**5.5. Thin-layer chromatography**

The technique of column chromatography helps us to know if the fractions recovered from the column chromatography are pure, otherwise it tells us how many components we Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles… http://dx.doi.org/10.5772/intechopen.78226 37

**Figure 9.** Chromatographic plates of the fruit extract.

They were used 300 g of dry leaves of *Taxodium mucronatum*. Milled with methanol, which were later packed for extraction in a glass column, where methanol was passed until the

The study was also carried out with fruit, for which 467 g of fresh fruits of *Taxodium muncronatum* (**Figure 8**) were milled with methanol, which were subsequently packed for extraction in a glass column, where methanol was passed until the material was exhausted. The extractives were evaporated at reduced pressure in a rotary rotator at a temperature of 50°C.

Column chromatography was used to separate the compounds. For this purpose, 1 g of sample was taken as a result of the rotations of the extracts and placed in a column (measures 20 cm long, 3 cm in diameter), prepacked with 30 g suspended in hexane. Silica gel was used

Three solutions were prepared at different proportions of hexane and ethyl acetate. The first solution containing 40 ml of hexane plus 10 ml of ethyl acetate, the second 20 ml of hexane plus 20 ml of ethyl acetate, and the third 10 ml of hexane plus 40 ml of ethyl acetate. To pack the column, 30 g silica gel were mixed with 100 ml of hexane, then poured into the column. Hexane is passed through the column to have a uniform packing. Then it was proceeded to repeat this operation several times to achieve a homogeneous packing. 3 ml of hexane was left on the surface of the silica, and then the sample was prepared. It is homogenized with 2 g of silica and rotavapor for a few minutes to evaporate solvent residues, and is added little by little to the column, leaving at least 1 ml of hexane remaining on the surface. Elute with the prepared solutions. Collect in a flask each fraction of the column (different color layers), evaporate each of the fractions in a rotavapor under reduced pressure and at a temperature of 50°C. Seven fractions of each of the columns were obtained for

both the fruits and leaves. These were analyzed by thin-layer chromatography and HPLC.

The technique of column chromatography helps us to know if the fractions recovered from the column chromatography are pure, otherwise it tells us how many components we

material was used up.

**5.4. Column chromatography**

**Figure 8.** Fruits and leaves of *Taxodium mucronatum.*

36 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

**5.5. Thin-layer chromatography**

to complete the separation of the components of the sample.

have in these fractions. Column chromatography also helps us to know the polarity of our compounds present in the sample, and therefore it is necessary to make several different solvent systems, this is vitally important because the good or bad purification of our compounds will depend on it. For thin-layer chromatography, the following components were used: A stationary phase: Silica gel 60F 254 Merk 0.25 mm thick, with a ceric sulfate developer solution, and the following solvent systems were tested: chloroformmethanol (7:3) (9:1) (1:9) (5:5), hexane-ethyl acetate (9:1) (5:5) (1:9), methanol-acetone (4:6) (9:1) (3:9), chloroform-ethyl acetate (3:7), methanol-hexane (3:7), chloroform-acetone (9:1), and ternary systems were also tested: ethyl acetate-chloroform-methanol (2:7:1), acetonechloroform-methanol (2:7:1).

In **Figure 9**, the separation of components of the fruit extract is observed, where at least six components are distinguished, the solvent mixture was hexane/ethyl acetate in a 9:1 ratio. **Figure 10** shows the results for the leaf extract, where at least three main components are identified.

In **Figure 11**, other plates are shown chromatography's with a different mixture of solvents, without success in the separation of components. Observing the results of the chromatography, we know that most of our compounds have an intermediate polarity, this based on our solvent system that is the best for the separation of compounds. Another interesting fact about our results is that we have several compounds with very similar characteristics, this is deductible because although you can see spots individually (each corresponding to a different compound) they are too close together.

**Figure 10.** Chromatographic plates of the leaf extract.

**Figure 12.** Taxol standard.

**Figure 13.** Fraction 4 of the preparative column of silica with leaf extract of *Taxodium mucronatum.*

Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles…

http://dx.doi.org/10.5772/intechopen.78226

39

**Figure 11.** Chromatographic plates with other solvent mixtures.

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

The identification of compounds is a task for liquid chromatography of high resolution, as long as our sample meets the necessary characteristics. The extracts obtained by means of column chromatography were analyzed by means of HPLC, in a Varian chromatograph with a C18 column an isocratic development acetonitrile-water was made 70:30 at a rate of 1 ml/min, and the injection volume was 20 μl. The identification of Taxol, in this case our metabolite of interest, was carried out by means of an external standard from the *Taxus brevifolia* (**Figure 12**), a retention time of 4.65 min is observed.

Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles… http://dx.doi.org/10.5772/intechopen.78226 39

**Figure 12.** Taxol standard.

**Figure 11.** Chromatographic plates with other solvent mixtures.

The identification of compounds is a task for liquid chromatography of high resolution, as long as our sample meets the necessary characteristics. The extracts obtained by means of column chromatography were analyzed by means of HPLC, in a Varian chromatograph with a C18 column an isocratic development acetonitrile-water was made 70:30 at a rate of 1 ml/min, and the injection volume was 20 μl. The identification of Taxol, in this case our metabolite of interest, was carried out by means of an external standard from the *Taxus brevifolia* (**Figure 12**),

**5.6. High-resolution liquid chromatography**

**Figure 10.** Chromatographic plates of the leaf extract.

38 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

a retention time of 4.65 min is observed.

**Figure 13.** Fraction 4 of the preparative column of silica with leaf extract of *Taxodium mucronatum.*

**Figure 14.** Fraction 5 of the silica preparative column with leaf extract 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 can also deduce that there is presence of other taxoids.

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

*Pestalotiopsis. Microespora* 1.487 1998

Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles…

http://dx.doi.org/10.5772/intechopen.78226

41

**Isolation source Fungus Concentration (μg/L) Year** *T. brevifolia T. andrene* 0.024–0.025 1993 *T. wallaiciana Pestalotiopsis. Microespora* 60–70 1996 *T. baccata Monochaetia* sp. 0.102 1996 *T. baccata Fusarium lateritium* 0.13 1996 *T. cuspidata Alternaria* sp. 0.157 1996 *T. cuspidata Pestalotiopsis. Microespora* 0.268 1996 *T. wallaiciana Pestalotiopsis. Microespora* 0.5 1996 *T. Sumatrana Phitomyces* sp. 0.095 1996 *T. baccata Pestalotia bicilia* 1.081 1996 *Wollemia nobilis P. guepinii* 0.481 1997

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

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.

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

controllable, which is important for its industrial scaling.

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

**6.1. High-resolution liquid chromatography**

allows the production.
