Herbaceous Peony (Paeonia lactiflora Pall.) as an Alternative Source of Oleanolic and Ursolic Acids

Oleanolic acid (OA) and ursolic acid (UA) have been proven to possess many biological activities, and much attention is focused on the search for plants which are rich in OA and UA. In this report, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony (Paeonia lactiflora Pall.) and were followed in three cultivars over different developmental stages as measured by high performance liquid chromatography (HPLC). OA and UA levels in leaves and stems demonstrated an overall upward trend from May 1 to September 15 except for UA in the leaves of “Hong Feng”. The maximum values of OA and UA in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 852.98, 575.60, 290.48 μg/g FW and 924.94, 827.36, 432.67 μg/g FW, respectively. The maximum values of OA and UA in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 359.28, 90.49, 43.90 μg/g FW and 326.86, 82.25, 56.63 μg/g FW, respectively. OA and UA contents in leaves of 47 different herbaceous peony cultivars ranged from 66.73–618.12 and 36.23–665.14 μg/g FW, respectively, with average values of 171.62 and 227.57 μg/g FW, respectively. The results suggested that the aboveground parts of herbaceous peony may be used as an alternative source of OA and UA for medicinal purposes in addition to its ornamental purposes.

In this study, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony and specifically followed in three cultivars over different developmental stages based on high performance liquid chromatography (HPLC). The results may provide a theoretical basis for herbaceous peony as an alternative source of OA and UA.

OA and UA Contents in Leaves and Stems of Herbaceous Peony at Different Developmental Stages
Herbaceous peony is usually divided into medicinal and ornamental types. Medicinal herbaceous peony is cultivated mainly for harvesting roots, while ornamental herbaceous peony is planted as cut flowers or pot plants. Therefore, investigation of OA and UA contents in herbaceous peony leaves and stems at different developmental stages, and determination of a reasonable harvest time, will be of great significance in ensuring comprehensive utilization of these resources.

OA and UA Contents in Leaves of Herbaceous Peony at Different Developmental Stages
The tendency for OA and UA in the leaves to change at different developmental stages is shown in Figure 1. The OA and UA contents changed little between May 1 and July 1 for all three cultivars, and a small difference were found between the cultivars. Then OA and UA contents sharply increased from July 15. In the following four developmental stages, significant differences between cultivars were observed, and the highest and lowest OA and UA contents were found in -Yangfei Chu Yu‖ and -Hong Feng‖, respectively, with the OA and UA contents of -Fen Zhu Pan‖ being between those of the two other cultivars. The changes of OA and UA contents in the leaves of herbaceous peony were generally similar among the three cultivars. The overall trend was upward from May 1 to September 15 with a small trough on July 15 for -Yangfei Chu Yu‖ and -Fen Zhu Pan‖. For -Hong Feng‖, the small trough of OA and UA in the leaves appeared earlier than the two other cultivars on July 1, with very little change in OA content, and by September 15, OA content maintained a relatively stable level, while the UA level even decreased.
The The ratio of UA to OA in the leaves obviously changed at the different developmental stages. For instance, the UA content in leaves of -Yangfei Chu Yu‖ was significantly higher (3.5-11.3 fold) than OA in the first three sampling times (May 1 to June 1) and then the ratio of UA to OA decreased to 1.1-2.1 fold, except for the samples on July 15 ( Figure 2). The OA and UA contents in stems of the three cultivars significantly changed at different developmental stages as shown in Figure 3. There was an overall upward trend of OA and UA contents from May 1 to September 15. The contents of OA and UA were lower than 20 μg/g FW before June 1 for all three cultivars. Then OA and UA contents in the stems of -Yangfei Chu Yu‖ accumulated more quickly than those of -Fen Zhu Pan‖ and -Hong Feng‖ from June 1, especially after July 15 with a more pronounced increase. For -Fen Zhu Pan‖ and -Hong Feng‖, the OA content difference was very little before July 15, after which the content of OA in -Fen Zhu Pan‖ was higher than that in -Hong Feng‖. However, the UA content of both cultivars had little difference during the whole development stage.

OA and UA Contents in Leaves of Different Herbaceous Peony Cultivars
Great differences existed between the OA and UA contents of leaves of 47 investigated herbaceous peony cultivars. The contents of OA and UA were 54.26-618.12 (average value 171.62) and 36.23-665.14 (average value 227.57) μg/g FW, respectively. The highest contents of OA and UA in leaves were 11.39 and 18.34 fold of the lowest levels, respectively. Among 47 cultivars, -Hong Xiu Qiu‖ and -Yangfei Chu Yu‖ contained the highest OA and UA contents, with 618.12 and 665.14 μg/g FW, respectively, while the lowest OA and UA contents were observed in -Fen Zi Lu Jin‖ and -Jiangshan Ru Hua‖, with 54.26 and 36.23 μg/g FW, respectively (Table 1). The UA content in leaves was generally higher than OA with some exceptions among the 47 cultivars. OA content was mainly concentrated between 100-200 μg/g FW, while the UA content was more than 200 μg/g FW in over half the cultivars. The ratio of UA to OA in leaves of the different herbaceous peony cultivars ranged from 0.52-3.72, with an average value of 1.43.
Similarly, the total OA and UA content in leaves of the different cultivars also largely differed with a range of 102.60-1251.38 μg/g FW (Table 1). From the distribution plot of total amount of OA and UA in leaves ( Figure 5), herbaceous peony can be divided into high-(>1000 μg/g FW), medium-(700-1000 μg/g FW) and low-content (<700 μg/g FW) types. Two cultivars, -Hong Xiu Qiu‖ and -Yangfei Chu Yu‖, belonged to high-content type, with values of 1251.38 and 1194.08 μg/g FW, respectively. The medium-content type included five cultivars, -Fen He Piao Xiang‖, -Xishi Fen‖, -Hong Ling‖, -Fen Zhu Pan‖ and -Hong Feng‖, with an average content of 801.18 μg/g FW. The remaining 40 cultivars were classified as low-level type, with an average concentration of 307.83 μg/g FW.

OA and UA Distribution in Herbaceous Peony at Different Development Stages
Lu et al. [32] found that the UA content of old leaves (1.21%) of Guangxi Kudingcha was higher than that of young leaves (0.57%). Zou [33] reported that OA and UA levels in ripened fruits of Forsythia suspensa (0.5490% and 0.406%) were significantly higher than those in young (0.3805% and 0.3275%) and mature fruits (0.417% and 0.346%). Our study indicated that OA and UA contents of old leaves and stems of herbaceous peony were also higher than those of young leaves and stems. Therefore, a mode of OA and UA content change may exist in plants such that the contents of OA and UA in old organs are higher than those in young ones during development.

OA and UA Distribution in Different Cultivars of Herbaceous Peony
The contents of OA and UA in the same part of different cultivars differed in varied degrees. Wang et al. [34] determined OA and UA contents in fruits of jujube cultivars and wild jujube, and the results showed that the contents in -Fupingdazao‖, -Junzao‖ and -Yazao‖ were higher than those in -Sanbianhong‖, -Popozao‖, -Jianzao‖ and -Jinsixiaozao‖. The research results of Zhou et al. [21] demonstrated that OA and UA contents in flower of five loquat cultivars, -Dahongpa‖, -Baozhu‖, -Dayeyangdun‖, -Jiajiao‖ and -Ruantiaobaisha‖, differed slightly, with the highest UA and OA contents in -Dahongpa‖, and the lowest UA and OA contents in -Ruantiaobaisha‖. In our study, the data demonstrated that great differences existed in UA and OA contents of the leaves between different herbaceous peony cultivars. The contents of OA and UA were 54.26-618.12 (171.62) and Ⅰ Ⅱ Ⅲ 36.23-633.26 (227.57) μg/g FW, respectively. According to the total amount of OA and UA in leaves, herbaceous peony can be divided into high-(>1000 μg/g FW), medium-(700-1000 μg/g FW) and low-content (<700 μg/g FW) types, and about five-sixths of the cultivars belonged to the low-content type.
There were some reports about the relationship between flower color and the OA and UA contents in plants. Huang [35] found significant differences in OA and UA content of azalea flower between cultivars of different color. The pink flower had higher OA and UA contents, white flower had lower OA and UA contents, and OA and UA contents in red flower were between the two cases of pink and white flowers. In our study, OA and UA levels in leaves of herbaceous peony may have some correlation with flower color. In general, triterpenes was relatively low in the cultivars with deep color (purple) flowers, while they were relatively high in the cultivars with pale color (pink, pale pink, white) flowers, which may be related to the balance of secondary metabolism in plants. However, further study is needed to assess whether flower color is closely related to OA and UA contents.

Comparison of OA and UA Contents in Herbaceous Peony with Other Plants
OA and UA are widely distributed in plants with great variations between different families, genera and species. The results of our experiment showed that the contents of OA and UA in herbaceous peony leaves were in the range of 54.26-618.12 μg/g (0.0054%-0.0618% FW) and 36.32-633.26 μg/g (0.0036%-0.0633%) based on fresh weight, respectively. Considering the roughly 90% water content of plant leaves, OA and UA contents in dry leaves would increase ten-fold over that in fresh samples, with about 0.054%-0.618% and 0.036%-0.633%, respectively; these levels are higher than or similar to those of Callicarpa nudiflora leaves [36], Pterocephalus hookerie underground and aboveground parts [37], Hedyotis diffusa whole plant [38], Fructus chaenomeles fruits [39], and Ligustrum lucidum fruits [40].

Chemicals
OA (TCM-031, purity ≥ 98%) and UA (TCM-036, purity ≥ 98%) standards were purchased from Nanjing TCM Institute of Chinese Materia Medica (Nanjing, Jiangsu, China). HPLC grade methanol was obtained from Caledon Laboratories Co. (Georgetown Ont., Canada). The deionized distilled water (ddH 2 O) was obtained with a Milli-Q water purification system from Millipore (Bedford, MA). All other reagents used in the present study were of analytical grade.

Plant Materials
Herbaceous peony (Paeonia lactiflora Pall.) was planted in the Germplasm Nursery of Herbaceous Peony at College of Horticulture and Plant Protection, Yangzhou University, Jiangsu Province, China (32°30′N, 119°25′E). The leaves and stems of -Yangfei Chu Yu‖, -Fen Zhu Pan‖ and -Hong Feng‖ were picked every 15 days from May 1 to September 15, 2009. The leaves of 47 cultivars were collected on September 1, 2009. All the samples were frozen in liquid nitrogen immediately after taking back to laboratory, and then stored in refrigerator at −20 °C until analysis.

Extraction
For preparation of crude extract, a suitable amount of plant materials of each sample was collected and fully grinded with liquid nitrogen. Subsequently, 2 g of sample was weighed and transferred to a 10 mL centrifuge tube, and 5 mL ethanol was added in and mixed homogeneously, and then extracted for 30 min with KQ-500B ultrasonic cleaning machine (Kunshan Ultrasonic Equipment Co. Ltd., Kunshan, Jiangsu, China). The sample was centrifuged at 8000 g for 10 min, and then the filtrate was collected. Precipitates were extracted again and both filtrates were pooled and evaporated to dryness at 35 °C . The residue was dissolved in 1.5 mL methanol and transferred to a 2 mL tube. The crude extract was filtered through a 0.22 μm micro-filter before HPLC analysis. Each sample was repeated in triplicate.

HPLC Analysis
Quantifications of OA and UA were performed on a Waters-2695 HPLC system (Beckman coulter, USA) equipped with Waters 2695 pump and 2487 UV detector according to the method of Zhou et al. [20] with some modifications. The column was a Lichrospher C18 (4.6 × 250 mm, 5 µm) column (Hanbon Sci. and Tech, China) equipped with a guard column with the same stationary phase. All these two compounds were detected at 210 nm at room temperature with a flow rate of 1.0 mL/min and an injection volume of 20 μL. The mobile phase consisted of methanol (A) and 0.1% phosphoric acid solution (B) with a ratio of 85:15 (A:B, v/v) for simultaneous detection of OA and UA. The contents of both compounds were calculated according to the standard curves obtained with a series of standard solutions (0, 25, 50, 75, and 100 μg/mL).

Statistical Analysis
All data are means of three replicates with standard deviations. Microsoft Excel (Microcal Software Inc., Northampton, MA, USA) was used to calculate standard deviations.

Conclusions
Traditional cultivation of both medicinal and ornamental herbaceous peony often causes a serious waste of resources due to discarding the aboveground biomasses. For medicinal purpose, herbaceous peony roots are usually harvested in August or September. During the same period, the aboveground parts of ornamental herbaceous peony will complete assimilate transportation and start to wilt in August to September. The OA and UA contents in the aboveground parts, mainly including the stems and leaves, reached a higher level from August to September. Therefore, the aboveground parts of herbaceous peony may be used as an alternative medicinal source of OA and UA in addition to its ornamental purposes.