Introduction

Taxus cuspidata produces the terpenoid paclitaxel (or taxol). This best-selling anticancer drug far surpasses many other antitumor drugs in annual global sales (Kwon et al. 1998). Paclitaxel, known as “plants gold”, inhibits the proliferation of tumor cells and has distinct therapeutic effects in the prevention and treatment of various cancers. However, Taxus is a slow-growing plant with extremely low content of paclitaxel that cannot meet current market demand (Liu and Ye 1999; Li 2009; Zhou et al. 2002). Because Taxus cuspidata is under state protection, it is imperative that we use modern technology to obtain paclitaxel through different pathways on the premise of protecting Taxus. Cell culture production of paclitaxel has many advantages and is becoming more and more popular with researchers (Tang and Zhong 2006; Gao et al. 2011; Seki et al. 1997; Zhong 2002). Most research has focused on basic theory (Zhai et al. 2009; Zhai and Fu 2010; Yu et al. 2013; Li et al. 2009; Zhao and Yang 2014; Kan et al. 2009); studies on the development of technology for efficient production of paclitaxel efficiently has been limited. In particular, work on systems for large-scale industrial production has not been reported. In this paper, Taxus cuspidata was used as a raw material. By optimization of the detection technology, callus induction, and screening for high-yield cell lines, we established a cell suspension system to improve paclitaxel production by adding and adjusting metabolic factors. This study established a good foundation for large-scale, commercial, industrial production of paclitaxel using Taxus cuspidata, providing technical support for reasonable and efficient use of wild medicinal plants of Changbai Mountain, which has significant, important, far-reaching social benefits.

Materials and methods

Materials

Taxus cuspidata (source in Changbai Mountain) callus was obtained through repeated merit-based screening during subculture.

Test methods

Suspension culture

Suspension culture medium: improved B5 liquid medium, containing basic B5 liquid medium (Zhang 2009), 20 g/L sucrose, 2 mg/L glycine, 7.5 g/L citric acid, 2.0 mg/LNAA (naphthylacetic acid), 0.5 mg/L 6-BA (6- benzyl adenine). Culture conditions: temperature: 25 ± 1 °C, speed: 120 r/min, dark. Oscillation culture in a constant temperature oscillation incubator (Shanghai yiheng Scientific Instrument Co., Ltd., Shanghai).

Extraction of paclitaxel

  1. (1)

    Extraction of intracellular paclitaxel Cells plus liquid suspension culture medium were centrifuged using LD 4-2A low speed centrifuge (Beijing Reble centrifuge Co. Ltd., Beijing) at 5000 r/min for 20 min. The resultant supernatant containing Taxus cells was incubated at 40 °C, dried to constant mass and ground to a powder with a mortar; there was 100 mg of the cell powder added to 2 mL of methanol and sonicated for 1 h. The mixture was filtered through the filter, repeating this filtration 3–5 times.

  2. (2)

    Extraction of extracellular paclitaxel (Qiao et al. 2009; Kan et al. 2009; He et al. 2010) A quantity of cell suspension culture medium was centrifuged using LD 4-2A low speed centrifuge (Beijing Reble centrifuge Co. Ltd., Beijing) at 5000 r/min for 20 min, and 10 mL of the resultant supernatant placed in a test tube, adjusted to pH 7.0 using 1 N NaOH or 1 N HCl and 3 mL of ethyl acetate added. The mixture was thoroughly shaken and allowed to separate through standing, before collection of the ethyl acetate layer. The extraction step was repeated twice, then the 9 mL of ethyl acetate from the three extractions was dried under reduced pressure and dissolved in 5 mL ethanol.

Quantification of paclitaxel content

The crude paclitaxel extract was separated with thin layer chromatography. At the Rf expected for paclitaxel, the silicone was scraped off and placed in 5 mL of ethanol, centrifuged for 15 min at 16,000 r/min to extract the paclitaxel. Absorbance of the sample was measured with a TU-1810 ultraviolet visible spectrophotometer (Beijing Purkinje General Instrument Co. Ltd, Beijing) using ethanol as a blank at the maximum absorption peak (228 nm). Paclitaxel content was obtained by reference to a paclitaxel standard curve.

Single factor experiments

On the first 7 days of culture of the cell suspension, different factors and concentrations of elicitors, precursors, or metabolic inhibitors were added to the medium. The corresponding concentration of each additive is shown below.

Methyl jasmonate (μmol/L): 20, 50, 100, 200, 500; salicylic acid (mg/L): 2.5, 5, 10, 20, 50; chitosan (mg/L): 5, 20, 50, 100, 200; phenylalanine (mg/L): 100, 200, 300, 400, 500; sodium acetate (mg/L): 5, 20, 50, 100, 200; GA3 (mg/L): 0.5, 1, 2, 5, 10; cinnamic acid (mg/L): 5, 10, 15, 20, 25.

Suspension cells were harvested after culturing for 28 days. Cell mass and unit production of intracellular paclitaxel were measured.

Multi-factor experiments

In the single factor experiments, several substances significantly increased paclitaxel production by the suspension culture. A quadratic general rotary combined design was used.

Results and discussion

Effect of elicitors, precursors and metabolic inhibitors on cell growth and paclitaxel production

Effect of elicitor on cell growth and paclitaxel production

As seen from Figs. 1, 2 and 3, as the elicitor concentration increased, the fresh cell mass of Taxus cuspidata showed a downward trend, that is, cell growth was inhibited; unit production of paclitaxel increased initially before reaching a maximum and then decreased. This result is due to the elicitor stimulating the plant defense system, thus promoting greater paclitaxel synthesis (Gao et al. 2011; Laskaris et al. 1999; Raskin 1992; Mei et al. 2001). However, the elicitor was somewhat toxic as shown by the gradual decrease in fresh mass with increasing elicitor concentration.

Fig. 1
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The effect of methyl jasmonate on cell growth and paclitaxel production

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Fig. 2
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The effect of salicylic acid on cell growth and paclitaxel production

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Fig. 3
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The effect of chitosan on cell growth and paclitaxel production

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On the basis of the fresh mass of cells and the highest paclitaxel yield per unit, the optimal concentrations of elicitors were: 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 100 mg/L chitosan with the yield of paclitaxel per unit of the respective elicitors, respectively, 4.28 times, 3.82 times, and 1.34 times the yield of the control group.

Effect of precursor on cell growth and paclitaxel production

As shown in Figs. 4 and 5, as the precursor concentration increased, the fresh mass of cells and unit production of paclitaxel increased slowly at first, then plateaued. Therefore, addition of the correct kind and concentration of precursors can increase the fresh mass of cells in suspension culture and improve the unit yield of paclitaxel. The optimal concentration of phenylalanine was 400 and 100 mg/L for sodium acetate; the yield of paclitaxel per unit was, respectively, 2.09 times and 1.51 times the yield of the control group.

Fig. 4
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The effect of phenylalanine on cell growth and paclitaxel production

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Fig. 5
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The effect of sodium acetate on cell growth and paclitaxel production

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The effect of metabolic inhibitor on cell growth and paclitaxel production

As seen in Figs. 6 and 7, the different metabolic inhibitors had different effects on the fresh mass of the cells in suspension culture. Increases in the GA3 concentration promoted cell growth, but increases in cinnamic acid inhibited growth. For both compounds, as their concentration was increased, unit production of paclitaxel increased at first, then reached a maximum, and then decreased slightly. This pattern was due to inhibition of branching pathways unrelated to synthesis of paclitaxel, so the reaction tended to the direction of paclitaxel synthesis. Gibberellin with auxin promotes cell division and can improve cell fresh mass; however, cinnamic acid has somewhat toxic effect on cell growth (Li et al. 2009). Optimal concentration of gibberellin and cinnamic acid was respectively 2 and 15 mg/L, the yield of paclitaxel per unit was, respectively, 1.37 times and 2.08 times compared with that of the control group.

Fig. 6
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The effect of GA3 on cell growth and paclitaxel production

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Fig. 7
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The effect of cinnamic acid on cell growth and paclitaxel production

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Optimization of paclitaxel synthesis by Taxus cuspidata cells

Design and results

On the basis of the results of the single factor tests, the four compounds that yielded a relatively large increase in production of paclitaxel by cells were selected for further tests: methyl jasmonate, salicylic acid, phenylalanine and GA3. Interactions among these four substances were studied by adopting a quadratic general rotary combined design. Analysis of the results is shown in Table 1.

Table 1 Test results analysis of the quadratic general rotary combination
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Statistical analysis software DPS v9.50 (Home Page, China) was used for processing data from Table 1; the regression equation was as follows:

$$ \begin{aligned} Y = 4.32000 - 0.12000X_1 + 0.06667X_2 + 0.10000X_3 - 0.03333X_4 - 0.40667X_1^{2} \hfill \\ - 0.32167X_2^{2} - 0.22167X_3^{2} - 0.57667X_4^{2} + 0.00500X1X_2 + 0.02500X1X_3 \hfill \\ + 0.01000X_1X_4 - 0.01000X_2X_3 + 0.01500X_2X_4 - 0.00500X_3X_4 \hfill \\ \end{aligned} $$

As can be seen from Table 2, the regression equation is highly significant, and lack of fit is not significant, indicating that the equation fit well with the actual situation. After excluding items that were not significant at α = 0.10, the simplified regression equation was as follows:

$$ \begin{aligned} Y = 4.32000 - 0.12000X_1 + 0.06667X_2 + 0.10000X_3 - 0.03333X_4 - 0.40667X_1^{2} \hfill \\ - 0.32167X_2^{2} - 0.22167X_3^{2} - 0.57667X_4^{2} + 0.02500X_1X_3 + 0.01500X_2X_4 \hfill \\ \end{aligned} $$
Table 2 Analysis of variance of the test results
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The effect of single factor analysis

When three of the four factors were fixed at the zero level, a single-factor model was used to describe the effect of changes in a single factor on paclitaxel production. The single factor effect analysis is shown in Fig. 8.

Fig. 8
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Analysis diagram of single factor effect

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As can be seen from Fig. 8, univariate effects of four factors showed a parabolic shape. Because the coefficients of the quadratic terms of four factors are negative, the parabola opens downward, indicating that there were maximums in this test. The total yield of intracellular paclitaxel increased initially and then decreased as the concentration of the four additives increased.

Interaction analysis

As can be seen from the regression equation and Table 2, the interactions in Fig. 9 among X 1 (methyl jasmonate), X 3 (phenylalanine), X 2 (salicylic acid) and X 4 (GA3) are significant. The point values on the interaction effect diagram agreed with the results of the single factor effect analysis, illustrating that the test and the equation were accurate.

Fig. 9
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Interaction effect diagram

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Optimization of extraction and validation process

As can be seen in Table 3, when the values of X 1, X 2, X 3, X 4 were −0.262 to −0.093, −0.076 to −0.313, −0.020 to −0.460, −0.203 to −0.135, respectively, intracellular paclitaxel production may be more than 3.14 mg/L with a 95 % possibility. The actual concentrations of methyl jasmonate, salicylic acid, phenylalanine and GA3 were 94.76–101.86 μmol/L, 19.62–21.565 mg/L, 399–423 mg/L and 1.8985–2.0675 mg/L respectively. The total yield of intracellular paclitaxel was more than 3.14 mg/L in ten repeated tests in the same conditions, confirming that the above analysis was accurate.

Table 3 Frequency distribution optimization of each variable value
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Y max, the maximum of intracellular paclitaxel yield, was 4.32 mg/L when the values of X were between −2 and 2. As mentioned above was the best solution of regression model. The highest value for each combination of factors is shown below. When the concentration of methyl jasmonate is 100 μmol/L, salicylic acid is 20 mg/L, phenylalanine is 400 mg/L and GA3 is 2 mg/L, the total production which depends on suspension cell biomass of Taxus cuspidata and unit production of paclitaxel reaches the maximum of 4.32 mg/L.

Conclusions

By single factor test studying effects of several elicitors, precursors and metabolic inhibitors on paclitaxel production by Taxus caspidata cell culture, methyl jasmonate, salicylic acid, phenylalanine and GA3 were screened out for their relatively large influence.

The test with the factors of methyl jasmonate (X 1), salicylic acid (X 2), phenylalanine (X 3), and GA3 (X 4) was conducted by adopting the quadratic general rotary combined design. The regression equation was simplified as:

$$ \begin{aligned} Y = 4.32000 - 0.12000X_1 + 0.06667X_2 + 0.10000X_3 - 0.03333X_4 - 0.40667X_1^{2} \hfill \\ - 0.32167X_2^{2} - 0.22167X_3^{2} - 0.57667X_4^{2} + 0.02500X_1X_3 + 0.01500X_2X_4 \hfill \\ \end{aligned} $$

The optimal combination of additive concentration was determined for methyl jasmonate 100 μmol/L, salicylic acid 20 mg/L, phenylalanine 400 mg/L and GA3 2 mg/L.

When adding adjustment factor on the 7th day of suspension cells growth, extracellular and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased respectively by 6.08 and 11.57 %.