flowering, and approximately sixfold higher at full ripeness than that of commercially available E. officinalis (0.21–0.28%) [16].

Fig. 4

Contents of evodiamine and rutaecarpine according to harvest time in the three Evodia species. (a) E. rutaecarpa, (b) E. officinalis, and (c) E. hupehensis (n = 3 each), Values are the measurement error for the same sample, Vertical lines indicate SD

Fruit rutaecarpine content 1 week post-flowering was 0.03–0.04% in E. rutaecarpa, 0.07–0.11% in E. officinalis, and 0.02–0.04% in E. hupehensis. Two weeks post-flowering, the rutaecarpine contents were 0.12%, 0.13–0.16%, and 0.07–0.09% in E. rutaecarpa, E. officinalis, and E. hupehensis, respectively. Three weeks after flowering, the concentrations were 0.24–0.28%, 0.13–0.23%, and 0.07–0.11% in E. rutaecarpa, E. officinalis, and E. hupehensis, respectively. After the ripe stage, the rutaecarpine content was 1.38–1.59% for E. rutaecarpa, 0.38–0.56% for E. officinalis, and not measurable in E. hupehensis. Compared with the rutaecarpine content of commercially available E. rutaecarpa (0.09–0.20%) [16], our investigation revealed levels that were slightly lower 1 week after flowering, roughly the same 2 and 3 weeks after flowering, and approximately fourfold higher at full ripeness. The rutaecarpine content in the present study was slightly lower 1 week post-flowering, roughly the same 2 and 3 weeks post-flowering, and approximately twofold higher at full ripeness than that of commercially available E. officinalis (0.07–0.29%) [16].

Thus, the content of evodiamine in fruits tended to gradually increase in the order E. hupehensis < E. rutaecarpa < E. officinalis from 1–3 weeks after flowering, while that of rutaecarpine content increased in the order E. officinalis < E. rutaecarpa from 3 weeks post-flowering to the ripening stage.

These results demonstrate that the contents of evodiamine and rutaecarpine in the fruits varied by species, with the differences becoming particularly evident at the ripe stage. Furthermore, considering the evodiamine and rutaecarpine content ratio in these fruits, evodiamine tended to be higher in E. officinalis and E. rutaecarpa, whereas rutaecarpine was higher in E. hupehensis. Additionally, we observed that the content of evodiamine and rutaecarpine increased with the number of days elapsed since flowering, indicating that the longer the time from flowering, the higher the content of both compounds. Particularly, the content was extremely higher at the fully ripe stage than that at other periods after flowering. For instance, E. rutaecarpa at the fully ripe stage had approximately 13.1-fold more evodiamine and approximately 5.9-fold more rutaecarpine than that at 3 weeks post-flowering. When fully ripe, the amount of evodiamine and rutaecarpine was approximately 2.5- and 2.7-fold higher than that at 3 weeks post-flowering.

Moreover, when examining the component composition by the harvest time of the fruit, the evodiamine content of E. officinalis tended to be almost equal to or slightly higher than that of rutaecarpine from 1 week after flowering until the fully ripe stage. Nevertheless, the component composition did not significantly change throughout the entire harvest period. Contrastingly, E. rutaecarpa showed a tendency for rutaecarpine to be slightly higher than evodiamine until the fruit was ripe, but thereafter, the evodiamine content was clearly higher than the rutaecarpine content, confirming a change in the component composition.