Abstract: Callus cultures were established on callus derived from stems of young Kyaraboku (Taxus cuspidata Sieb. et Zucc. var. nana) trees and the effect of oligosaccharides on taxol production was investigated. The production was stimulated about 5.1-fold and 1.2-fold by the addition of chito-oligosaccharides (COS) and laminaran-oligosaccharides (LOS), respectively. To find the active compound that stimulates the taxol production, COS were separated into 8 fractions (Fr.1 - Fr.8) by ion-exchange chromatography. Fr.5 was found to be the most active fraction and chitoheptaose was its most active compound. The production was stimulated 8.2-fold on addition of chitoheptaose to the calli.
Introduction
Kyaraboku, Taxus cuspidata var. nana, with dark, linear and evergreen foliage is a popular garden tree in Japan (Uehara, 1969). Kyaraboku is a dwarf variety of ichii, Taxus cuspidata. We have examined the extractive of T. cuspidata var. nana leaves and found that taxol is present, although in small amounts (Tachibana et al., 1994a). Taxol, a diterpene alkaloid initially isolated from the bark of Taxus brevifolia by Wani et al. (1971), has strong cytotoxic activity against breast cancer, ovarian cancer, stomach cancer and other types of cancer (Gelmon, 1994). Therefore, for taxol to be utilized as a resource of T. cuspidata var. nana, its production needs to be increased.
At present, the commercial source of taxol is the bark of Taxus brevifolia, which grows slowly and yields relatively low amounts of taxol. Taxol is also found in Taxus spp. plants. Many attempts have been made to produce taxol by chemical synthesis (Nicolaou et al., 1994; Holton et al., 1994a,b), semi-chemical synthesis (Holton et al., 1995), plant tissue culture (Fett-Neto et al., 1992,1993,1994a, 1994b; Wickremesinhe et al., 1993; Tachibana et al., 1994b; Ketchum et al., 1995; Wang et al., 1997; Son et al., 2000; Zhang and Xu, 2001) and fermentation (Stierle et al., 1993; Strobel et al., 1996). However, chemical synthesis requires multiple (over 20) steps and the total yield is very low. The most-promising methods for the large-scale production of taxol seem to be semi-chemical synthesis and plant tissue culture.
Production of taxol has been conducted by the callus culture of Taxus cuspidata (Fett-Neto et al., 1992,1993,1994a, 1994b), T. cuspidata var. nana (Tachibana et al., 1994b), T. cuspidata and T. media (Furmanowa et al., 2000) and four Taxus spp. (Wickremesinhe et al., 1993). However, there have been no reports about the stimulatory effects on taxol production of treatment with oligosaccharides in callus cultures of Taxus spp. Therefore, we examined taxol production in Taxus cuspidata var. nana callus cultures and the effect of oligosaccharides on the production.
Materials and Methods
General: The research project was carried out in the Faculty of Agriculture, Ehime University, Ehime, Japan during 1999 – 2001 to produce taxol from tissue cultures of Taxus cuspidate var. nana. Melting point of taxol was determined on a Yanagimoto MP micro melting point apparatus and is uncorrected. FDMS data were obtained with a Jeol JMS-700 mass spectrometer. Authentic taxol was purchased from Sigma Chemical Company as well as isolated from the leaves of T. cuspidata var. nana (Tachibana et al., 1994a). Chitosan and algal laminaran were purchased from Seikagaku Kogyo Ltd. and Tokyo Kasei Kogyo Co. Ltd., respectively. Tunicase was obtained from Wako Pure Chemical Industries, Ltd. Authentic chito-oligosaccharides (glucosamine to chitoheptaose as each respective hydrochloride) and polyvinylpyrrolidone were also obtained from Wako Pure Chemical Industries, Ltd.
Plant materials, tissue culture media: Fresh stems of young garden-grown trees T. cuspidata var. nana were used as explant material. The stems were collected in May 1999 from the Ehime Prefectural Garden Center located in Shigenobu Town, Ehime, Japan and from the garden of a house located in the suburbs of Matsuyma City, Ehime, Japan, respectively.
MurashigeSSkoog (MS) (Murashige and Skoog, 1962), Gamborg's B5 (Gamborg et al., 1968) and SchenkSHildebrandt (SH) (Schenk and Hildebrandt, 1972) media were prepared. In the case of the MS and SH media, the sucrose content was 30 g/l. In the case of B5, the sucrose content was 20 g/l. The pH of all three media was adjusted to 5.6. The growth regulators added to each medium were 2,4 D (4.0 mg/L) and kinetin (0.5 mg/L). Casamino acid (1.0 g/L) and polyvinylpyrrolidone (15g/L) were also added. The media were solidified with 0.9% w/v agar.
Callus initiation and cultures: Explants were sterilized by soaking in 70% ethanol for a few minutes and by immersing in sodium hypochlorite (1% active chlorine) for 20-30 minutes with gentle stirring followed by thrice washes with sterile water. Damaged tissue was removed and segments of the stems were transferred into each of the sterilized media.
Explants were placed on surface agar solidified medium (30ml) supplemented with 2,4 D, kinetin and casamino acid in 100-ml Erlenmeyer flasks, or on 20 ml of solidified medium in 9-cm petri dishes. Petri dishes were sealed with Nescofilm. Cultures were maintained at 25°C in dark. The calli (about 1g fresh weight) that formed were transferred to 200-ml Erlenmeyer flasks each having 70 ml of B5 medium supplemented with growth regulators, casamino acid (1.0 g/L) and agar (0.9%). To find the optimum concentration of growth regulators for the proliferation of callus, combinations [(A) - (C)] of two kinds of growth regulators were added to B5 medium. The combinations were: (A): 2,4 D (12.0 mg/L) and kinetin (1.5 mg/L); (B): 2,4 D (8.0 mg/L) and kinetin (4.0 mg/L) and (C): 2,4 D (4.0 mg/L) and kinetin (0.5 mg/L). The cultures were maintained at 25°C in the dark for 30 days. All calli were kept at 25°C and were routinely subcultured onto fresh agar-solidified B5 medium supplemented with 2,4SD (4.0 mg/L) and kinetin (0.5 mg/L) every 30 days at 25°C in dark.
Fresh weights of the calli were measured directly and dry weights after freeze-drying of tissue. The growth rate was calculated from the increase in the weight of calli relative to incubation time (day).
Extraction and isolation: Fresh calli (61.7g, dry weight 8.02g) subcultured three times on the B5 medium were freeze-dried for 5 days, ground with a pestle and extracted twice with methanol and water (1:1 v/v) (100ml) for 4 days at room temperature. The resulting extracts were then concentrated under reduced pressure and partitioned between dichloromethane and water (1:1 v/v). The dichloromethane layer was dried with Na2SO4 and evaporated to give dichloromethane solubles (0.54g). The dichloromethane solubles were then subjected to preparative thin layer chromatography repeatedly followed by recrystallization from methanol and water to give taxol as colourless crystals (0.8mg), m.p. 213 - 215°C. The isolated taxol was confirmed to be identical with the authentic taxol (mixed melting point and comparison of mass spectrum). FDMS m/z (rel. int.): 854 [M++H] (73), 853 [M+] (12), 836 (7), 794 (15), 568 (16), 211 (21), 210 (100), 105 (12), 61 (5), 43 (6).
Thin layer chromatography: Preparative TLC was carried out using a 1mm (preparative) layer of silica gel (Merck Kieselgel G F254). Taxol was located by UV light and by spraying with potassium dichromate in 40% sulfuric acid, then heating with a hot air dryer (McLaughlin et al., 1981).
Effect of media on growth of calli and taxol production in the calli: The fresh weights of the calli induced on MS, B5 and SH media were determined every ten days for a period of 60 days. Taxol was extracted from the calli derived from T. cuspidata var. nana stems by the method of Witherup et al. (1990). Each callus was freeze-dried, pulverized and extracted with methanol and water (1:1 v/v) for one week at room temperature. The resulting extracts were evaporated in vacuo to give crude residues. The crude residues were subsequently partitioned between dichloromethane and water (1:1 v/v) to give, after evaporation of the solvent and drying in vacuo, dichoromethane solubles. The amount of taxol in a dichloromethane soluble was determined by HPLC performed on a reverse phase column (Supelcosil TM LC F) in a Shimadzu LC 10A liquid chromatograph equipped with UV (ultraviolet) detector (wave length: 227nm) by isocratic elution with acetonitrile - tetrahydrofuran - water (17: 28: 55 v/v) mobile phase. The flow rate was 1.5 ml/min, and all chromatograms were plotted at the absorbance maximum of taxol, 227nm. The dichloromethane soluble obtained was dissolved in 5 ml of methanol, and 10Fl of the solution was injected into the column. The taxol in the dichloromethane soluble was identified by comparing the retention time with that of authentic taxol isolated from T. cuspidata var. nana (Tachibana et al., 1994a) and by comparing its mass spectrum with that of the authentic taxol. A calibration curve was obtained using authentic taxol. For comparison with the content of taxol in the cultured calli, intact stems were extracted similarly and were subjected to HPLC analysis.
Preparation of oligosaccharides by a partial hydrolysis of chitosan and algal laminaran: Chito-oligosaccharides (COS) were prepared by a partial hydrolysis of chitosan according to the method of Kikkawa et al. (1990). Laminaran-oligosaccharides (LOS) were prepared from algal laminaran with tunicase by the method of Kobayashi et al. (1993).
Fractionation and analysis of chito-oligosaccharides: Chito-oligosaccharides were separated into 8 fractions (Fr.1 S Fr.8) by ionSexchange chromatography (Dowex 50WSX8H) with an aqueous hydrochloric acid and water gradient (from 1.15N HCl to 4.10N HCl). Each fraction was neutralized with ionSexchange resin (Amberlite IRAS410) and dried in vacuo to give a residue. Fr.5 was analyzed by HPLC performed at 30°C on a reverseSphase column (Asahipak NH2 PS50) in a Shimadzu LCS10A liquid chromatograph equipped with a RI (refractive index) detector by isocratic elution with 100mM tetrapropylammonium hydroxide (pH 10) S acetonitrile (30: 70 v/v). The flow rate was 1.0 ml/min. Fr.5 was dissolved in 1 ml of water, and 10Fl of the solution was injected into the column. The constituents (glucosamine to chitoheptaose) in Fr.5 were identified by comparing the retention time with that of authentic chito-oligosaccharides (glucosamine to chitoheptaose) and by adding authentic samples. A calibration curve was obtained using authentic chito oligosaccharides.
Treatment of calli from T. cuspidata var. nana with oligosaccharides: Chito-oligosaccharides and laminaran-oligosaccharides were used. One milliliter of an aqueous solution of the chito-oligosaccharides (0.5mg, 1.0mg and 5.0mg), each fraction separated by ion-exchange chromatography (1.0mg), and standard samples (glucosamine to chitoheptaose) (1.0mg) were added to the fresh calli (1g). One milliliter of an aqueous solution of the laminaranSoligosaccharides (0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0mg) was added to the fresh calli (1g). After addition of the compounds, the calli were incubated for 15 days at 25°C in the dark. The amount of taxol in the calli treated with chitoSoligosaccharides, each fraction separated by ion-exchange chromatography, standard samples (glucosamine to chitoheptaose) and laminaran-oligosaccharides was determined as described above. Each experiment was repeated independently three times and the results shown are the average of three measurements, the deviation of each experimental value being within 13%.
Results and Discussion
Induction and proliferation of callus from T. cuspidata var. nana: Callus was produced from sterile stems of T. cuspidata var. nana on all three media supplemented with 2,4SD (4.0 mg/L) and kinetin (0.5 mg/L) solidified with agar (0.9 %) in the dark. No callus formed in any of the media lacking growth regulators. Each callus formed within 20 days of culture on the three media. The calli generally appeared white to yellowish brown. The contamination level during the initiation in each medium was about 40 % with garden-grown plant tissues. The growth rate of each callus was greatest on SH medium followed by B5 medium (85% of that on the SH
| Fig. 1: | Effect of medium on taxol production by calli from T. cuspidata var. nana |
| SH = Schenk and Hildebrandt medium | |
| B5 = Gamborg’s B5 medium | |
| MS = Murashige and Shook medium | |
| Fig. 2: | Effect of chito-oligosaccharides on taxol production by calli from T. cuspidata var. nana |
| Fig. 3: | Effect of laminaran-oligosaccharides on taxol production by calli from T. cuspidata var. nana |
| Fig. 4: | Effect of each fraction of chito-oligosaccharides on taxol production by calli from T. cuspidata var. nana. |
| Con = Control | |
| Cos = Chito-oligosaccharides obtained by a partial hydrolysis of chitosan medium), and then MS medium (45% of that on the SH medium). | |
Fett-Neto et al. (1992, 1993) and Son et al. (2000) reported that callus was induced from T. cuspidata on B5 medium. Wang et al. (1997) and Zhang et al. (2001) induced callus from T. chinensis on MS medium. Wickremesinhe et al. (1993) induced callus from Taxus spp. on SH medium. However, comparison of growth rate of each callus induced on three media (B5, MS and SH) was not conducted. From the results obtained here, we found that there was difference in the growth rate of each callus induced on the three media.
To find the optimum concentration of 2,4SD and kinetin for the proliferation of the calli derived from the stems, three combinations [(A) S (C)] of each growth regulator were added to B5 medium. The results are shown in Table 1. Callus growth by the combination C was the most largest in the three combinations (Table 1). The callus increased 9.7-fold for 50 days. From the results obtained here, the optimum concentrations of 2,4SD and kinetin for the proliferation of the calli were 4.0 and 0.5 mg/L, respectively. In addition, B5 medium supplemented with 2,4SD and kinetin supported growth without regeneration. Subculturing on this medium led to a reduction in the proliferation of calli after five cultures and ultimately, a complete loss of proliferation after ten cultures.
| Fig. 5: | Effect of each chito-oligosaccharide on taxol production by calli from T. cuspidata var. nana |
| Con = Control | Cos = Chito-oligosaccharides |
| C1 = Glucosamine | C2 = Chitobiose |
| C3 = Chitotroise | C4 = Chitotetraose |
| C5 = Chitopentaose | C6 = Chitohexaose |
| C7 = Chitoheptaose |
| Table 1: | Effect of growth regulator on proliferation of callus derived from Taxus cuspidate var. nana |
* Callus growth showed the growth ratios after incubation for 50 days, (%) of the starting callus as 100. | |
| Table 2: | Composition of chito-oligosaccharides and the separated fraction (Fr. 5). |
Chito-oligosaccharides prepared by partial hydrolysis of chitosan were separated into eight fractions (Fr. 1-8) by iron-exchange chromatography. | |
In the previous paper, Tachibana et al. (1994) reported that callus was induced from T. cuspidate var. nana on MS medium supplemented with 2,4-D and kinetin. The optimum concentration of 2,4-D and kinetin for proliferation of the calli were 4.0 and 0.5 mg/L, respectively. The callus growth increased about 4 to 5-fold for 30 days. However, the calli induced on the MS medium became brown color after three subcultures and completely stopped the proliferation after five subcultures.
Production of taxol by T. cuspidata var. nana: The amount of taxol in each callus derived from the stems on all three media is shown in Fig. 1. The taxol in the calli derived from the stems on B5 medium constituted 0.084% dry weight, as opposed to 0.061% for the intact stems. The taxol in the calli derived from the stems on the MS and SH media constituted 0.073 and 0.029%,
respectively. The amount of taxol in the calli derived from the stems on the B5 medium was about 1.4 times higher than that in the intact plant. In addition, no taxol was found in the three agar media. In the previous paper. Tachibana et al. (1994) reported that the content of taxol in the calli derived from stems of T. cuspidata var. nana by use of MS medium was almost the same as that in the intact plant, though the amount of taxol produced by the callus cultures in MS medium was lower than that in B5 medium as mentioned above. Fett-Neto et al. (1992) reported that the content of taxol produced by callus cultures of T. cuspidate in B5 medium was almost the same as that in the intact plant. The results obtained here was almost consistent with the previous results obtained by Fett-Neto et al. (1992) and Tachibana et al. (1994). Based on growth rate and taxol yield, the B5 medium was considered to be the best for production of taxol by callus cultures of T. cuspidata var. nana. As the content of taxol was increased by callus cultures of T. cuspidata var. nana, it was considered that the production of taxol by the cultures could be enhanced by several methods, for example, treatment with an elicitor.
Effect of oligosaccharides on the production of taxol in callus cultures of T. cuspidata var. nana: The production of taxol was stimulated significantly when COS were added to the calli (Fig. 2). The production was stimulated 5.1-fold compared to that of the control (no COS) when 1mg of COS was added to fresh calli (1g) and the mixture incubated on B5 medium for 15 days at 25°C in the dark.
The effect of LOS on the production of taxol is given in Fig. 3. The stimulation of production was less marked on treatment with LOS than COS, with only about 1.2-fold increase over the control (no LOS).
The results show that COS stimulate the production of taxol in callus cultures of T. cuspidata var. nana. This is the first report on the stimulation of production of taxol in callus cultures of Taxus spp. by treatment with oligosaccharides. In addition, Furmanowa et al. (2000) reported that chitosan (a polymer of glucosamine) enhanced the taxol yield by 1.5- fold in callus cultures of T. media. They also reported that the production of taxol was not enhanced in callus cultures of T. cuspidata treated with chitosan.
Muranaka et al. (1998) found that the production of podophyllotoxin in callus cultures of Juniperus chinensis was stimulated about 15-fold when COS was added. Kobayashi et al. (1993) reported that LOS had elicitor activity and stimulated the production of antifungal compounds in alfalfa. However, in case of callus cultures of T. cuspidata var. nana, the effect of LOS on taxol production was less than that of the antifungal compounds in alfalfa. Muranaka et al. (1998) found that the production of podophyllotoxin by callus cultures of J. chinensis was stimulated about 3.5-fold when LOS was added. In the case of callus cultures of T. cuspidata var. nana, the stimulation of taxol production by treatment with LOS was 2.9 times lower than that of the production of podophyllotoxin. These results suggest that COS and LOS act as elicitor, though the degree to which they affect taxol production may vary.
The active compound in chito-oligosaccharides that stimulates the production of taxol: The production of taxol was stimulated significantly when Fr.5 was added (Fig. 4). The production increased about 5.7-fold over that of the control (no COS) when 1 mg of Fr.5 was added to fresh calli (1g). Fr.5 was further analyzed by HPLC. The composition of Fr.5 is shown in Table 2. The main constituents are chitotriose, chitotetraose and chitoheptaose.
Effect of each constituent in Fr.5 on stimulation of taxol production in the callus cultures was examined and the results are shown in Fig. 5. Production increased about 8.2-fold compared with control (no chito-oligosaccharide) when 1 mg of chitoheptaose was added to fresh calli (1g). The results show that chitoheptaose is the most active compound in COS for stimulation of taxol production. There is no report on the stimulation of production of taxol in callus cultures of Taxus spp. by treatment with chitoheptaose. We also found that chitoheptaose stimulates the production of taxol in cell suspension cultures of T. cuspidate var. nana. The results will be published later.
Acknowledgments
We thank Mr. Koukichi Shinkai, Akinori Nakamura and the late Tatsuo Tamagawa for the fresh stems of T. cuspidata var. nana. Part of this work was supported (financially) by a Grant-in Aid for Scientific research (10556039) from the Ministry of Education, Science and Culture, Japan.