Introduction

The genus Taxus attracts significant levels of interest due to its content of diterpene alkaloids, particularly taxol. The anticancer properties of taxol were discovered in T. brevifolia (Wani et al. 1971; Schiff et al. 1979). A similar compound was discovered in T. baccata (Guenard et al. 1993) and in another Taxus species (Fang et al. 1993; Fuji et al. 1993; Nadeem et al. 2002). Taxanes belong to the group of mitosis inhibitors and are used as either monotherapy or in combination with other anticancer agents (Lo et al. 2010). One serious problem is the low concentration (0.001–0.05 %) of these compounds in the trees and the fact that the treatment of each cancer patient requires about eight 60-year-old yew trees (Cope 1998). Therefore, it is very important to select cultivars that have the richest taxane contents (Hook et al. 1999). Paclitaxel is probably produced in a 19-step process of biosynthesis (e.g. Koepp et al. 1995; Hezari et al. 1998; Walker et al. 2002), and many genes encoding key enzymes involved in this metabolic pathway have been identified (e.g. Jennewein et al. 2001, 2004; Chau et al. 2004; Hampel et al. 2009).

The very strong demand for taxanes led many researchers to seek ways to increase its yield. Currently, a common method for the production of drugs from the group of taxanes is their semi-synthesis using their relatively easily accessible precursor 10-deacetylbaccatin III (10-DAB) (Chau et al. 2004). This compound is present in relatively higher concentrations in T. baccata needles (Denis et al. 1988). The high variability in taxane contents was found not only between different species of yew, but also at the intraspecific level, between cultivars (Hook et al. 1999; van Rozendaal et al. 2000), and between male and female specimens (Mukherjee et al. 2002; Migas and Switka 2010). The diversity in taxane contents is influenced by the tissue type and age of needles (Kelsey and Vance 1992; Cameron and Smith 2008), and they also vary during the growing season (van Rozendaal et al. 2000; Cameron and Smith 2008; Ghassempour et al. 2009).

Little is known about the influence of light intensity on taxane concentrations, although in many cases it increases the production of other secondary metabolites in plants (Brzezińska and Kozłowska 2008; Karolewski et al. 2010). Significantly, high paclitaxel and cephalomannine concentrations were only found in the bark of shaded T. brevifolia individuals (Kelsey and Vance 1992), although exposure to UV radiation was found to increase the concentration of paclitaxel on the surface of the needles (Hajnos et al. 2001). Therefore, when planning the establishment of yew plantations for the production of taxanes, it is very important to take into consideration the light demands of this species. Yew trees are shade-tolerant species (Thomas and Polwart 2003; Iszkuło and Boratyński 2006). In the more continental climate of Central Europe, particularly near the eastern limit of its range (Jalas and Suominen 1973), this species is more exposed to damage due to low temperatures (Thomas and Polwart 2003). Intermediate shading causes an increase in minimum temperatures and reduces temperature variations, resulting in increased survival and productivity of yew trees (Iszkuło 2010).

The sex of yew trees may also have a significant impact on the content of taxanes. Females of dioecious plants usually have a greater reproductive effort compared to males (Ashman 1992; Obeso 2002; Iszkuło et al. 2011a; Sanchez-Vilas and Retuerto 2011). This phenomenon was found in the case of yew (Iszkuło et al. 2009; Cedro and Iszkuło 2011; Iszkuło et al. 2011b). A greater reproductive effort by females results in the intensification of gas exchange and consequently higher concentrations of carbon-based secondary metabolites (Danell et al. 1985; Jing and Coley 1990; Danell et al. 1991), including taxanes (Wani et al. 1971).

Despite the relatively numerous data on the diversity of taxane contents depending on species, variety, sex and the growing season, there are still no wider population studies offering opportunities for further treatments, leading to the individual selection of breeding varieties with high concentrations of taxanes. Therefore, the following research hypotheses were formulated: (1) populations of T. baccata have large variations in taxane contents within and between populations; (2) female individuals have higher taxane contents; (3) light intensity influences taxane contents.

Materials and methods

Plant material preparation

Five yew (Taxus baccata L. Taxaceae) populations from the Sudetian region (Lower Silesia, SW Poland) were sampled: Cisy (Bardo Mts., Central Sudetes), Cisowa Góra (Bardo Mts., Central Sudetes), Książ (Sudetian Foothills), Nowy Waliszów (Eastern Sudetes) and Wąwóz Myśliborski (Sudetian Foothills) (Table 1). Needles from three different parts of the crown (1-year-old twigs) were randomly gathered from 60 individuals per population (average values for each specimen were analysed).

Table 1 Characteristics of the Taxus baccata populations analysed
Full size table

The yew samples were examined for their taxane contents: paclitaxel (T) and 10-deacetylbaccatin III (10-DAB). The material was extracted three times in methanol; the crude extract was purified by the Solid Phase Extraction (SPE) method in the system RP-18/methanol + water. The taxane contents were determined by the High Performance Liquid Chromatography with Diode Array Detector (HPLC–DAD) method in the system RP-18/acetonitrile + water.

Extraction

The material was dried for 7 days at 30–35 °C in a dryer with a forced air flow. The raw material was ground in the laboratory mill and then sieved through a 1 mm mesh and placed in a freezer at -20 °C. Before performing the test the samples were brought to room temperature. Then, 4 g portions of dried and ground samples was added to 50 ml of methanol and placed in an ultrasonic bath at about 60 °C for 30 min. The remaining material was extracted twice more under the same conditions. The extracts obtained were combined and concentrated in a vacuum evaporator to a volume of 50 ml. The dry extracts were each dissolved in 50 ml of dichloromethane. The concentration of water in each sample was controlled via the gravimetric method after drying at 110 °C to a stable weight (Hajnos et al. 2001). SPE, using BAKERBOND™ SPE Octadecyl (C18) Disposable Extraction Columns and eluent containing 85 % methanol, was used to purify the crude yew extracts (Hook et al. 1999).

These solutions were chromatographed in an isocratic C18/acetonitrile + water (30 % and 50 % v/v) system using an HP 1100 chromatograph with a DAD detector. A C 18 Symmetry stainless steel column, 4.6 × 250 mm, dp = 5 μm (Waters, Milford, MA, USA), acetonitrile (for chromatography, E. Merck, Darmstadt, Germany) and bi-distilled water were also used. The following gradients of acetonitrile were applied: 0 min, 20 %: 16.65 min, 45 %: 33.35 min, 50 %: 34 min, 100 %.

Quantification of the taxanes was accomplished using external standards. Calibration curves for paclitaxel and 10-DAB (Sigma-Aldrich) were drawn on the basis of multiple measurements of different concentrations of the standard solutions.

Morphological variability

The area and length of the needles were measured prior to chemical analyses for the Nowy Waliszów (NW) and Wąwóz Myśliborski (WM) populations. The fresh needles were measured and analysed using WinSeedle software (Regent Inc.). Then, the needles were dried at 65 °C for 3 days and weighed to calculate the specific leaf area (SLA: leaf area/leaf dry mass, cm2 g−1). Samples were collected at the end of February and at the beginning of March. It was shown that the taxane contents, especially paclitaxel, were the highest at this time (Hook et al. 1999).

Statistical analysis

Analyses of variance were used to study the relationship between populations and sex in the analysed populations. The analyses were conducted using JMP software (SAS Institute).

Results

A highly significant effect of population and sex was found for the concentrations of 10-deacetylbaccatin III (10-DAB) and paclitaxel (T) in the samples analysed (Table 2). The highest levels of DAB and T were observed for the Nowy Waliszów (NW) population, followed by Wąwóz Myśliborski (WM) and Książ (Ks), and the lowest levels were found in the Cisy (Cs) and Cisowa Góra (CG) populations (Table 3). Females contained significantly higher taxane contents than males; the exception was a slightly higher content of T in male individuals in the Książ (Ks) population (Table 3).

Table 2 Analysis of variance of taxane contents according to population and sex
Full size table
Table 3 Average (and standard error) contents of taxanes in the populations studied divided for females and males
Full size table

No significant relationship was found between the coefficients indicating light intensity (SLA) and taxane concentrations of males and females in the Nowy Waliszów (NW) and Wąwóz Myśliborski (WM) populations (Fig. 1). Similarly, no association was found between length, area of needles and taxanes, and the average taxane contents according to the height above sea level (data not shown).

Fig. 1
figure 1

The relationship between specific leaf area (SLA) and taxane contents: DAB 10-deacetylbaccatin III: females (a) and males (b); T paclitaxel: females (c) and males (d) in two of the populations analysed: Nowy Waliszów (NW, open circles) and Wąwóz Myśliborski (WM, closed circles)

Full size image

Discussion

Our study confirmed earlier reports of high variability in taxane contents at the intra- and interpopulation level (Hook et al. 1999; Cameron and Smith 2008; Ghassempour et al. 2009). Previous studies showed significant differences in taxane contents between species and cultivars of yew, suggesting that these differences may be genetic (Kelsey and Vance 1992), which justifies the breeding work aimed at selecting individuals with the highest contents of selected taxanes.

Differences between sexes in terms of morphology and physiology in yew trees are well known (Iszkuło et al. 2009; Cedro and Iszkuło 2011; Iszkuło et al. 2011b). In our study, we also found that these differences applied to the contents of the two taxanes tested. Females were characterized by a greater concentration of these compounds than males. This can be explained by differences in the metabolism of the sexes. According to current knowledge, females of dioecious plants are predicted to grow slower, because of greater reproductive effort. But females have higher rate of gas exchange (providing a greater intensity of physiological processes) compared to males (Dawson and Ehleringer 1993; Laporte and Delph 1996). Consequently, they have higher concentrations of carbon-based secondary metabolites (Danell et al. 1985; Jing and Coley 1990; Danell et al. 1991; Iszkuło and Boratyński 2011), which include taxanes (Wani et al. 1971). Different results were obtained by Nadeem et al. (2002), who found a higher content of paclitaxel in the bark of Taxus walichiana in male individuals (approximately 64 %), but because of the small number of trees that were surveyed, these differences were not significant. After examining a larger number of individuals and populations of the same species, no differences were found between the sexes (Mukherjee et al. 2002). This could have been due to seasonal changes in the taxane contents (Migas and Switka 2010); therefore, the results were highly influenced by the sampling date (or the phenological shift, resulting from harvesting in areas with significant differences in height above sea level).

Our studies did not confirm the theory that populations located at higher altitudes tend to have a higher content of paclitaxel (Mukherjee et al. 2002). However, differences in height above sea level in the populations studied were relatively small and such effects cannot be excluded. An additional argument for the existence of such a trend are the results obtained by Hajnos et al. (2001), who reported an increased production of taxanes under the influence of UV light rays. This could have been be a secondary effect resulting from the adaptive response of yew trees to higher doses of UV radiation, which increases with altitude (5–23 % per 1,000 m) (Pfeifer et al. 2006).

An impact of shading/insolation on the production of secondary metabolites by plants was noted in several studies (e.g. Brzezińska and Kozłowska 2008; Karolewski et al. 2010), but little is known about this in the case of taxanes. Kelsey and Vance (1992) studied the effect of sunlight on the concentration of paclitaxel in the bark and leaves. They found a significantly increased concentration of paclitaxel in the bark of shaded trees. In our study, we did not find a relationship between taxane contents and SLA (specific leaf area), which indirectly indicates a lack of correlation between light intensity and taxane content. SLA is frequently used to describe the morphological adjustments of plants to variations in light availability. Under low light levels, plants frequently produce leaves with the largest possible surface area in order to maximize light capture for photosynthesis (Walters and Reich 1999; Reich et al. 2003). A strong relationship between SLA and light intensity was confirmed in the case of yew (Iszkuło 2010). Yew is a shade-tolerant species (Thomas and Polwart 2003) and it has highest biomass and survival rates under reduced light conditions (Thomas and Polwart 2003; Iszkuło 2010). The European yew is sensitive to low temperatures and under the more continental Central European climate it frequently suffers from frost (Thomas and Polwart 2003), which particularly affects specimens growing in open areas (Iszkuło 2010). Our results indicate that shading (natural or artificial) should not have a negative impact on taxane contents; furthermore, it could be a good protective measure against low temperatures.

Conclusions

Our study confirms the opportunities for individual selection (high concentration of taxanes), cloning and field production of European yew trees for taxane extraction. We also showed significantly higher taxane contents in females, making them better material for the production of these compounds. We found no negative impact of shading on the contents of taxanes, which may be very important for optimizing the production of yew trees.

Author contribution

G. Iszkuło wrote the manuscript, designed the experiment, ran the experiment, and analysed data. P. Kosiński wrote the manuscript, designed the experiment, and ran the experiment. M. Hajnos ran the experiment.