**6. Case study 2: alternative for obtaining natural products**

Secondary metabolites can also be produced by endophytic fungi, and this feature has opened the door to new research. Some of the most important advantages of this find are that no plant species will be threatened. The process for the production of natural products can be industrialized. Therefore, the following research is about the production of Taxol by means of endophytic fungi of *Taxodium mucronatum*.

The main source of obtaining Taxol until now is the extraction of trees of the genus *Taxus* (Tejo); however, it is estimated that the amount of purified Taxol required to treat only 500 patients with cancer is 1 kg, equivalent to the performance of near of 10 tons of bark or the felling of 700 trees. Therefore, the next step for science is to find new ways to obtain this drug. Some of the sources to obtain this medicine, are the semi-synthesis, from other Taxanes being the most used the 10-Deacetilbaccatina III obtained from the leaves of *Taxus*, the disadvantage that this method has, is the low yield and the high cost; by total synthesis of plant cell cultures of *Taxus* and cultures of microorganisms such as fungi and bacteria (**Table 4**). The production Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles… http://dx.doi.org/10.5772/intechopen.78226 41


**Table 4.** Production of Taxol by endophytic fungi [32].

**Figure 14.** Fraction 5 of the silica preparative column with leaf extract of *Taxodium mucronatum.*

**6. Case study 2: alternative for obtaining natural products**

can also deduce that there is presence of other taxoids.

40 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

endophytic fungi of *Taxodium mucronatum*.

In **Figure 13**, it is the chromatogram of the analysis of fraction 4 of the column of silica gel of 1 g of the leaf extract of *Taxodium mucronatum*, in it a peak is observed with the same retention time with the standard which indicates us Taxol inside the leaves. Observing the distance that exists between the signals that are close to the retention time characteristic of Taxol, one of these signals could also be about a mixture of taxoids. **Figure 14**, corresponding to fraction 5 of the extract of the leaves shows a characteristic retention time of Taxol, but as in fraction 4, it also presents signals with times very close to the retention time of Taxol, which is why we

Secondary metabolites can also be produced by endophytic fungi, and this feature has opened the door to new research. Some of the most important advantages of this find are that no plant species will be threatened. The process for the production of natural products can be industrialized. Therefore, the following research is about the production of Taxol by means of

The main source of obtaining Taxol until now is the extraction of trees of the genus *Taxus* (Tejo); however, it is estimated that the amount of purified Taxol required to treat only 500 patients with cancer is 1 kg, equivalent to the performance of near of 10 tons of bark or the felling of 700 trees. Therefore, the next step for science is to find new ways to obtain this drug. Some of the sources to obtain this medicine, are the semi-synthesis, from other Taxanes being the most used the 10-Deacetilbaccatina III obtained from the leaves of *Taxus*, the disadvantage that this method has, is the low yield and the high cost; by total synthesis of plant cell cultures of *Taxus* and cultures of microorganisms such as fungi and bacteria (**Table 4**). The production of Taxol, by means of microorganisms, represents a potential source of Taxol; due to its multiple advantages among them that no plant species is affected, the process is reproducible and controllable, which is important for its industrial scaling.

For this reason, the main purpose of this project is to isolate and select strains of endophyte microorganisms capable of producing Taxol and also develop a biotechnological process that allows the production.

The proceeding of isolating the fungi associated with *Taxodium mucronatum* was made by getting a collection of samples of microorganisms than was carried out in test tubes with nutritious broth, at room temperature. A short and deep cut was made in the bark of the selected tree; in the cut with an applicator, three samples were taken. Later in the laboratory, the preparation of five culture media was made: Czapeck medium, Sabourod, PDA, YPD, Agar Plate count; microorganisms were seeded from each in tubes in five different culture media. This was realized with the purpose of observing in which agar these microorganisms grow better and make a cellular differentiation.

Then proceeded to cultivate the fungi; using 2 ml of saline containing the contents of a fungal Petri dish in 250 ml of PDA were inoculated and incubated in a shaker at 250 rpm and 27°C for 7 days. After this time, the culture broths were filtered to remove the biomass, and extractions of each Erlenmeyer flask were carried out with ethyl acetate. The organic phase was separated and dried with anhydrous sodium sulfate and filtered; then the organic phase was evaporated in a rotatory evaporator until the solvent was removed at 50°C and in vacuum. The extract was resuspended in 1 ml of acetonitrile HPLC grade with 0.01% acetic acid to avoid esterification of Taxol and was placed in Bakelite tubes and kept in refrigeration at 4°C for future analysis.

#### **6.1. High-resolution liquid chromatography**

All the extracts were analyzed in HPLC, in a Varian chromatograph 8090 mod. (USA) with a C18 column under isocratic conditions and in an 80:20 acetonitrile-water mixture at a flow rate of 1 ml/min, the injection volume was 20 μl. The identification of Taxol was carried out

**Figure 15.** Chromatogram of the culture extract of strain 1.

by means of a Taxol standard of sigma Aldrich (Toluca, México) from *Taxus brevifolia* which presented a retention time of 4.65 min.

**Figure 16.** Chromatogram of the culture extract of strain 17 in potato broth.

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**Figure 17.** Chromatogram of extract of strain 17.

**Figure 12** shows the chromatogram corresponding to the Taxol standard of Sigma Aldrich from *Taxus brevifolia*. **Figure 15** is the chromatogram corresponding to strain 1, and this strain is matter of interest because it produces very few natural products, although it does not show any time characteristic of Taxol. Therefore, the purification process would be easier and identify these products. **Figure 16** shows the chromatogram corresponding to strain 17, in which we observe a signal with the same retention time as the characteristic signal of Taxol.

Based on our chromatogram of the extract of strain 17 and identifying Taxol in our extract, we conducted another experiment in which we enriched our culture medium using brown sugar, which contains different types of salts as well as different carbon sources (sucrose, glucose, etc.). In the chromatogram of **Figure 17**, we observe the effect of enrichment of the medium, our corresponding to Taxol increases its area and volume; however, the rest of our compounds also increase significantly. The result of Taxol production was confirmed by adding 0.5 ml of standard as internal control To 0.5 ml of sample **Figure 18**.

In the identification of the Taxol-producing endophytic fungus, lacto phenol blue staining was performed for its microscopic morphological structure. **Figure 19** shows the endophytic fungus with Taxol production capacity, in the image, the growth in petri box and its microscopic view is appreciated.

Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles… http://dx.doi.org/10.5772/intechopen.78226 43

**Figure 16.** Chromatogram of the culture extract of strain 17 in potato broth.

**Figure 15.** Chromatogram of the culture extract of strain 1.

42 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

as internal control To 0.5 ml of sample **Figure 18**.

scopic view is appreciated.

presented a retention time of 4.65 min.

by means of a Taxol standard of sigma Aldrich (Toluca, México) from *Taxus brevifolia* which

**Figure 12** shows the chromatogram corresponding to the Taxol standard of Sigma Aldrich from *Taxus brevifolia*. **Figure 15** is the chromatogram corresponding to strain 1, and this strain is matter of interest because it produces very few natural products, although it does not show any time characteristic of Taxol. Therefore, the purification process would be easier and identify these products. **Figure 16** shows the chromatogram corresponding to strain 17, in which we observe a signal with the same retention time as the characteristic signal of Taxol. Based on our chromatogram of the extract of strain 17 and identifying Taxol in our extract, we conducted another experiment in which we enriched our culture medium using brown sugar, which contains different types of salts as well as different carbon sources (sucrose, glucose, etc.). In the chromatogram of **Figure 17**, we observe the effect of enrichment of the medium, our corresponding to Taxol increases its area and volume; however, the rest of our compounds also increase significantly. The result of Taxol production was confirmed by adding 0.5 ml of standard

In the identification of the Taxol-producing endophytic fungus, lacto phenol blue staining was performed for its microscopic morphological structure. **Figure 19** shows the endophytic fungus with Taxol production capacity, in the image, the growth in petri box and its micro-

**Figure 17.** Chromatogram of extract of strain 17.

**Author details**

Mexico

**References**

Nadia Mendoza and Eleazar M. Escamilla Silva\*

\*Address all correspondence to: eleazar@iqcelaya.itc.mx

Department of Chemical Engineering, Technological Institute of Celaya, Celaya, Guanajuato,

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**Figure 18.** Chromatography of the extract of strain 17 with the addition of 0.5 ml of standard.

**Figure 19.** Image of the Taxol producing endophytic fungus.
