TERPENES OF EUPHORBIA UMBELLATA LATEX ARE ......2021/01/21 · obtained from the hexane extract of...

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26

Andrade et al. World Journal of Pharmacy and Pharmaceutical Sciences

TERPENES OF EUPHORBIA UMBELLATA LATEX ARE INVOLVED

IN CYTOTOXIC EFFECT AGAINST MELANOMA CELLS

Evelyn Assis de Andrade1*, Luiza Stolz Cruz

1, Isadora Machinski

1, Ana Carolina Terso

Ventura1, Bruna Carletto

1, Kátia Sabrina Paludo

2, Leandro Cavalcante Lipinski

3,

Eduardo César Meurer4 and Flávio Luís Beltrame

1

1Department of Pharmaceutical Science, State University of Ponta Grossa, Ponta Grossa, PR,

Brazil.

2Department of Structural Biology, Molecular and Genetics, State University of Ponta

Grossa, Ponta Grossa, PR, Brazil.

3Department of Medicine, State University of Ponta Grossa, Ponta Grossa, PR, Brazil.

4Laboratory Fenn of Mass Spectrometry, Federal University of Paraná, Jandaia do

Sul, PR, Brazil.

ABSTRACT

Diluted latex of Euphorbia umbellata is empirically used to treat

several diseases, including different types of cancer, in southern Brazil.

This study aimed to evaluate the in vitro cytotoxicity of the fractions

obtained from the hexane extract of E. umbellata latex; to evaluate the

in vivo anticancer action of the most active fraction; and to correlate

this effect with the chemical composition. The hexane extract and its

fractions were tested using cytotoxic assays (MTT and neutral red)

against the B16F10 cell line; the IC50 and selectivity index were

determined for these samples. The most active in vitro sample of the

latex was evaluated using an in vivo anticancer assay. LC-MS/MS

analysis was performed to qualify and estimate the presence of the

phorbol esters in the latex samples. The dichloromethane fraction presented the highest

toxicity against the B16F10 cell line; the partioning process increased the cytotoxic effect and

the selectivity index of the fraction obtained from the hexane extract (IC50 = 2.82 ± 0.88

µg/mL, SI = 3.4 and IC50 = 18.02 ± 1.07 µg/mL, SI = 1.1, respectively). The dichloromethane

fraction did not show a significant difference regarding the volume of the tumour in relation

to the tested concentrations and posology compared to the negative control. The LC-MS/MS

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 10, Issue 4, 26-37 Research Article ISSN 2278 – 4357

*Corresponding Author

Evelyn Assis de Andrade

Department of

Pharmaceutical Science,

State University of Ponta

Grossa, Ponta Grossa, PR,

Brazil.

Article Received on

21 Jan. 2021,

Revised on 11 Feb. 2021,

Accepted on 03 March 2021

DOI: 10.20959/wjpps20214-18624


27


analysis made it possible to identify phorbol esters, mainly in the dichloromethane and

ethanol fractions. The ethnopharmacological use of E. umbellata latex to treat cancer may be

related to the presence of terpenes in the phytocomplex of the vegetal matrix.

KEYWORDS: Skin, diterpenes, janaúba, antitumour, phorbol esters.

INTRODUCTION

Melanoma is a type of cancer that originates in transformed melanocytes. It represents 10%

of skin cancer in Brazil and is considered the most lethal type (responsible for up to 70% of

deaths from skin cancer), mainly due to its high probability of metastasis.[1,2]

As the incidence of melanoma continues to increase - mainly due to resistance to existing

pharmacological treatments - the understanding and application of new approaches becomes

ever more important in the clinical context.[2,3]

Phytopharmaceuticals have been the mainstay of cancer chemotherapy for several years and

more than 3,000 plants worldwide have been reported due to their cytotoxic/anticancer

properties. However, in practice, a much larger number of unofficial natural products, mainly

plants, are known and are used due to their ethnopharmacological knowledge. This popular

use has contributed to the discovery of more than 70% of the drugs used for the treatment of

cancer.[4]

Euphorbia umbellata (Pax) Bruyns belongs to the Euphorbiaceae family and its latex is

popularly used in Brazil as a homemade preparation (garrafada), which is obtained by

diluting 18 drops of latex in 1 litre of water (the recommended dose is 1 cup (30 mL) 3 times

per day). This preparation is widely administered orally for the treatment of various diseases,

and particularly to treat different types of cancer.[5,6]

The concentrated latex and its diluted form (identical to popular usage) have already been

evaluated against a melanoma cell line (B16F10), presenting in vitro cytotoxic activity and

demonstrating a significant reduction in the tumour mass of melanoma in mice.[5]

This

cytotoxic effect was related to the presence of terpenes, mainly triterpenes.[6-8]

However, the diterpenes of the tiglian nucleus, such as esters of phorbol, are considered the

chemical markers in the Euphorbiaceae family and they are also related to cytotoxic

activity.[9,10]


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Considering that this latex previously presented interesting anticancer results, this study

aimed to evaluate the fractionated samples of the latex of E. umbellata against a melanoma

cell line to correlate the biological activity with chemical compounds present in the latex

phytocomplex of this vegetal species.

MATERIALS AND METHODS

Plant material

Latex from E. umbellata (synonyms: Euphorbia pseudograntii Bruyns; Synadenium grantii

Hook.f.; Synadenium umbellatum Pax; and Synadenium umbellatum var. puberulum

N.E.Br.),[11]

which is popularly known as janaúba and African milk bush, was collected in

the city of Ponta Grossa, Brazil (25° 4'24” S 50° 9'53” W) between October and December of

2018, through incisions in the petioles and stem of the species. The material was stored under

refrigeration (5 ºC) until the moment of use. A voucher was registered at Maria Eneida P.

Kauffamnn Fidalgo Herbarium (No. 453920).

Extraction of plant material

The latex (11.3 g) was adsorbed on silica and placed in Soxhlet equipment. The vegetal

matrix was subsequently extracted for 5 h with 200 mL of each solvent (hexane, chloroform,

ethyl acetate and methanol, which were all added subsequently). The hexane extract (HE, 1.5

g) was subjected to fractionation using the same methodology described above; however, the

solvents used were dichloromethane, ethanol and methanol. The solvents were removed

under vacuum at 40 °C and the dried samples were stored under refrigeration (5 ºC) until the

moment of use.

In vitro evaluation

Cell cultures

Murine melanoma cells (B16F10, code No. 0342) and murine fibroblast cells (3T3, code No.

0017) were obtained from the Rio de Janeiro Cell Bank, Brazil. The cell cultures were

maintained by continuous expansion in complete culture medium (RPMI®

1640 medium (pH

= 7.4), which was supplemented with 10% fetal bovine serum (FBS), 100 μg/L streptomycin

and 100 IU/L penicillin and maintained at 37 ºC and 5% CO2.

MTT assay

Medium containing 3x103 cells/well was seeded in 96-well plates. Subsequently, the medium

was removed and replaced with a complete culture medium containing different


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concentrations (0.2-100 µg/mL) of the different samples (hexane extract and fractions). After

48 h the medium was replaced with the MTT reagent (0.5 mg/mL). The plates were incubated

at 37 ºC for 1-2 h. The formazan crystals were solubilised in dimethyl sulfoxide (DMSO, 100

µL) and the absorbance was determined at 570 nm. Negative control was performed using the

medium, and for the sample’s medium with 0.1% of DMSO was used. The blank was

obtained by performing the MTT technique in a well without cells. All the samples were

tested in triplicate and the tests were repeated 2-4 times. The data obtained were transformed

into % of cell viability according to Equation:

% viability = (absorbance of the treated well - absorbance of the blank/absorbance of the

mean of the negative control wells - absorbance of the blank) x 100.

Neutral red assay

The cells were seeded (3x103 cells/well) in 96-well plates and incubated for 24 h. After this

period, the medium was removed and replaced with complete culture medium containing the

samples (hexane extract and fractions) in different concentrations (0.2-100 µg/mL) and

incubated for 48 h. Subsequently, the wells were washed with RPMI®

, and 200 μL of neutral

red solution (40 µg/mL of neutral red in RPMI®

) was added. The plates were incubated for 1-

2 h at 37 ºC. The neutral red solution was subsequently removed, and the cells were fixed

(0.5% formaldehyde and 1% CaCl2 in PBS) for 1 min, followed by dilution with 200 μL of

neutral red solubilising reagent (50% ethanol and 1% acetic acid in PBS). The absorbance

was determined at 520 nm using a UV/Vis spectrophotometer. The blank was obtained by

performing the NR technique in a well without cells. The samples were tested in triplicate

and the tests were repeated 2-4 times. The controls were performed in the same way as

described for the MTT experiment, as was the calculation of % of cell viability.

IC50 and selectivity index (SI)

The cell viability results obtained in the neutral red assays were used to estimate the IC50 of

the treated cells. These were calculated as the % viability/logconc ratio by non-linear

regression using GraphPad Prism® (version 6.01) software. The IC50 values were used to

calculate the SI from the ratio between the IC50 values calculated for non-tumorous cells

(3T3) and the IC50 calculated for tumorous cells (B16F10).

In vivo evaluation

The experimental protocol was approved by the Committee of Animal Use and Ethics of the

State University of Ponta Grossa, under registration number 06476/2019. The animals (36


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male C57BL/6 mice, weighing approximately 25 g and 60 days old) were kept at the

Advanced Centre for Life Studies at the State University of Ponta Grossa, with free access to

water and feed. The B16F10 cells were cultured as described above; an amount of 1x105 cells

were inoculated in the dorsal region of the C57BL/6 mice [12]

. After 10 days the animals were

randomly divided into the following three experimental groups: the control group (vehicle,

suspension of saline + DMSO (0.1%); group 1 (1 mg of dichloromethane fraction (DIF) per

kg of the animal/day); and group 2 (10 mg of DIF per kg of the animal/day). The treatment

was carried out once a day, in the morning, via gavage (100 μL/animal) for 15 days. The

animals were then sacrificed to remove the tumours and calculate their volume according to

Equation:

Tumour volume (mm3) = longitudinal measurement (mm) x (transverse measurement)

2 (mm)

x 0.52

LC-MS/MS analysis

A C18 column (Acquity BEH HSS T3 (1.8 μm, 2.1 x 50 mm)) and a gradient elution

condition with methanol:formic acid (99.9: 0.1, v/v, phase B) in water:formic acid (99.9: 0.1,

v/v, phase A) were used with the following steps: 50% to 75% B for 2.0 min; increased from

75% to 90% of B in 2.4 min; maintained at 90% B for 2.0 min; increased to 100% B for 1.0

min; an isocratic run was maintained at 100% B for 1 min and a reverse gradient applied to

50% B (0.01 min). An isocratic condition was maintained for 4 minutes before each new

injection. The flow rate was 0.3 mL/min., and the injection volume was 5 μL. The hexane

extract and fractions were dried under nitrogen flow and then resuspended in acetonitrile (1

mg/mL) and filtered (0.22 µm) before injection. Ionisation was carried out using an

electrospray ionisation (ESI) source in a positive mode. The following probe conditions were

used: source temperature of 150 °C; capillary of 2.5 KV; cone of 25 V; collision energy of 15

eV; desolvation gas flow of 700 L/h; and desolvation temperature of 500 °C. The mass

spectrometric MRM analysis used the following monitoring: m/z 539.26 → 311; 545.64 →

311; 617.40 → 311; 669.43 → 311 and 311 → 293. Tetradecanoyl phorbol acetate (TPA)

was used as positive control and to prepare a calibration curve by plotting the peak area of the

phorbol fragment in relation to the concentration of the compound. The TPA (27.6 mg) was

precisely weighed and dissolved in 6 mL of methanol (stock solution of 4.6 mg/mL).

Aliquots of the stock solution were pipetted and made up to 2 mL in volume to obtain

methanol calibration solutions (230-4,600 µg/mL). The calibration curve solutions were

prepared in triplicate.


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Statistical analysis

The data were analysed using GraphPad Prism® software (version 6.01) and expressed as

mean ± standard error of the mean, with a significance level of 5% (p ≤ 0.05). For the in vitro

and in vivo tests, the results obtained were subjected to the Shapiro-Wilk and Bartlett

normality tests. When the results were parametric, they were evaluated by analysis of

variance (one-way ANOVA) followed by Tukey's post-test. When the results were non-

parametric, they were analysed by the Kruskal-Wallis and Dunn's post-hoc tests.

RESULTS AND DISCUSSION

The use of solvent in the extraction of plants (or part of their vegetal material) is intended to

fractionate different metabolites present in these matrices. It is necessary to carry out a

separation procedure using different solvents capable of segregating the compounds of

interest, based on their solubility and partition coefficient. This procedure can help to

decrease the chemical complexity of the precursor material and to facilitate the identification

of the compounds responsible for the biological effect.[13,14]

Previous studies have found that samples obtained by polarity-based fractionation

demonstrated more successful cytotoxic effects in different types of cells.[7,15]

This may be

because the fractionation process specifically uses solvents to extract the bioactive

compounds, which supports the higher effectiveness of enriched fractions in comparison to

the extracts. It could also be due to an increase in the concentrations of the

phytoconstituents.[16-18]

The latex and its diluted form (garrafada) of E. umbellata have already demonstrated

anticancer activity against the B16F10 cell line, reducing the tumour size of the melanoma in

mice.[5]

Consequently, fractionation of the latex was performed, followed by the partitioning

of the hexane extract - the most active extract of E. umbellata latex.[8]

The three fractions

obtained from this extract were initially tested against the B16F10 cell line using in vitro

cytotoxicity assays.

The dichloromethane fraction (DIF) presented higher cytotoxicity than the hexane extract

(HE) against B16F10 cells (Figure 1), reinforcing the theory that non-polar compounds

present in these samples, and initially in the latex, could be related to the studied activity.[17]


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Figure 1: Effect of hexane extract (HE) and dichloromethane fraction (DIF) on cell

viability of B16F10 cell line (neutral red assay). Data are expressed as mean ± standard

error of mean of triplicates.

However, an increase in the cytotoxic effect against cancer cells does not simultaneously lead

to an increase in the cytotoxic effect against normal cells [19]

, so the selectivity index (SI) was

determined. The DIF presented an increase in the SI value when compared with the precursor

extract (HE) (Table 1).

Table 1: IC50 values (μg/mL) and selectivity index (SI) of E. umbellata hexane extracts

(HE) and dichloromethane fraction (DIF) using neutral red assay.

B16F10 3T3 SI

HE 18.02 ± 1.07 19.49 ± 2.08 1.1

DIF 2.82 ± 0.88 9.60 ± 0.13 3.4

Values represent the mean ± standard error of the mean of triplicates.

For the tested fractions, the DIF showed a decrease in cell viability as the tested

concentrations were increased. Furthermore, cell viability was below 50% in concentrations

above 50 μg/mL (MTT assay, data not shown).

SI values greater than 2 suggest that the sample presents a safe profile with antitumour

potential,[20]

therefore, the DIF demonstrated the ability be a good candidate to be evaluated

in an in vivo anticancer studies. In addition, previous results have shown that DIFs have

presented interesting data against other different tumor cell lines.[7]


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After 15 days treating the animals once a day via gavage, different macroscopic aspects were

observed in the tumours. They had a gelatinous aspect, disintegrated easily, and had

yellowish colouration, characterising jaundice (group 1, data not shown). However, the

volumes of the tumours in the three groups did not present significant statistical differences

(Figure 2).

Figure 2: Tumour volume after 15 days of treatment with dichloromethane fraction.

CG = control group; G1 = group 1; G2 = group 2. (n=4).

The present study also observed the presence of black spots in the spleen, lymph nodes and

liver, suggesting the process of metastases in all the tested groups.[5]

It can be suggested that

the absence of differences in tumour volumes may have been related to the posology used in

this experiment, which was different to the posology that is commonly used, and which was

previously described,[5]

and which observed a reduction in tumor size.

To correlate the chemical compounds, present in the DIF and other non-polar samples, a

chemical evaluation using liquid chromatography–mass spectrometry (LC-MS/MS) was

performed. The MRM analysis enabled us to propose a tentative identification of some

phorbol structures in the E. umbellata latex hexane extract and the fractions (Figure 3).

Phorbol esters have unusual structural characteristics, which suffer neutral loss of mono-, di-

or tri-esters through different fragmentation mechanisms. These produce the m/z 311 ion,


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which is related to the basic phorbol skeleton, and the m/z 293 ion, which is related to the loss

of a water molecule due to the presence of hydroxyl groups in the phorbol structure.[21,22]

Figure 3: (A) LC-MS/MS of the DIF of Euphorbia umbellata latex. (B) Proposed

fragmentation behaviour of phorbol esters: m/z 539.26 → 311; m/z 545.4 → 311; m/z

617.8 → 311; m/z 669.47 → 311.

Other authors observed that the fractionation process of hexane extracts could increase the

concentration of terpenes into the fractions [7]

. This was confirmed in the present study,

which identified the presence of diterpenes (phorbol esters), mainly in the DIF (Table 2).


35


Table 2: Qualification of phorbol ester strutuctures in hexane extract and fractions

obtained from E. umbellata latex

Sample mg of phorbol/g of extract

HE 56.0

DIF 1,245.0

EF 462.0

MF 143.0

HE = hexane extract; DIF = dichloromethane fraction; EF = ethanol fraction; MF =

methanol fraction.

The presence of phorbol esters, mainly in the DIF and EF, reinforces the biological potential

of these samples. Different mechanisms for triterpenes and diterpenes are described in the

literature and their prescence can help to explain the cytotoxic activity atributed to the latex

of E. umbellata.[23,24]

CONCLUSIONS

Based on the in vitro analyses, it can be concluded that this plant matrix presented cytotoxic

potential against the B16F10 strain, with relevant results for the hexane extract, and

particularly for its fractionated samples.

Regarding the in vivo results, the dichloromethane fraction did not show a significant

difference in relation to the tumour volume between the groups, and metastases were

observed for all the tested groups at the evaluated dosage.

The dichloromethane and ethanol fractions appeared to present terpenes, corroborating the

important relationship between diterpenes and triterpenes in the cytotoxic activity observed in

this study for melanoma cells.

The results so far support the characterisation of the anti-tumour ethnopharmacological

potential of E. umbellata latex. They particularly suggest a correlation between the biological

activities and the chemical components of the plant matrix.

ACKNOWLEDGEMENTS

The authors are grateful to the technical support of the Multi-user laboratory of the Biology

and Health Science Centre (LABMU-SEBISA) of the State University of Ponta Grossa


36


(UEPG). We are also grateful to Dr. Sean Stroud for checking and correcting the English

version of the text.

Funding

The authors received a research grant (number 02/2013) from FINEP-CT/Infra, and the

corresponding author received the scholarship from CAPES.

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