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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.45 no.2 Santiago  2012

http://dx.doi.org/10.4067/S0716-97602012000200009 

Biol Res 45: 171-175, 2012

ARTÍCULOS DE INVESTIGACIÓN

Variation in concentrations of major bioactive compounds in Prunella vulgaris L. related to plant parts and phenological stages

Yuhang Chen1,2, Zaibiao Zhu1, Qiaosheng Guo1*, Lixia Zhang1 And Xiaoming Zhang1

1Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing 210095, PR China
2College of Pharmaceutical Sciences, Chengdu Medical College, Chengdu 610083, PR China.


ABSTRACT

Prunella vulgaris L. (Labiatae) contains a variety of structurally diverse natural products, primarily rosmarinic acid (RA), ursolic acid (UA) and oleanolic acid (OA), which possess a wide array of biological properties. In the present study, P. vulgaris was harvested at three developmental stages (vegetative, full-flowering and mature-fruiting stages), dissected into stem and leaf tissues and assayed for chemical contents using high performance liquid chromatography. Significant changes in the concentrations of the major secondary metabolites (RA, UA and OA) were observed at the different development stages. The highest concentrations of RA, UA and OA were found at the full-flowering stage (15.83 mg/g dry weight (DW) RA, 1.77 mg/g DW UA and 0.65 mg/g DW OA). Among the different aerial parts of the plant, the concentrations of RA, UA and OA were higher in the leaves than in the stems at the different developmental stages. These results suggest that the full-flowering stage is characterized by the highest concentrations of bioactive compounds. Therefore, this stage may be the optimum point for harvesting P. vulgaris plants. In additional, the leaves of P. vulgaris demonstrated higher RA, UA and OA concentrations than the stems, suggesting higher utilization potential.

Key terms: Prunella vulgaris L.; ontogenetic and morphogenetic variation; rosmarinic acid; ursolic acid; oleanolic acid.


 

1. INTRODUCTION

Prunella vulgaris L. (Labiatae), also known as the "self-heal", is a perennial herb widely distributed throughout eastern Asia and Europe (Flora of China, 1977). In China, P. vulgaris is primarily located in the southern provinces, particularly in the Anhui, Jiangsu, Henan and Zhejiang provinces (Chen et al., 2010). The dried spica of P. vulgaris, Prunellae Spica, is a standard medicinal material in the Chinese pharmacopoeia (''Pharmacopoeia of the People's Republic of China'', 2010) and is occasionally used as an herbal medicine for alleviating sore throat, reducing fever and accelerating wound healing (Pinkas et al., 1994; Marková et al., 1997; Cheung and Zhang, 2008). Recent studies have demonstrated that methanol or water extracts of this herb exhibit many bioactive properties, including antihyperglycemic activity (Zheng et al., 2007), UV-A radiation photoprotection (Psotova et al., 2006), systemic anaphylaxis inhibition (Kim et al., 2007), immune modulation (Fang et al., 2005) and antioxidative, antiviral and antibacterial effects (Psotova et al., 2003). In addition to the pharmaceutical uses of P. vulgaris, the air-dried plants are used as an additive in some functional beverages, and the fresh leaves are also edible and often show up in vegetable recipes in southeast China (Chen et al., 2011; Chen et al., 2012).

P. vulgaris is rich in phenolic acids (Psotova et al., 2003). Rosmarinic acid (RA: Fig.1), the major phenolic component of P. vulgaris, which exhibits a wide spectrum of biological activities (Holzmannova, 1996), including lipoperoxidation suppression (Laranjinha et al., 1994), scavenging superoxide radicals (Osakabe et al., 2002) and antioxidant (Psotova et al., 2003) and anti-inflammatory (Osakabe et al., 2004) effects. The Chinese Pharmacopoeia 2010 considers RA to be the only criterion for the quality control of Prunellae Spica. In addition, triterpenes are the dominant compounds in P. vulgaris (Cheung and Zhang, 2008). Of the triterpenes, ursolic acid (UA: Fig.1) and oleanolic acid (OA: Fig.1) are most prevalent in P. vulgaris and exhibit many bioactivities, including hepatoprotection, anti-hyperglycemia and antifungal, antitumor and anti-inflammatory effects (Liu, 1995). There is increasing evidence that UA is the main active substance in anti-diabetic medicines due to its ability to lower blood sugar, urine sugar, water intake and urine volume. In addition, OA has been reported to be the main compound that protects liver sickness (Liu et al., 2003).

Previous phytochemical studies on the bioactive components of P. vulgaris have reported the isolation of triterpenoids, phenolics, flavonoids, tannins, caffeic acids, the anionic polysaccharide prunelline and essential oils (Lamaison et al., 1991; Xu et al., 1999; Wang et al., 2000; Ryu et al., 2000; He et al., 2007; Zhang et al., 2011). Because secondary metabolites are particularly prone to qualitative and quantitative variations depending on factors such as genetic drift, physiological conditions, season and harvesting time, as well as analytical methods and sample preparation techniques, numerous investigations concerning the influence of the aforementioned factors have been conducted (Guo et al., 2010; Wu et al., 2010; Liao et al., 2010; Chen et al., 2011; Wang et al., 2011). The phenological stage of the plant is another source of variance that considerably influences its concentration of secondary metabolites (Liu et al., 2010). However, the knowledge of the potential contribution of the phenological stage is still lacking, and the few papers that have been published regarding this topic have focused on particular plant components and/or species (Wang et al., 1994; Luo et al., 2005; Jiang et al., 2009).

We reasoned that the developmental stage of P. vulgaris at harvest might influence the concentration of secondary metabolites in the aerial parts of the plant and affect the quality of the harvested plant material. Therefore, the aim of the present study was to analyze the RA, OA and UA content of different aerial parts of P. vulgaris at various stages of development.

2. METHODS

2.1. Plant material

Seeds of P. vulgaris were collected from Queshan County, Henan Province, P. R. China. Twenty seeds of approximately the same size were sown in separate pots on October 7, 2008 and germinated in the greenhouse. One month later, the seedlings were transplanted to an experimental field located at a flat land site (117°23¸, 31°24'N, 48.64 altitude) in Lujiang County, Anhui Province, P. R. China. The spacing was 30 cm between the rows and 25 cm between the plants within a row. The field was cultivated using conventional commercial methods from October 2008 to June 2009.

The plant material of P. vulgaris was collected from April to June 2009. The material represented 20 randomly gathered plants from three phenological stages: vegetative, full-flowering and mature-fruiting. Newly emerged shoots (5-6 months old) with leaves were harvested at the vegetative stage (April 10). At the full-flowering stage, only shoots with fully opened flowers were harvested (May 20). At the mature-fruiting stage, only shoots that had red-brown spicas were harvested (June 15). The top of 2/3 of the plant was harvested between 11:00 AM and 1:00 PM. After collection, 10 individuals were kept as whole plants. The rest of the plants were dissected into leaf and stem tissues, dried at room temperature (25 ± 2°C), ground and passed through a 0.3 mm sieve and assayed for RA, UA and OA content using HPLC.

2.2. Chemicals and reagents

HPLC-grade methanol was purchased from Fisher Scientific (Fisher Scientific Co., Fair Lawn, NJ, USA). Deionized water was purified using the Milli-Q system (Millipore, Bedford, MA, USA). Analytical-grade phosphoric acid and ethanol were purchased from Nanjing Liudu Fine Chemicals Co. Ltd. (Nanjing, China). Three authentic standards of RA, UA and OA were purchased from the National Institute for Control of Biological and Pharmaceutical Products (Beijing, China).

2.3. Apparatus and analytical conditions

HPLC was performed with a chromatographic pump (LC-20AT), a manual sample injector, and a diode array detector (SPD-M20A, Shimadzu, Kyoto, Japan). The HPLC fingerprint was performed on a C18 column (Shimadzu, ODS-C18, 4.6 mmx250 mm, 5 µm) at 25°C, with a sample injection volume of 10 µL. The data were collected with a model N2000 chromatography workstation (Zhejiang University, Hangzhou, China).

The chromatographic conditions used to detect the UA and OA components were as follows: the detection wavelength was 210 nm for the analysis; the flow rate of the mobile phase, consisting of methanol and 0.5% ammonium acetate (90:10, v/v), was 0.6 mL-min-1; and the column temperature was maintained at 25°C. The chromatographic conditions for the RA component were as follows: the detection wavelength was 330 nm; the flow rate of the mobile phase, consisting of methanol and 0.01% aqueous phosphoric acid (52:48, v/v), was 1.0 mL-min 1; and the column temperature was maintained at 25°C.

2.4. Calibration curves of active constituents

Stock solutions of RA, UA and OA were prepared in deionized water and diluted to six different concentrations using 75% methanol to construct the calibration plots. The linearity of each standard curve was confirmed by plotting the peak area (y) against the corresponding concentration (x, µg·mL-1) of the analytes. The regression equations are listed in Table 1.


2.5. Preparation of sample solutions

The samples for HPLC were prepared using a modification of the methods of Fang et al. (2010). Powdered spica samples (0.10 g) were mixed with 20 mL of 75% methyl alcohol for 30 min, extracted through a 30 min ultrasonic treatment at 20°C and centrifuged at 10,000 rpm for 10 min. The supernatant of the centrifugate was filtered through a 0.45 ìÃç organic membrane before it was injected into the HPLC system.

2.6. Statistical analysis

All the data were analyzed using SPSS 11.5 for Windows. The data were initially compared by an analysis of variance (ANOVA), and differences between means were detected using the Duncan test. p values < 0.05 were considered significant.

3. RESULTS AND DISCUSSION

3.1. Phenological changes in the RA, UA and OA concentrations of whole plants

Phenological changes in the RA, UA and OA concentrations of whole plants were found to be significant (p < 0.05) in P. vulgaris. The concentrations of RA, UA and OA in samples of the whole plant increased during the course of ontogenesis. The highest level of those compounds was observed at the full-flowering stage (15.83 mg/g DW RA, 1.77 mg/g DW UA and 0.65 mg/g DW OA). After the development of flowers, the concentrations of bioactive compounds decreased with the advancement of reproductive development (Table 2).

These results are in agreement with most of the previous work on the topic, which has reported that the full-flowering stage is characterized by the highest content of secondary metabolites. For example, Ayan et al. (2007) reported that the total phenol content reached its highest level at full-flowering in Hypericum pruinatum. Additionally, Verma and Kasera (2007) indicated that the peak concentration of phenols was observed at the flowering stage in Boerhavia diffusa. Similar results were also found by Sellami et al. (2009) in Origanum majorana, in which the highest levels of volatile terpenes occurred at the budding and full-flowering stages. These results could be explained by the low rate of biosynthesis of bioactive compounds during the vegetative stage, which may be due to the partial inactivation of enzymes necessary for the biosynthesis of certain compounds. Indeed, in previous studies conducted on members of the Lamiaceae family (primarily Herba rabdosia Serrae and Rabdosia rubescens), Chen et al. (2003) and Wu et al. (2004) reported that the UA and RA concentrations increased during plant development and reached their maximum levels during the flowering stage. Our findings in the present study confirmed this observation. The RA, UA and OA concentrations of whole plants increased during several ontogenetic development phases, and the highest level of those compounds was reached at the full-flowering stage in P. vulgaris.

The observed variations of phenolic acid and triterpene concentration during the growth of P. vulgaris affirmed the influence of phenological stages on the production and release of these metabolites. The peak amounts of the accumulation of RA, UA and OA observed during the full-flowering stage may be because, during this stage, the protection of the plant is achieved primarily by these phenolics and triterpenes. Indeed, it is well known that an important function of triterpenes and phenolic acids is their activity in plant defense mechanisms (Dixon and Paiva, 1995; Grassmann et al., 2002). However, the accumulation of triterpenes and phenolic acids during the full-flowering stage might also be related to ecological functions, such as the intensification of antifungal defenses and the attraction of pollinators (Langenheim, 1994). Regarding these variations in the accumulation of secondary metabolites in P. vulgaris, it is possible that the developmental stage of the plant affects the choice of the optimal harvesting time. For the maximization of RA, UA and OA concentrations, P. vulgaris plants should be harvested at the full-flowering stage.

3.2. Phenological changes in the RA, UA and OA concentrations of different aerial parts

The differences in the RA, UA and OA concentrations of stem and leaf tissues during P. vulgaris development were also found to be significant (p < 0.05). In general, the highest levels of these compounds were achieved at the full-flowering stage in all the plant parts. The highest concentration of RA (Fig. 2C) was found at full-flowering stage, for example, but the concentration was higher in the leaves (28.16 mg/g DW) than in the stems (21.70 mg/g DW). Similar results were obtained for these plant organs during both the vegetative and mature-fruiting stages. The RA content in the leaves of both the vegetative and mature-fruiting stages was found to be 38.79% and 83.92% higher than that found in the stems, respectively.


UA and OA (Fig. 2A-B) reached their highest concentrations during the full-flowering stage in leaves (3.32 and 1.90 mg/g DW, respectively). Specifically, the UA levels of the leaves were 112.50% higher in the vegetative stage, 46.26% higher in the full-flowering stage, and 66.25% higher in the mature-fruiting stage, respectively, in comparison with the stem concentrations. Similarly, in comparison with the stem concentrations, the OA concentrations in the leaves were 70.63% higher in the vegetative stage, 80.05% higher in the full-flowering stage, and 54.89% higher in the mature-fruiting stage, respectively.

In the full-flowering stage, the proportions of RA, UA and OA in the stems and leaves of P. vulgaris were at their maximum measured levels, and the amounts of these bioactive components were significant higher in the leaves of the plant than in its stems. The aerial portions of P. vulgaris grew vigorously during this experiment. Large amounts of anabolites were produced, accumulated and transported to the reproductive parts of the plant, which also grew quickly. It was speculated that the RA, UA and OA in P. vulgaris were synthesized initially in the leaves and then transported to the inflorescences for storage via the phloem of the veins and the stem vascular bundles. Liu et al. (2010) reported that the highest UA and OA content in the stems and leaves of P. vulgaris plants occurred at the full-flowering and post-flowering stages. Similarly, Zhang et al. (2007) demonstrated that the RA content of P. vulgaris leaves was higher than that of the stems at the flowering and mature-fruiting stages. Similar results were also found in other species of the Lamiaceae family, including in Rabdosia eriocalyx (Dunn) Hara (Gao and Yuan, 2009), Lavandula angustifolia Mill (Zou, 2010) and Mesona chinensis Benth (Leng, 2011).

In the mature-fruiting stage of P. vulgaris, the concentration of RA, UA and OA in the stems and leaves decreased dramatically. This phenomenon may be due to the rapid consumption of nutrients by the developing reproductive organs, which would cause a rapid decrease of these bioactive components in the stems and leaves. We determined that the RA, UA and OA levels in mature P. vulgaris spicas were 37.64, 2.58, and 0.78 mg/g, respectively (Chen et al., 2012).

These observations indicated that the decrease of RA, UA and OA content in the stems and leaves is closely related to the reproductive growth of P. vulgaris. These results are in agreement with the existing literature (Liu et al., 2010).

These results indicate high levels of variability, which are associated with the stage of plant development, in the content of the investigated bioactive compounds. According to Luckner (1980), as plant maturation progresses, there appears to be an ordered expression of the genome such that particular enzymes or group of enzymes are synthesized or activated at particular stages of plant development. According to our results, it appears that the aerial parts of P. vulgaris should be harvested during the full-flowering stage, during which they are the richest in RA, UA and OA.

ACKNOWLEDGEMENTS

This research was supported by the programs of National Nature Science Foundation of China (No. 30772730 and 81072986).

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* Corresponding author: Qiaosheng Guo, Ph.D. Nanjing Agricultural University. Institute of Chinese Medicinal Materials. Nanjing 210095. P. R. China. Tel: +86-25-84395980. Fax: +86-2584395980. E-mail:gqs@njau.edu.com.

Received: March 5, 2012 Accepted: May 9, 2012.

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