BRPI1003652A2 - pharmaceutical composition for the treatment of cancer from active ingredients of psidium guajava l. - Google Patents

Abstract

PHARMACEUTICAL COMPOSITION FOR CANCER TREATMENT FROM ISOLATED ACTIVE PRINCIPLES OF PSIDIUM GUAJAVA L. The present invention relates to a pharmaceutical composition comprising the guajadial molecule (mlz = 474, base ion = 271) obtained from the plant species Psidium guajava L (popular name: guava) of the Myrtaceae family, which has anticancer activity.

Description

"PHARMACEUTICAL COMPOSITION FOR CANCER TREATMENT FROM ISOLATED ACTIVE PRINCIPLES OF PSIDIUM GUAJAVA L.

FIELD OF INVENTION

The present invention relates to a pharmaceutical composition which

comprises the guajadial molecule (m / z = 474, base ion = 271) obtained from the plant species Psidium guajava L. (popular name: guava) of the family Myrtaceae, which has anticancer activity. BACKGROUND OF THE INVENTION Despite the scientific and technological progress achieved by

Chemical synthesis, drugs obtained from natural products have shown that this is an important source for research and development of new drugs (da Rocha et al., 2001; Diwanay et al., 2004; Newman and Cragg, 2009). Nature is a rich source of candidates for therapeutic compounds because of the immense chemical and biological diversity found in it. Many pharmaceutical agents have been discovered through the search for natural sources like plants, marine organisms and microorganisms. For many organisms, this chemical diversity reflects the impact of evolution on the selection and conservation of defense mechanisms that represent strategies employed to repel or destroy potential predators or competitors (da Rocha et al., 2001). Forests offer great opportunities for the discovery of promising drugs, which are a source of chemical and biological richness, and are therefore called by many as a vegetable pharmacy. Once isolated, these substances are evaluated by pharmacological activity tests that may lead to the discovery of a new drug or possibly to compounds that can be chemically improved (Mans et al., 2000).

Cancer is the second most common cause of death in the world second only to cardiovascular disease (Reddy et al., 2003). The community has benefited from studies based on research from natural sources (da Rocha et al., 2001). Of the drugs used to treat cancer, about 60% come from natural products or directly derived from them (Newman & Cragg, 2009) and, for example, taxol, doxorubicin, vincristine, vinblastine, etoposides, among others.

It is important to emphasize the importance of encouraging the expansion of nature research as a source of new active compounds that will possibly be the basis for the elaboration of drugs for human diseases. The constant challenge of developing therapies that are doubly effective and with high specificity for cancer cells or tumor tissue is reiterated (Newman & Cragg, 2007). Possible compounds and active ingredients should ideally be selective for cancer cells versus normal cells, not cause toxicity, have minimal or no side effects, and should not interfere with any other treatment therapies (Diwanay et al., 2004).

For the research of new anticancer drugs, the improvement of the cell culture methodology has allowed the development of several cell lines from several human tumors, enabling the development of in vitro screening methodology. For this purpose, the National Cancer Institute (NCI) maintains a panel of cancer cells with more than 60 strains from eight types of solid tumors (lung, melanoma, breast, kidney, colon, prostate, ovary and brain) and the hematopoietic system (leukemia). This methodology allows the evaluation of pharmaceutical compositions, extracts and active principles in several neoplastic cells, enabling the discovery of drugs with greater specificity. It also has the fastest and most efficient method, which evaluates a large number of drugs in a short period of time (Rubistein et al., 1990; Skehan et al., 1990).

The guava tree, belonging to the Myrtaceae family, originates from the tropical regions of Central and South America, being the Psidium guajava L. species the most important of the genus Psidium. Widely scattered in Brazilian forests, many of their species are cultivated for their edible fruits, ornamental purpose, source of wood and firewood, or as a source of essences of commercial value (Lima et al, 2010). Previous phytochemical analyzes of Sunttornusk1 2005 indicate that P. guajava is rich in tannins, phenols, triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fiber and fatty acids in both leaves and fruits. Of the various isolated compounds of P. guajava described in the literature, quercetin is the most active constituent considered for this plant species. Quercetin flavonoid has spasmolytic and antidiarrheal activity, justifying the traditional use of this plant for gastrointestinal disorders (Ubóh ef ai., 2010). US2009 / 0004270 A1, US20050255569 A1,

EP1080205 B1, US20090252796 A1, W02006 / 090206 A1, W02007 / 053865 A1, W02008 / 002460 A2, PI0605658-0 refer to similar uses, but differ in their treatment of distinct molecules and with other therapeutic activities. Such documents refer to the use of crude extracts without discrimination of their specific active components, use of other plant sources and non-leaves as in the present invention, or suggest action of other secondary metabolite classes, obtained from phytochemical processes that purify compounds of polarity different from that used herein or different therapeutic activities of the compounds such as antimicrobial, antibiotic, antiinflammatory, antidiabetes and others. Therefore, the present invention differs from the state of the art in both the methodology for obtaining the compound and the object of protection. BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising the guajadial molecule (m / z = 474, base ion = 271) obtained from the plant species Psidium guajava L. (popular name: guava) of the family Myrtaceae, which has anticancer activity.

The molecule of interest was obtained from the dichloromethane crude extract of Psidium guajava L. (Popular name: guava, Family: Myrtaceae), from which an enriched fraction containing guajadial molecule isomers was obtained. From the enriched fraction was performed a biofractionation that allowed the isolation of the active principle from the crude dichloromethane extract of P. guajadial L. for inclusion in a pharmaceutical composition with anticancer activity. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: Flowchart of the Extracting Process

cold and hot grains of the plant species Psidium guajava L. (PG).

FIGURE 2: Flowchart of the extraction and isolation process of active ingredients from P. guajava L. plant material (leaves)

FIGURE 3: Flowchart of the methodology used in the in vitro test for evaluation of antiproliferative activity of samples.

FIGURE 4: In vitro anticancer activity of doxorubicin chemotherapy, used as positive control against cell panel

human tumors.

FIGURE 5: In vitro anticancer activity of hot crude extract

guajava dichloromethane (EBQD).

FIGURE 6: Overlap of the chromatogram expansions of

PG (F2) and PG (F3) (t R = 43-55 min).

FIGURE 7: In vitro anticancer activity of PG (F2) obtained from

of the 1st fractionation of Psidium guajava. FIGURE 8: In vitro anticancer activity of PG (F3) obtained from

of the 1st fractionation of Psidium guajava.

FIGURE 9: Overlap of the chromatograms PG (F2-3). (F6) to PG. (F2-3). (F9), fractions from the 2nd Psidium guajava fractionation.

FIGURE 10: In vitro anticancer activity of PG.F (2-3). (F6), obtained from the 2nd fractionation of Psidium guajava.

FIGURE 11: In vitro anticancer activity of PG.F (2-3). (F7), obtained from the 2nd fractionation of Psidium guajava.

FIGURE 12: In vitro anticancer activity of PG.F (2-3). (F8), obtained from the 2nd fractionation of Psidium guajava. FIGURE 13: In vitro anticancer activity of PG.F (2-3). (F9), obtained

from the 2nd fractionation of Psidium guajava. FIGURE 14: Chromatograms of PG (F2-3). (F6-F9). (F4) fraction from the 3rd P. guajava fractionation.

FIGURE 15: In vitro anticancer activity of PG.F (2-3). (F6-F9). (F4), obtained from the 3rd Psidium guajava fractionation. FIGURE 16: Chromatogram of PG fraction (F2-3). (F6-

F9). (F4). (F7), of the 4th fraction of P. guajava.

FIGURE 17: Expanded chromatogram of PG (F2-3). (F6-F9). (F4). (F7), fraction from the 4th fraction, t R = 40 to 48min.

FIGURE 18: Chromatogram of sample PG. (F2-3) (F6-F9). (F4). (F7) obtained by the GC-Tof technique.

FIGURE 19: Fragmentogram of the peak with tR = 12.75 from sample

PG (F2-3). (F6-F9). (F4). (F7).

FIGURE 20: H1 NMR Spectrum for Enriched Fraction

PG (F2-3). (F6-F9). (F4). (F7). FIGURE 21: Chemical structure of the guajadial meroterpenoid,

described by Yang et al. (2007).

FIGURE 22: Schematic of the in vivo experimental model used in

Ehrlich Solid Tumor.

FIGURE 23: Ehrlich Solid Tumor volume variation for the negative (saline), positive (doxorubicin) and guava-treated control groups at doses of 10, 30 and 50 mg / kg. * = Duncan test, p <0.05 (ANOVA).

FIGURE 24: Body weight variation in different groups of Ehrlich Solid Tumor mice during the 21 days of the experiment. * = Duncan test, p <0.05 (ANOVA).

FIGURE 25: Analysis of gross (left) and relative tumor (right) weight, assessed at the end of the experiment (D21). * = Duncan test, p <0.05 (ANOVA), * * = Duncan test, p <0.01 (ANOVA).

FIGURE 26: Assessment of uterine weight at the end of the Ehrlich Solid Tumor experiment (D21). * = Duncan test, p <0.05 (ANOVA). DETAILED DESCRIPTION OF THE INVENTION The plant species Psidium guajava L. has been selected because of its widely described action for antiparasitic activity. In addition, the biological action of the plant species has also been proven for antidiarrheal, antibacterial, antimutagenic, antimicrobial, antiinflammatory, antioxidant, hypoglycemic, hypotensive and hypocholesterolemic activity. For the extraction process, preliminary evaluations were performed: 1. Extraction of the essential oil; 2. Cold extraction of plant material; 3. Extraction of hot plant material.

The material used consisted of the aerial parts of Psidium guajava L. (Myrtaceae) obtained from the experimental field of CPQBA (Center Iuridisciplines of Chemical, Biological and Agricultural Research) - Unicamp (State University of Campinas). The species desiccate is in the CPQBA - Unicamp Herbarium under CPMA (Collection Entry Number) 1814 and exsiccate number 1335. From the collected material, an aliquot of fresh material was

separated for essential oil extraction through Clevenger hydrodistillation. The remaining leaves were dried and ground to allow the extraction process with different organic solvents. The moisture content of the plant species was in the range of 50 to 60%, which was calculated by drying an aliquot of the leaves in a greenhouse at a temperature between 100 and 110 ° C, preferably 105 ° C for 22 to 26 hours, preferably 24 hours, until become brittle. The dried and ground plant material of P. guajava leaves was hot extracted by the Soxhlet method in a 1: 5 ratio with dichloromethane (CH 2 Cl 2) and 90% ethanol (EtOH), preferably 95% (Figure 1).

The crude dichloromethane extract (EBD) extracted for 65 to 75h, preferably 72h, was collected and rotary evaporated under vacuum at a temperature between 35 and 45 ° C, preferably 40 ° C until complete removal of the solvent. The vegetable residues of this extraction process were used for the 90 to 99% ethanol, preferably 95% ethanol extraction process, in the same proportion for 65 to 75h, preferably 72h. The crude ethanolic extract (EBE) was lyophilized after the rotary evaporation process. Extraction yields were calculated (extract mass obtained / dry plant mass used) and aliquots of each crude extract obtained were separated for the in vitro test on human tumor cell culture (Table 1).

Table 1: Extraction yields of each phytochemical process

to which the plant material of Psidium guajava L. was submitted.

P. guajava Sample Yield (%) Extraction essential oil OE 0.22 Cold extraction EBFD 6.00 EBFE 10.88 Hot extraction EBQD 8.44 EBQE 9.71

The extraction of guava essential oil showed the following compounds: alpha-humulene = 37.58%, transcariophilene = 20.47%. Such compounds showed no relevant anticancer activity. The comparison of the two techniques made it possible to verify the best efficiency in obtaining compounds with anticancer activity from the dichloromethane crude extract than from the ethanolic crude extract. The hot Soxhlet extraction method yielded a higher yield than cold, 8.44% and 6% respectively, as well as a reduction in solvent usage as it is a cyclic process, with renewal of the solvent used instead. of the complete replacement.

The fractionation was performed from the hot dichloromethane crude extract (EBQD) of Psidium guajava L. to obtain the compounds of interest. The EBQD was fractionated following the following steps (FIGURE 2):

1st stage: Filter column fractionation

2nd stage: Classical column fractionation 3rd stage: Classical column fractionation 4th stage: Filter column fractionation until the fraction enriched with the active ingredient of interest (guajadial and its isomers) is obtained.

After the fractionation process, the F7 enriched fraction obtained after the 4th fractionation was identified and characterized by: GC-MS, GC-Tof1 1H-NMR and optical rotation.

The compounds present in the active fractions and the active principle were identified and characterized by GC-MS (Gas Chromatography Mass Spectrometry) under the following conditions: Samples were analyzed under AA Series conditions (injector: 240 to 260 ° C, preferably 250 ° C) ; detector: 290 to 310 ° C preferably 300 ° C; HP-5MS column: 110 ° C - 280 ° C, raising the temperature by 5 ° C / min, remaining at 280 ° C for 20-30 minutes, preferably 26 minutes, run time between 50 minutes and 10 hours, preferably 1h, carrier gas He1 carrier flow 1 ml / min) with a 5% Phenyl Methyl Siloxane HP-5MS Column (30cm χ 0.25mm χ 0.25pm).

GC-Tof (Time of Flight Mass Spectrometry) analyzes were performed with an HP-5MS column (30cm χ 0.25mm χ 0.25μητι), injection volume 1μΙ, ti = 1min, 220 ° C, temperature between 3 ° C and 300 ° C. Nuclear magnetic resonance was performed (NMR-1H - Nuclear Magnetic Resonance) with the sample diluted in deuterated chloroform. optical rotation was performed using the Perkin-Elmer Polarimeter 341 equipment at a concentration of 2.2700g / 100ml.

The fractions were evaluated in vitro for evidence of their anticancer activity, as shown in Table 2. The samples obtained were evaluated against a panel of eleven human tumor lines: K562 (leukemia), MCF-7 (breast), OVCAR-3 (ovary). , NCI-ADR (multidrug-resistant ovary), NCI460 (lung), UACC62 (melanoma), PC03 (prostate), HT29 (colon), U-251 (glioma), 786-0 (kidney) and a normal VERO strain (kidney). Table 2: List of human tumor lines used in the in vitro test for evaluation of anticancer activity of crude extracts and fractions.

Lineage Histological Origin Cell Type Cell Density Inoculation 1 M CF-7 Tumoral Breast 6 χ 104 cells / mli 2 OVCAR-3 Tumoral Ovary 7 χ103 cells / ml 3 NCI-ADR Multiple Drug Resistant Tumor 5 χ 104 cells / m! 4 P C-3 Tumor Prostate 5 χ 10 10 cells / ml 786-0 Tumor Kidney 4.5 χ 104 cells / mil 6 UACC-62 Tumor Melanoma 5 χ 10 "cells / ml 7 HT-2 9 Tumor Colon 4x10" cells / ml 8 NCI-460 Oral Tumi Lung 4 χ 10 * cell Ias Ias-TnI 9 U-251 Tumor Glioma 5x10 "cells / mt K 562 Tumor Leukemia 4 χ 10a cells s / m I 11 VERO Kidney 5 x 10a cells / ml

The evaluation of anticancer activity in vitro was performed by

of 100μΙ cells / well plating in 96-well plates

compartments, homogenized in RPMI / SFB / PEN-STREP medium.

Samples were diluted with sodium dimethyl sulfoxide (DMSO)

1:10 (1mg sample: 10 μΙ DMSO). For the addition in culture of

cells, these solutions were diluted at least 400 times in

RPMI / 5% SFB / PEN-STREP, thus avoiding DMSO toxicity. The sign

T0 control was fixed by the addition of 50 μΙ / trichloroacetic acid compartment

50% (TCA) to determine the amount of protein 24 hours after the

cell plating (ie at the time of sample addition). Nas

Treated plates, samples were added at concentrations of 250; 25; 2.5 and 0.25 μg / ml in serial dilution with 100 μ / compartment and in triplicate (Figure 4) and then incubated for 48 hours. As positive control, the chemotherapy drug doxorubicin was used, at concentrations of 0.025; 0.25; 2.5 and 25 μ9 / Γπί, 100 μΙ / compartment and in triplicate. After this period, the cells were fixed with 50 μΙ of 50% trichloroacetic acid (TCA). To complete cell fixation, plates are incubated for 1 hour at 4 ° C and then subjected to four consecutive washes with distilled water to remove TCA, medium, SFB and secondary metabolite residues. After washing, the plates are kept at room temperature until complete drying.

The plates were stained by the addition of 50nl / well of 0.4% sulforhodamine B protein dye (SRB) (Sigma Chemical Co®, St. Louis, MO, USA) and incubated for 30 minutes at 4 ° C. After this time, the plates are washed 4 consecutive times with 1% acetic acid (CH3COOH) solution. After drying at room temperature, cell proteins bound to the SRB sulforhodamine B) dye are solubilized with Trizma Base solution (10μΜ, pH 10.5) for plate reading.

Spectrophotometric absorbance reading was performed at 540 nm in a microplate reader (ELISA, Molecular Devices®, model Versa Max), using the Soft Max Pro 4.0 software. Tumor cell survival was assessed by their absorbance percentage compared to the control plate survival percentage (untreated T0 plate). Sulforhodamine B is a protein dye that binds to the basic amino acids of cell proteins that were viable at the time of fixation. Therefore, the greater the amount of SRB bound to the particulate, the lower the cytotoxic activity of the test sample (Skehan et al, 1990). Figure 5 schematically represents the method

employed for in vitro testing of samples obtained from plant species.

In vitro assays for cytotoxicity and antiproliferative activity evaluation yield preliminary results that assist in the selection of plant extracts with potential therapeutic activities whose in-depth study is interesting (Cardellina et al, 1999). As the different cell lines present different sensitivities to cytotoxic compounds, other strains were used for the detection of substances with selective activity (Skehan et al., 1990). Moreover, the combination of different in vitro assays not only leads to the screening ability of active compounds, but also directs possible mechanisms of action and therapeutic effects (Kintzios & Barberaki, 2004).

With the calculated growth percentages, graphs of percent growth or cell death (y axis) versus sample concentration (χ axis) were plotted. The doxorubicin chemotherapy was

used as a positive control. Values below 50 and above zero represent growth inhibition (cytostatic activity), and values that reach zero represent total growth inhibition (TGI = total growth inhibition) and negative values (below zero) represent cell death. (cytotoxic activity).

In addition, from graphs of cell growth as a function of

At the sample concentration, TGI (Total Growth Inhibition) was calculated using the Origin 7.5 program. TGI values, expressed in pg sample / ml, maintain a relationship inversely proportional to the sample selectivity for a given tumor lineage, since reduced values

TGI levels indicate greater selectivity and the ability to totally inhibit the growth of

particular lineage.

Figures 6 and 7 show, respectively, the results of in vitro antiproliferative activity for doxorubicin (used as a positive control) and for EBQD, which was more active against cells.

human tumors.

From the EBQD, other more powerful and selective fractionation steps were performed, as described below: 1st fractionation: a) Phytochemistry:

- Method = filter column (hexane / dichlor / methanol 0-5%)

- Sample ratio: silica = 1: 3

- Sample: EBD (m = 25, Og) * The filter column used in the first fractionation was performed at a ratio of 1 sample: 3 silica, and the collected samples were stored in numbered vials, resulting in a total of 24 fractions. Once the fractionation process was completed, a CCD was performed with the parent fraction (EBD, here called F0) and the fractions obtained in the fraction (F01 - F024) and these were grouped according to the 1% dichlor / methanol CCD profile. , by qualitative similarity between the compounds visualized in the stationary phase, as follows: PG. (F1) = F0I to F04 PG. (F2) = F05 PG. (F3) = F06 to F07 PG. (F4) = F08 to F09 PG (F5) = F010 to F014 PG. (F6) = F015 to F017 PG. (F7) = F018 to F020 PG. (F8) = F021 to F022 PG. (F9) = F023 to F024

GC-MS analysis of each of the fractions grouped in the 1st fraction was also performed. By overlapping the expanded chromatograms (tR = 43 - 55 minutes) obtained by this technique, it was found that PG. (F2) and PG. (F3) are very similar in composition (Figure 8) b) In vitro pharmacology: Separated aliquots of the parent fraction (FO) and the fractions obtained

in fractionation after pooling, PG. (F1) to PG. (F9), for the in vitro antiproliferative test in human tumor cell culture. The aim was to compare the potential anticancer activity of each fraction and to determine the active fractions responsible for such activity. Percent growth graphs were obtained in

concentration function for each fraction tested from the 1st fraction, PG. (F1) to PG. (F9) and the doxorubicin chemotherapy was used as a positive control against the tumor lines. Of all fractions evaluated, PG. (F2) and PG. (F3) showed the most promising results in vitro (Figures 9 and 10, respectively). Since in vitro graphs were similar in activity, and the CCD of the fractions had already indicated similarity between the compounds of both fractions, a fact confirmed by the overlap of the chromatograms, these fractions were grouped to continue biofractionation techniques. 2nd fraction: a) Phytochemistry:

- Method = classic column (hexane / dichlor / methanol 1-8%)

- Sample ratio: silica = 1:30

- Sample: PG (F2-3) (m = 1.8g)

The classic column used in the 2nd fractionation was performed at a ratio of 1 sample: 30 silica, and the collected samples were stored in numbered flasks, resulting in a total of 37 fractions. Once the fractionation process was finished, the fractions were grouped according to the 20:80 CCD hexane / dichloromethane profile, by qualitative similarity between the compounds visualized in the stationary phase, as follows: PG. (F2-3). (F1) = F0I to F05 PG. (F2-3). (F2) = F06 to F08 PG. (F2-3). (F3) = F09 to F011 PG. (F2-3). (F4) = F012 PG. ( F2-3). (F5) = F013 to F014 PG. (F2-3). (F6) = F015 to F019 PG. (F2-3). (F7) = F020 to F027 PG. (F2-3). (F8) = F028 to F030 PG. (F2-3). (F9) = F031 to F035 PG. (F2-3). (F10) = F036 to F037 Similarly to the first fractionation,

GC-MS analysis of each of the grouped fractions. Again, some fractions were similar to the composition in the sample: PG. (F2-3). (F6) to PG. (F2-3). (F9). The overlap of the chromatograms of such fractions is shown in Figure 11. b) In vitro pharmacology: Similar to that performed for the first fractionation, it was separated

10 - 20mg aliquots of the fractions obtained in the fractionation after pooling, PG.F (2-3). (F1) to PG.F (2-3). (F10), for in vitro cell culture antiproliferative test human tumors. Of all fractions evaluated, PG.F (2-3). (F6) to PG.F (2-3). (F9) were those that were active in the in vitro anti-poliferative test (Figures 12, 13, 14 and 15, respectively). As CCD had suggested similarity between such fractions, a statement corroborated by the overlap of the chromatograms, their contents were grouped in order to give continuity to the 3rd fraction. 3rd fraction: a) Phytochemistry:

- Method = classic column (hexane / dichlor / methanol 0-5%)

- Sample ratio: silica = 1:30

- Sample: PG (F2-3). (F6-9) (m = 1.0g)

Similar to the 2nd fraction, the classic column used in the 3rd fraction was performed at a ratio of 1 sample: 30 silica, and the collected samples were stored in numbered vials, resulting in a total of 40 fractions. Once the fractionation process was finished, the fractions were grouped according to the 15:85 CCD hexane / dichloromethane profile, by qualitative similarity between the compounds visualized in the stationary phase, as shown below: PG. (F2-3) (F6-F9). (F1) = F0I to F09 PG. (F2-3). (F6-F9). (F2) = F0IO to F012 PG. (F2-3). (F6-F9). ( F3) = F013 PG. (F2-3). (F6-F9). (F4) = F014 to F017 PG. (F2-3). (F6-F9). (F5) = F018

PG (F2-3). (F6-F9). (F6) = F019 to F024 PG. (F2-3). (F6-F9). (F7) = F025 PG. (F2-3). (F6 (F8). (F8) = F026 to F027 PG. (F2-3). (F6-F9). (F9) = F028 PG. (F2-3). (F6-F9). (F10) = F029 a F031 PG. (F2-3). (F6-F9). (F11) = F032 to F040

GC-MS analysis of PG.F (2-3). (F6-F9). (F4) revealed the presence of 3 large classes of compounds, as can be seen in Figure 16.

(b) in vitro pharmacology:

Aliquots of 10 - 20mg were separated from the fractions obtained in

fractionation after pooling, PG.F (2-3). (F6-F9). (F1) to PG.F (2-3). (F6-F9). (F11) for in vitro antiproliferative culture test of human tumor cells. Of all fractions evaluated, PG.F (2-3). (F6-9). (F4) was the most active in vitro (Figure 17). So this was elected the fraction to give

continuity of the 4th fraction. Table 3 indicates the EBI TGI values (pg / mL) and the

fractions of P. guajava, following the chronological order of the

fractions used for enrichment of fractions.

Table 3: TGI values (pg / mL) of EBD and active fractions of

P. guajava.

20

U251 UACC62 MCF7 NCIADR 7860 NCIH460 PC-3 OVCAR-03 HT29 K562 VERO EBD 65.05 60.79 48.71 43.81 64.39 28.60 25.83 43.04 32.20 PG. (F2-F3) 0.31 31.45 13.75 32.35 28.42 27.76 0.64 19.78 20.29 PG. (F2) F6) 12.81 10.53 1.58 28.53 11.91 27.10 21.33 12.91 0.78 PG (F2-F3). (F7) 20.02 8.12 0.8 27.62 15.77 26.97 18.74 17.69 7.83 PG. (F2-F3). (F8) 19.43 16.34 0.04 33.21 45.20 29.61 26.09 23.26 1.74 PG. (F2-F3). (F9) 15.68 15.57 0.25 16.34 12.29 13.62 19.19 12.19 15.25 ------- PG. (F2-F3). (F6-9). (F4) 7.48 36.71 8.38 3.96 27.56 5.05 12.32 1.01 4.70 2.01 7.30

4th fraction: a) Phytochemistry:

- Method = filtering column (hexane / dichloro) - Sampling ratio = 1: 3 -Sample: PG. (F2-3). (F6-9). (F4) (m = 380mg) The classic column of the 3rd fractionation used 1 sample: 3 silica ratio, the collected samples were stored in numbered vials, resulting in a total of 43 fractions. CCD was performed with all obtained fractions and, once the fractionation process was finished, the fractions were grouped according to the 30:70 hexane / dichloromethane CCD profile, by qualitative similarity between the compounds visualized in the stationary phase:

PG, (F2-3). (F6-F9). (F4). (F1) = F0I to F0IO PG. (F2-3). (F6-F9). (F4). (F2) = F011

PG (F2-3). (F6-F9). (F4). (F3) = F012 to F018 PG. (F2-3). (F6-F9). (F4). (F4) = F019 to F021 PG (F2-3). (F6-F9). (F4). (F5) = F022 to F027 PG. (F2-3). (F6-F9). (F4). (F6) = F028 to F033 PG. (F2-3). (F6-F9). (F4). (F7) = F034 to F039 PG. (F2-3). (F6-F9). (F4). (F8) = F040 to F041 PG (F2-3). (F6-F9). (F4). (F9) = F042 to F043

GC-MS analysis of PG.F (2-3). (F6-F9). (F4). (F7) performed at the CPQBA-Unicamp Division of Organic and Pharmaceutical Chemistry showed that through the 4th fractionation, It is possible to separate the last group of compounds found in the active sample from the 3rd fraction, PG. (F2-3). (F6-F9). (F4), and leaving at tR = 40 to 50min. Figure 18 shows the chromatogram of the fraction PG.F (2-3). (F6-F9). (F4). (F7), and Figure 19 the

40 to 48min expanded chromatogram. The comparative analysis of the fragmentograms obtained for the 4

major compounds of the PG (F2-3). (F6-F9). (F4). (F7) chromatogram indicated that the 4 peaks refer to compounds of the same molecular weight (m / z 474) and same base ion (m / z 271, 100%), and are therefore isomers. Table 4 presents the retention times (tR) of the 4 major peaks, as well as their percentage representativeness in the evaluated sample. Table 4: Majority peaks found in the chromatogram of the fraction PG (F2-3). (F6-F9). (F4). (F7), by Psidium guajava.

Retention Time (tR) Percentage <%) 43,282 min 17.56% 44,351 min 24.02% 4: 688 ιτΐίϊη 45.46% 45 ~ 407 min 7.20%

Since such peaks together account for 94% of the sample, it was found that the fractionation processes were effective in purifying the initial crude extract, and at this point a well-enriched fraction of the active compounds was already in hand. interest. Thus, it was started for the chemical elucidation of the sample. For this purpose, we used various chemical techniques of structural elucidation, carried out in collaboration with the ThoMSon Mass Spectrometry laboratory, IQ - Unicamp. The results presented below refer to the data obtained through GC-Tof (Figure

19 and 20), 1 H-NMR (Figure 20) and optical rotation.

The analysis of the major peaks present in this sample by GC-Tof technique (more accurate than GC-MS) confirmed that these are isomeric molecules. Peak fragmentograms with tR = 12.75, tR = 13.60 and tR = 14.09 always indicate molecules with molecular weight m / z 474 and base ion

m / z 271, as exemplified in Figure 21.

The 1 H NMR spectrum obtained from the PG (F2-3). (F6-F9). (F4). (F7) fraction is shown in Figure 22. The optical rotation results indicate that the enriched fraction has [a] = 0.008, not being treated,

therefore a racemic mixture.

The joint analysis of the data obtained by the different techniques

Chemicals allowed us to perform a thorough search in the literature to check if there was any molecule described with the same characteristics as the one we found. The isolated molecule in the present project corresponds to that described by Yang et al. (2007), a meroterpenoid called guajadial (Figure 23), chemical formula C30H35O5, weight [M + H] + m / z 475,2484 and base ion m / z 271. Molecular weight m / z 475 (not m / z 474 ) as found for our analysis, reflects the technique used by the group of Yang et al. (2007), which adds an H + to molecular weight. Thus, it is the same molecule and the active principle with which it was worked was identified.

The meroterpenoid classification, in which the guajadial molecule fits, was first proposed by Cornforth (1968) as "mixed biogenesis compounds, containing fragments of terpenoid origin and also structures of different non-terpenoid biosynthetic origin". These compounds are commonly found in natural sources such as plants, microorganisms, and marine invertebrates and mostly consist of a quinone or hydroquinone structure bound to terpene moieties ranging from one to nine isoprenic units (Simon-Levert et al., 2010). . The pharmacological activity of marine meroterpenoids has been reported to inhibit certain enzymes (Shiomi et al., 1999) as well as terrestrial meroterpenoids with antiρroIiferative activity under cells.

tumors (Marion et al., 2006).

In conclusion, we have the graph for the active fraction PG. (F2-F3). (F6-F9). (F4), from the 3rd fraction shown in Figure 16 and the TGI (Total Growth Inhibition) values for the sample. guava activity against each of the human tumor cell lines from the cell culture panel broken down in Table 3 clearly demonstrates the increased EBQD activity for the enriched fraction PG (F2-F3). (F6-F9). (F4) , fact explained due to the enrichment of the tested sample in terms of active principle. Thus, it is concluded that biofractionation was effective in the separation of active compounds, and this was reflected in an increase in antiproliferative activity against tumor lines. The further enriched PG. (F2-F3). (F6-F9). (F4). (F7) fraction was isolated and then analyzed by GC-MS (Figure 18 and 19), GC-TOF ( 20 and 21) and 1 H-NMR (Figure 22). These analyzes allowed us to conclude that this sample is composed of 94% of three isomeric molecules, of molecular weight m / z 474 and base ion in m / z 271, whose bibliographic survey made it possible to identify the molecule as "guajadial", extracted and described. by Yang et al, 2007 (Figure 22), but to which no biological activity has been attributed.

However, it is noteworthy that the sample PG. (F2-F3). (F6-F9). (F4). (F7) still has 2 isomers, whose chemical elucidation is in progress. Thus, the present technology highlights the evaluated biological activity for a molecule that had not previously been described with (guajadial) activity, as well as proposing the discovery of two more isomeric molecules whose chemical structure has not yet been elucidated. Molecules have great potential as an active ingredient in medicinal plants for the development of a possible new chemotherapeutic drug.

In addition to the phytochemical purification process and validation of its in vitro pharmacological activity, this potential therapeutic activity was evaluated in vivo. For this evaluation we used the murine Ehrlich Solid Tumor model, first described by Paul Ehrlich in 1906. It is a transplantable mouse neoplasm originating from a

breast adenocarcinoma (Ehrlich, 1906).

The ascitic or solid forms of its growth occur according to the place of its inoculation (Wainstein, 1999). When inoculated into the abdominal cavity, tumor cells grow in their ascitic form, developing a peritoneal carcinomatosis, with large accumulation of fluid in the abdomen (ascites). Ehrlich's tumor in solid form, in turn, presents as a palpable mass and firm consistency, being described morphologically by the presence of cells with high degree of atypias (anaplasia), characterized by numerous and evident nucleoli, condensed chromatin, atypical or aberrant mitoses and greater nucleus / cytoplasm ratio than normal cells (Dagli, 1989). One of the advantages of using this model is that it is possible to standardize the number of cells to be inoculated into animals for both ascitic and solid growth, as cells present in ascites fluid can be easily

(Matsuzaki, 2004).

Ehrlich's tumor was maintained in ascites form by

weekly ticks on Balb / c mice. Weekly, ascitic fluid was taken from animals bearing this tumor and immediately inoculated into the peritoneal cavity of recipient animals of the same lineage to subsequently act as donor animals from Ehrlich cells.

------- 10 "inoculated in solid form. After inoculation, recipient animals were

kept in individual isolators properly identified with water and ration ad libitum.

The Ehrlich Solid Tumor test lasted 21 days, with 7 animals (female Balb / C mice) per group, at doses previously determined from toxicity tests. A total of 2.5 χ 106 Ehrlich Tumor cells were inoculated into the right hind footpad of the mouse. The number of tumor cells for solid form inoculation was obtained by direct counting of cells obtained from ascites fluid in the Neubauer chamber. Treatment was given intraperitoneally every 3 days from the 3rd day after cell inoculation.

A 0.9% saline group was used as the negative control (G1), a 0.25 mg / kg doxorubicin group was the positive control (G2), and 3 other groups were treated with the enriched guava fraction PG. (F2- (F6-F9) at the doses of 10, 30 and 50mg / kg (G3, G4 and G5, respectively). On the day of cell inoculation (OD), each day of treatment (D3, D6, D9, D12, D15 and D18) and on the day of sacrifice (D21), animal weight and tumor volume (with Digital Plethysmometer, WE7500, Panlab), as well as the survival rate of animals / group. At the end of the experiment, the animals were sacrificed for cervical dislocation and the organs will be collected (brain, stomach, liver, kidney, heart, lung, spleen, uterus) as well as the paws affected by the tumor for histopathological analysis (more detailed in item 3). From the parameters evaluated during the 21 days of testing, the following assessments were made: tumor volume / day, gross tumor weight (animal weight / day), relative tumor weight (tumor weight / animal weight). Weight variation and differences in tumor size were statistically analyzed using the Duncan Test (p <0.05). Figure 25 is a schematic of the experimental model followed, with the day of commencement of the test (OD), days of treatment (D3, D6, D9, D12, D15 and D18) and the day of sacrifice.

(D21).

At the end of the experiment, the following assessments were made: tumor volume / day (Figure 23), animal weight / day (Figure 24), gross weight and relative tumor weight (Figure 25),% tumor inhibition (Table 5) . Statistical analysis of each of these parameters was performed using the

Duncan (p <0.05).

According to the results obtained by the analysis of the graph of

In tumor volume (Figure 26), tumor growth inhibition was observed for the groups receiving guava at the 3 doses (10, 30, 50mg / kg), as well as for the group treated with doxorubicin chemotherapy (positive control) when compared to saline treated group (negative control). There was no statistical difference between the groups treated with guava in the 3 doses, nor this one regarding the positive control group at the end of the experiment (D21). The antiproliferative activity of doxorubicin chemotherapy in relation to the saline group occurs more effectively from day 9, maintaining the control of tumor development until the end of the experiment. The tumor was aggressive in its growth, with a rapid increase in size between the days of treatment, especially from day D12, where even the chemotherapy is not able to contain the increase in cancer.

Although the acute toxicity test (presented in the student's final IC project activity report, Fapesp Case 08 / 50026-8) did not show dramatic signs of toxicity, such an effect may be apparent when the sample is administered at repeated doses, as in this long-term experiment and multiple dose treatment. The animals 10

treated with 30 and 50mg / kg guava showed drastic weight loss until the D12 day of the experiment, with subsequent gradual weight gain from that day. This gradual weight gain was not accompanied by doxorubicin-treated animals in which weight loss was continuous, leading to

animals to a state of cachexia.

Figure 24 represents the evolution of weight gain and weight loss.

animals during the experiment. There was no weight variation in the 10 mg / kg guava group, which presented a similar profile to the negative control (saline). The higher dose groups (guava 30 and 50mg / kg) proved to be toxic, "showing considerable variation in body weight. The toxicity of chemotherapeutic agents is somewhat expected, since they act on cells in cell division, also affecting cells. in this case, and the criterion used in the clinic, is the risk-benefit assessment, verifying if the tumor reduction justifies the

any signs of toxicity observed.

Tumor weight analysis, assessed at D21 by the difference between

the tumoral and non-tumoral paw indicates that the guava sample at the 3 doses tested (10, 30 and 50mg / kg) was able to significantly contain tumor growth. However, relative tumor weight assessment seems to

more appropriate than gross weight, since relative weight is calculated by the ratio of tumor weight to animal weight, and therefore takes into account the drastic weight loss experienced by doxorubicin-treated animals, thus resulting in a more consistent assessment. as observed. Figure 25 represents, respectively, the gross and relative weight of the tumor.

The percentage of tumor inhibition assessed at the end of

The experiment (D21) was calculated by the following relation: (V tumor treatment group - V saline) / V saline. Table 5 presents the tumor reduction percentage of guava-treated animals in relation to the negative control group (saline).

Table 5: Percent tumor reduction for treated animals

with guava compared to those treated with negative control (saline). Growth inhibition (%) Guava 10 mg / kg 20; 19% ± 6.46 Guava 3Gm g / kg 22.12% ± 10.66 Guava SOnntg / kg 31.94% ± 1 4.09

According to the results obtained by analyzing the tumor volume graph (Figure 23) and calculating the percentage of tumor reduction. (Table 5), tumor growth inhibition was observed for the guava-treated groups at 3 doses, with inhibition of 20.19% of the tumor in the 10mg / kg-treated group, 22.12% in the 30mg / kg-treated animals and of 31.94% in the group treated with 50mg / kg guava. The higher percentage of tumor reduction presented by the animals of the highest guava dose does not justify the drastic weight loss suffered by the animals of the group and the signs of toxicity observed in the peritoneal organs. Moreover, as shown in the tumor volume graph, the three doses (10, 30 and 50 mg / kg) do not differ statistically, thus justifying the use of the lowest dose of the sample to

obtaining the same effect.

From the organs collected (brain, stomach, liver, kidney, heart,

lung, spleen, uterus) at the end of the experiment, none of them except the uterus showed changes in morphology, weight or macroscopic appearance in guava-treated animals 10 or 30mg / kg. The animals treated with the highest guava dose (50mg / kg) showed signs of toxicity, with adherence of the peritoneal organs. This finding, coupled with the observed weight loss, confirm the toxicity of the sample at this higher dose, corroborating the use of lower doses, since they had the same statistical effect. The enriched guava fraction tested, PG. (F2-F3). (F6-F9), showed satisfactory tumor inhibition result. However, it would be interesting to evaluate purer samples or, ideally, the active principle (guajadial or the mixture of isomers thereof), in order to verify if the anticancer activity is amplified and the signs of toxicity reduced.

The liver and kidneys are important parameters for toxicological evaluation of new potential drugs, as they are responsible, respectively, for metabolism and filtration in the body. There was no evidence of hepatic and renal toxicity in guava 10 and 30mg / kg groups, which presented statistically equal weight to animals treated with saline (vehicle), and whose histopathological examination showed no necrosis or

other possible morphological changes. The uterus, in turn, presented

relevant size increase, visually and macroscopically noticeable

discussed in detail below.

Additionally, histological sections of the tumors were performed.

Ehrlich solid removed from right hind paws of mice

Balb / C and the uterus, an organ that has been shown to increase in size. The

materials were fixed in 4% buffered paraformaldehyde for 24 hours, including

paraffin, sectioned to 5 μηη thickness and stained with hematoxylin-

eosin.

The histolopathology of the cross sections of Ehrlich Solid Tumor presents as the most striking feature the presence of large areas of necrosis, superficial and deep, interspersed with also extensive areas of high cell proliferative neoplastic cells. Tumor cells inoculated in the plantar cushion showed intense local proliferation, invading and adjacent tissues, such as the connective and the musculature. Poorly differentiated rounded and bulky tumor cells were observed in all groups, with evident cellular pleomorphism, nuclear hyperchromatism and irregularly distributed chromatin. Several mitotic figures can be visualized, as well as foci of inflammatory infiltrates. There are cells in the process of apoptosis, located scattered and punctually. The stroma is quite scarce, with little vascularization. The proliferation of neoplastic cells (marked by the reaction of PCNA) was widely diffused and widespread, not showing preponderance by certain region of the tumor (central or peripheral). The proliferating areas are characterized by being very large, without clear delimitation, and embedded in the areas of necrosis (discussed below), and several easily characterizable mitotic figures are also observed. The tumor epithelium has very marked cells, which was already expected for this region due to its constant renewal.

The areas of necrosis (marked by TUNEL reaction in situ), as well as the areas of cell proliferation, were very extensive, with no definite delimitation between them. Both regions are intrinsically embedded, tending to larger necrotic areas in the central regions of the tumor. The presence of necrosis is most likely related to lack of oxygen (Assis, 2007) and other nutritional factors, especially in the central region of the tumor. Tumor cells have the ability to produce growth factors that stimulate angiogenesis, and when the growth rate is very high and the production of these factors is not proportional to the need, the tumor cells suffer from hypoxia and die from necrosis (Montenegro & Franco, 1999). The central regions of the tumor are more subject to this nutritional and oxygen deficiency, since they are located farther from the blood vessels that

nourish the tumor.

The most evident change at the end of the experiment when the organs were collected and evaluated for obvious signs of toxicity was that presented by the uterus, notably increased in size in guava-treated animals. This increase was clear macroscopically for the three doses of guava (10, 30 and 50mg / kg). Figure 26 shows the weight of the uterus presented by the groups (negative, positive control and the three guava doses) at the end of the experiment. With the analogous reasoning to the weight of the tumor, the assessment of relative weight of the uterus seems more appropriate also for this parameter, O 26/29

since it takes into account the weight of the animals at the end of the experiment.

The increase in the size of the uterus observed at the end of the in vivo experiment was an unexpected finding, but it opened several possibilities for bibliographic survey and hypothesis about the action of the guava sample. Similar to the one performed for the Ehrlich tumor paw slides, histological sections at 5pm were stained with hematoxylin-eosin from the uteri of the animals used.

during the experiment. _______ _____ ______ ~

------ ---- The general histology of the uterus reveals its classical structure formed

of lining epithelium, endometrium with secretory glands and myometrium.

Analysis of the uterus HE slides allowed us to conclude that the increase in

for this organ in guava-treated animals is due to

changes in the morphology of the lining epithelium, whose cells

were higher, with increased epithelial surface and

hyperplasia. Normal (non-neoplastic) mitotic figures are also

Gifts The wombs of the doxorubicin-treated group, in turn,

atrophy of the epithelium, a clear reflection of the weakened condition of the

animals due to the chemotherapy effect, which also presented significant

weight loss.

Guava-treated animals, despite having the tumor

Ehrlich solid, presented this significant enlargement of the uterus, a curious fact, since the tumor itself already weakens the animal, being common to observe a general atrophy of the organs due to the state of the animal before the neoplasia. The influence on the uterus, a hormone dependent organ, suggests the action of guava as phytohormonium, possibly with an estrogen-Iike effect. Growth inhibition of Ehrlich Solid Tumor, a neoplasm that has its histopathological origin in breast adenocarcinoma, suggests that this neoplasm may retain hormone-dependent characteristics. The hypothesis that some hormones regulate the incidence of

Neoplasms was first postulated by Bitter et al (1948), but there is still controversy about the genesis of hormone-dependent neoplasms. Hormones induce cell proliferation with consequent genetic mutations that will give rise to the neoplastic cell (Meuten, 2002). Carreno et al (1999), however, states that the participation of hormones in carcinogenesis is restricted to the proliferation of cells already transformed by other carcinogens. Hormone-dependent neoplasms include breast, uterus, ovary, testis, prostate, thyroid and osteosarcoma neoplasms. These neoplasias share the same mechanism of carcionogenesis, but under the action of specific hormones (Henderson et al, 1993; Henderson & Feigelson1 2000). Breast neoplasms are of special interest to researchers because of the similarities to human breast cancer, undergoing strict regulation of estrogen, prolactin (Deng & Brodie, 2001), progesterone (Vorherr, 1987), androgens (Kodama & Kodama, 1970). ) and even thyroid hormones (Nogueira & Brentani, 1996).

Although the use of hormones and antihormonal drugs is still unrepresentative in the treatment of hormone-dependent neoplasms in animals (Morris et al, 1993), such therapy has shown good results in the treatment of some human hormone-dependent neoplasms. Inhibition of growth of Ehrlich Solid Tumor by guava treatment, coupled with growth observed in the womb, suggests that the sample has a similar action to tamoxifen, a chemotherapy clinically used against breast and ovarian cancer, and also has this effect. combat breast cancer while stimulating the proliferation of breast

uterus.

Tamoxifen is an antiestrogenic drug used in menopausal women to prevent breast cancer recurrences and metastases (Kostoglou-Athanassiou et al, 1998). This drug suppresses myoepithelial cell proliferation in estrogen receptor neoplasms (Shao et al, 2000). The mechanism of action of tamoxifen as well as that of other drugs with antiestrogenic action is not completely elucidated, but it is believed to be related to the reduction in the number of estrogen receptors, with the stimulation of compressors (TGFp) 1 with inhibition of co-activators (IGF and TGFÎ ±) and rapid estrogen inactivation (Norman & Litwack1 1997; Ingvarsson1 2001). This hormone, in turn, promotes cell growth by stimulating the release of tumor growth factor α and by inhibiting the fact of tumor growth β (Normann & Litwack, 1997). Tamoxifen action (in combination with epirubicin) has been reported by Õzcan Arican et al. (2005), attesting a reduction in the proliferation

of Ehrlich's Ascitic Tumor.

Despite the clinically proven results of tamoxifen observed for breast cancer patients, the highest risk of treatment refers to concomitant stimulation of the uterus, in the most severe cases, possibly causing endometrial cancer. Endometrial cancer stems from exposure of the endometrium to cumulative estrogen action (Vorherr, 1987), which even at low concentrations induces hyperplasia.

(Henderson et al., 1993).

The inhibitory effect of the guava sample on the Solid Tumor of

Ehrlich, assuming that it still has hormone-dependent characteristics, coupled with the observed increase in uterus size, can be explained by the stimulating effect of guava on estradiol and progesterone levels in female rats, reported by Uboh et al. (2010). Tests conducted by this research group found increased estradiol and progesterone levels in female rats treated with aqueous guava extract as well as testosterone in males. Thus, it is strongly suggested that this plant species present stimulatory activity on the reproductive system and may also be indicated for organisms with hormonal disorders.

Although the administration of hormones and anti-hormonal drugs has good results, their use requires the evaluation of risks and benefits (Henderson et al., 1993). In general, its use seems to cause lower intensity and severity side effects when compared to chemotherapy drugs (da Silva et al., 2004). Thus, a larger number of tests are needed to validate the efficacy and / or risk of treatment with such drugs. Thus, knowing the expression of hormone receptors in neoplasms of animal and human models can help significantly in the search for the best treatment.

Thus, it is concluded that the active fractions of P. guajava, enriched in the guajadial active principle and its isomers, have promising anticancer activity both in vitro and in vivo. This finding, coupled with the fact that the sample did not show toxic effects of weight reduction at a dose of 10mg / kg, but nonetheless presented significant tumor reduction, ^ is of relevant importance and opens great possibilities of industrial applicability regarding the production of new chemotherapy drugs,

even justifying the use of reduced doses.

The guajadial molecule (m / z = 474) obtained from the plant species Psidium guajava L. can be added to pharmaceutically acceptable excipients for an anticancer pharmaceutical composition.

Claims (8)

1. Method for obtaining the guajadial molecule and its isomers, comprising the following steps: • Selection of the plant species Psidium guajava L .; • Obtaining dichloromethane hot crude extract (EBQD) from Psidium guajava L .; • Fractionation of Psidium guajava L. Dichloromethane hot crude extract (EBQD); • Identification and characterization of the enriched fraction PG (F2-3) (F6-F9). (F4). (F7). Method for obtaining the guajadial molecule according to Claim 1, characterized in that the dichloromethane hot crude extract (EBQD) of Psidium guajava L is obtained as follows: the leaves were dried and ground, the moisture content the plant species was in the range of 50 to 60%, which was calculated by drying the leaves in a greenhouse at a temperature between 100 and 110 ° C, preferably 105 ° C for 22 to 26 h, preferably 24 h, until brittle; the dried and ground plant material of P. guajava leaves was hot extracted by the Soxhleti method at a 1: 5 ratio with dichloromethane (CH 2 Cl 2) and 90% ethanol (EtOH), preferably 95% (Figure 1); crude dichloromethane extract (EBD), extracted for 65 to 75h, preferably 72h, was collected and rotary evaporated under vacuum at a temperature between 35 and 45 ° C, preferably 40 ° C until complete solvent removal; The vegetable residues of this extraction process were used for the 90 to 99% ethanol, preferably 95% ethanol extraction process, in the same proportion for 65 to 75h, preferably 72h; The crude ethanolic extract (EBE) was lyophilized after the rotary evaporation process. Method for obtaining the guajadial molecule and its isomers according to claims 1 and 2, characterized in that the fractionation step of the dichloromethane hot crude extract (EBQD) takes place in four stages, as follows: • 1st stage: Filter Column Fraction • 2nd Stage: Classic Column Fraction • 3rd Stage: Classic Column Fraction • Step: _ Filter Column Fractionation until the fraction enriched with the active ingredient of interest (guajadial and its isomers) is obtained. Method for obtaining the guajadial molecule according to claims 1 to 3, characterized in that the step Identifying and characterizing the enriched fraction PG. (F2-3). (F6-F9). (F4). (F7 ) are GC-MS, GC-Tof, NMR-1H1 and optical rotation, using the following conditions: • GC-MS (Gas Chromatography Mass Spectrometry): samples were analyzed under AA Series conditions (injector: 240 at 260 ° C preferably 250 ° C, detector: 290 to 310 ° C preferably 300 ° C HP-5MS column: 1100 ° C - 280 ° C raising the temperature by 5 ° C / min, remaining at 280 ° C for 20 to 30 minutes , preferably 26 minutes, run time between 50 minutes and 1 hour and 10 minutes, preferably 1h, He carrier gas, 1 ml / min carrier flow), with a 5% Phenyl Methyl Siloxane HP-5MS Column (30cm χ 0, 25mm χ 0.25pm). • GC-Tof (Time of Light Mass Spectrometry): samples were analyzed with an HP-5MS column (30cm χ 0.25mm χ 0.25μιτι), injection volume 1μΙ, ti = 1min, 220 ° C, temperature between 3 ° C ° C and 300 ° C. • NMR-1H (Nuclear Magnetic Resonance): was performed with the sample diluted in deuterated chloroform. • Optical rotation: was performed by Perkin-Elmer equipment, Polarimeter 341, at a concentration of 2.2700g / 100ml. Use of the guajadial molecule obtained by the method described in claims 1 to 4 for producing a pharmaceutical composition for the treatment of cancer. Compound characterized in that it is obtained by the method described in claims 1 to 4. Pharmaceutical composition comprising the guajadial molecule and its pharmaceutically acceptable isomers and excipients. Pharmaceutical composition according to Claim 1, characterized in that they are arranged in the selected forms of: capsules, pills, tablets, dragees, paste, or liquid.