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Cytotoxicity and Chromatographic Fingerprinting of Euphorbia Species Used in Traditional Medicine



Setlabane Tebogo Michael Mampa, Samson Sitheni Mashele and Mamello Patience Sekhoacha
 
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ABSTRACT

Background and Objective: Chromatographic fingerprinting of plant species play an important role in species identification and standardization of plant based health products. Some of the Euphorbia species are used in folk medicine, yet majority of these exhibit various degrees of toxicity. It becomes a challenge to distinguish the toxic from the non-toxic species. The study aimed to evaluate cytotoxicity and to determine the method for fingerprinting the chemical constituents of the selected Euphorbia species to identify markers of toxicity. Materials and Methods: Hexane, DCM, ethyl acetate and methanol extracts of E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida indigenous and E. horrida var. were examined in mammalian vero cell line using MTT cell viability test assay. The presence of secondary metabolites and proteins were assessed in the plant extracts and thin layer chromatography was used to identify toxicity markers. Results: The hexane and DCM extracts of E. arabica, E. bupleurifolia and the DCM extract of E. horrida var. exhibited the highest cell growth inhibition reaching IC50 at a concentration of 10 μg mL1. Both polar and non-polar extracts of E. enopla exhibited cell growth inhibition with the hexane extract reaching IC50 at a concentration of 10 μg mL1. Euphorbia gorgonis and E. horrida indigenous were not active against the vero cell line. Secondary metabolites were detected, however, proteins were not detected in all six Euphorbia species. The TLC profiles of toxic extracts revealed additional bands which were absent in non-toxic species. Conclusion: It is concluded that the TLC method developed in this study can be used as a quick screen method to possibly distinguish toxic from non-toxic species, as well as in identifying the studied species.

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Setlabane Tebogo Michael Mampa, Samson Sitheni Mashele and Mamello Patience Sekhoacha, 2020. Cytotoxicity and Chromatographic Fingerprinting of Euphorbia Species Used in Traditional Medicine. Pakistan Journal of Biological Sciences, 23: 995-1003.

DOI: 10.3923/pjbs.2020.995.1003

URL: https://scialert.net/abstract/?doi=pjbs.2020.995.1003
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Application of analytic chromatographic techniques for fingerprinting of plant species has brought solutions to challenges of species identification, qualitative and quantitative analysis of plants’ constituents, as well as standardization of plant-based health products. Long before introduction of western medicine, plants were considered a valuable source of medicine. Medicinal plants are still preferred in their natural form as they are claimed to have less side effects compared to pharmaceutical drugs1,2. However, it remains a major problem to distinguish between similar species and to determine the desired constituents that may be present at the effective levels. Inadequate scientific information on standardization of plant extracts affects the efficacy of treatments and consistency of treatment outcomes. It is necessary to have a proper identification of plants through chemical fingerprinting in order to have a library that can be used to identify plant species for quality and quantity assessment3.

Euphorbia is a highly diverse genus of flowering plants in the family Euphorbiaceae4. Euphorbia species have been reported to be toxic and this toxicity is mostly found in the white milky sap called latex, which has been reported to be harmful to humans and livestock5-7. Literature has reported that the latex and the aerial parts of Euphorbia species have historically been used to treat different ailments including cancer, wounds, warts and headaches8,9. Some species in this genus have some pharmacological properties such as; antiviral, anticancer, antimicrobial and anti-fungal properties5.

The widespread use of some Euphorbia species in folk medicine necessitates that the toxic species and non-toxic species and/or medicinally useful species can be distinguished in order to categorize the species for their suitable applications.

Euphorbia species have also been reported to contain biologically active proteins such as proteases, chitinases, oxidases and lectins and have various phytochemicals such as, steroids, phenolic, cerebrosides, glycerols, flavonoids, glycosides, tannins, saponins, alkaloids, pentose, anthraquinones, phytosterols, terpenes including; diterpenes and triterpenes10-13. The presence of both phytochemical constituents and proteins implies different extraction and identification methods for these plant species. It is therefore necessary that different application methods are applied to obtain the ones suitable for fingerprinting the chemical constituents of these species to identify markers responsible for toxicity.

This study evaluated cytotoxic effects of Euphorbia species (E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida var and E. horrida indigenous) on vero cell line in vitro. The chemical fingerprinting will assist in quick screening of most Euphorbia species to determine whether the species is toxic or not and to help determine whether the tested species contains the necessary chemical composition for the intended application.

MATERIALS AND METHODS

Plant collection and extraction: The study was conducted at the Central University of Technology and University of the Free State in Bloemfontein, South Africa, from July, 2018-September, 2019. Six species of Euphorbia were collected from KwaZulu-Natal province of South Africa and Lesotho. Plants were authenticated by a botanist at University of the Free State. Fresh plants were chopped into small pieces, left to dry at room temperature and ground to fine powder. Crude extract was obtained by homogenizing 10 g of powdered material with 100 mL of organic solvents in their increasing order of polarity starting with hexane, dichloromethane, ethyl acetate and methanol.

Mixtures were left on a shaker for 48 h (FMH instruments, sepsci), then filtered with a filter paper (Whatman® Maidstone). Filtrates were dried by rotary evaporation (Buchi, labortechnik Switzerland) at 45°C, then placed under fume hood until dry. Dried extracts were stored at 4°C until further use. The percentage yields were calculated.

Phytochemical screening: The powdered plant materials of E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida var. and E. horrida indigenous were screened for phytosterols, pentose, tannins, glycosides, triterpenoids, anthraquinones, saponins, flavonoids and alkaloids based on the protocols14,15.

Cytotoxicity screening: The mammalian vero cell line was obtained from cellonex, South Africa. Cells were cultured in complete medium; DMEM supplemented with 10% Fetal Bovine Serum (FBS) and maintained in an incubator (NUVE EC 160) at 37°C including 5% CO2. Cells were sub-cultured at 90% confluency by trypsinization. Cells were centrifuged at 800 rpm for 5 min to obtain a cell pellet. Cells were re-suspended in 5 mL of the medium, viability of the cells was assessed using trypan crystal blue dye and cells were counted using automated cell counter (countess FL, life technologies). The concentration of the cells was calculated to obtain 1×105 cells mL1 for plating in 96 well plates. Plates were incubated for 24 h at 37°C temperature. Following incubation, cells were treated with 100 μL of test extracts added in triplicates. The stock solutions of the test samples (20 mg mL1) were prepared in DMSO, diluted to concentrations of 100, 10 and 1 μg mL1 in complete medium. Emetine was used as control standard drug. The plates were then incubated at 37°C for 48 h. Cell viability was measured using the MTT assay16. Results were analyzed using Microsoft excel.

Protein detection: Methanol extracts of the plants were dissolved and prepared in warm distilled water. The extracts were tested for presence of proteins using biuret and xanthoproteic tests. For Biuret test, Sodium Hydroxide (NaOH) and a few drops of Copper Sulfate (CuSO4) solutions were added to the sample solution17. A violet or pink colour was observed. For xanthoproteic test, concentrated sulfuric acid (H2SO4) was added to the sample solution. A white precipitate was formed. In both tests, egg white was used as positive control.

Plant extract fingerprinting by TLC: Silica gel on thin aluminium plates (5×10 cm) was used as stationary phase. For mobile phase, three different solvent systems: Toluene-acetone (8:2) (non-polar solvent), Toluene-chloroform-acetone (40:25:35) (Semi-polar solvent) and n-butanol-glacial acetic acid-water (50:10:40) (Polar solvent) were used in elution18. Dried extracts were reconstituted (2 mg mL1) in the solvent used for extraction. The plates were developed in the appropriate mobile system. The TLC plates were visualised under ultraviolet (UV) light. The retention factors were calculated for every spot visible on the TLC plate. The Rf values were used to compare the chemical profiles of plant extracts to identify the presence/absence of toxicity markers in different plant species of Euphorbia.

Statistical analysis: The values are presented as the mean±standard deviation (SD).

RESULTS

The percentage yield of the dried plant extracts was calculated and results are summarised in Table 1. Generally, DCM and methanol had the highest yields in all plants extracted.

All six Euphorbia species confirmed the presence of phytosterols, glycosides, triterpenoids and flavonoids. Pentose was only found in E. horrida indigenous and E. horrida var. Saponins were detected in E. bupleurifolia, E. horrida indigenous and E. horrida var. Alkaloids were present in most species; E. bupleurifolia, E. enopla, E. gorgonis and E. horrida var. (Table 2).

Table 1:
Yields (%) of six Euphorbia species following extraction with different solvents
DCM: Dichloromethane, MeOH: Methanol, EtOAc: Ethyl acetate

Table 2:Phytochemical screening of Euphorbia species
+: Present, -: Absent

Fig. 1:
Cell growth inhibitory effects of E. arabica extracts on vero cells

Fig. 2:Cell growth inhibitory effects of E. bupleurifolia extracts on vero cells

Fig. 3:Cell growth inhibitory effects of E. enopla extracts on vero cells

Cytotoxicity screening: The following graphs show cell growth inhibition effects of extracts of E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida indigenous and E. horrida var. Extracts that attained an IC50 at a concentration of 10 μg mL1 and below were considered active.

Fig. 4:
Cell growth inhibitory effects of E. gorgonis extracts on vero cells

Hexane extracts of E. arabica exhibited the highest cell growth inhibition reaching IC50 at all concentrations tested, the DCM extract reached IC50 at 10 μg mL1. The methanol and ethyl acetate extracts of this Euphorbia didn’t show any activity (Fig. 1).

Figure 2 shows that all four extracts of E. bupleurifolia showed varying cytotoxicity, with hexane and DCM extracts showing IC50 values at concentrations of 1 and 10 μg mL1, respectively.

Only the highly non-polar hexane extract of E. enopla exhibited considerable cell growth inhibition, at a concentration of 10 μg mL1. Interestingly, proliferation of cells was observed at concentrations of 1, 10 and 100 μg mL1 for methanol extracts (Fig. 3).

The DCM and ethyl acetate extracts of E. gorgonis reached the IC50 value only at a concentration of 100 μg mL1 (Fig. 4).

Hexane and DCM extracts of E. horrida indigenous reached IC50 only at a concentration of 100 μg mL1. Again, proliferation of vero cells was observed at a concentrations of 1, 10 and 100 μg mL1 for methanol extracts (Fig. 5).

The DCM extract of E. horrida var. reached IC50 at a concentration of 10 μg mL1. Hexane and ethyl acetate extracts showed activity only at a concentration of 100 μg mL1 (Fig. 6).

Protein detection: Euphorbia species have been reported to contain biologically active proteins. In this study, presence of proteins in the plant extracts was detected using biuret and xanthoproteic tests. For biuret test, a violet or pink colour was not observed as indicated in Table 3. For xanthoproteic test, a white precipitate was not formed as indicated in Table 3. Positive control results are shown in Fig. 7-8. Table 3 shows protein detection results for E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida indigenous and E. horrida var.

Fig. 5:
Cell growth inhibitory effects of E. horrida indigenous extracts on vero cells

Fig. 6:
Cell growth inhibitory effects of E. horrida var. extracts on vero cells

Table 3:Protein detection results of six Euphorbia species The whole plant was tested
In all the plant species, proteins were not detected using both tests

Fig. 7:Biuret test using egg white

Fig. 8:Xanthoproteic test using egg white

Figure 9 shows the TLC profiles of all extracts. The toxic extracts, based on the cell culture results revealed additional bands which were absent in non-toxic species.

Thin layer chromatography: The TLC profiling results of hexane extracts showed that E. bupleurifolia had the highest number of bands followed by E. enopla and E. horrida var. with 6 bands; E. arabica and E. horida indigenous with 4 bands; E. gorgonis with no bands. The number of bands produced when visualized under UV light and the Rf values determined (Table 4).

The TLC profiling results of DCM extracts showed that E. enopla and E. horrida indigenous had the highest number of bands, followed by E. bupleurifolia and E. horrida var. with 11 bands each; E. arabica with 7 bands and E. gorgonis with no bands (Table 5).

Fig. 9(a-c): TLC profiling results of (a) Hexane extracts, (b) DCM extracts and (c) Methanol extracts
  Euphorbia species, A: E. arabica, B: E. bupleurifolia, C: E. enopla, D: E. gorgonis, E: E. horrida indigenous and F: E. horrida var. ethyl acetate and water extracts were not done due to inadequate plant material

TLC profiling results of methanol extracts showed that E. arabica had the highest number of bands followed by E. enopla with 8 band; E. bupleurifolia, E. horrida indigenous and E. horrida var. with 7 bands each and E. gorgonis with 1 band (Table 6).

Table 4:TLC profiling results of hexane extracts

Table 5:TLC profiling results of DCM extracts

Table 6:TLC profiling results of methanol extracts

DISCUSSION

The study suggested classification of cytotoxic and non-cytotoxic species of Euphorbia based on cytotoxicity screening, phytochemical screening and profiling. Phytochemical analysis confirmed the presence of phytosterols that have been reported to have potential health benefits19. Pentose was only detected in E. horrida indigenous and E. horrida var. and this has been reported to reduce cytotoxicity of plant extracts20. This could be due to the sugar providing nutrients to the cells. This supported the low activity observed in these two species. Previous studies21-24 reported that tannins cause regression of tumors that are already present in tissue, implying their potential in anti-proliferation of cancer cells activity. Euphorbia gorgonis did not show the presence of tannins in which could have added to the lack of anti-proliferation activity observed.

All six Euphorbia species showed the presence of triterpenoids, glycosides and flavonoids. These have been reported to exhibit innumerable biological and pharmacological activities such as antioxidant, anti-inflammatory and anti-cancer properties. These secondary metabolites have also implicated growth inhibition of cell lines through induction of apoptosis25-28. E. arabica, E. enopla, E. horrida indigenous and E. horrida var. showed the presence of anthraquinones. Literature has reported that anthraquinones detected in plant extracts are increasingly used for pharmaceuticals due to their therapeutic and pharmacological properties29.

Toxicity is regarded as a secondary function of alkaloids30, which support cytotoxicity exerted by E. bupleurifolia, E. enopla and E. horrida var. Although Euphorbia species are reported to contain biologically active proteins13, in this study proteins were not detected in all six Euphorbias. The cytotoxicity of the plants could result primarily from the presence of secondary metabolites (phytochemicals). The results from this study suggested that the cytotoxic molecules in the studied Euphorbia plants are non-polar, since only the non-polar extracts showed activity while the more polar extracts were not active. Furthermore, the study focused on fingerprinting of phytochemical constituents of studied Euphorbia species, which can be used for identification of species for quality control purposes31.

Species with the highest bands produced in the TLC profiles imply high amount of chemically varied phytochemicals. Based on the results obtained, Euphorbia extracts with less Rf values were considered more polar, which means that they stick to the stationary phase a lot stronger than Euphorbia extracts with more Rf values and therefore, moves slower in the mobile phase. Due to presence of various phytochemicals within extracts, it is difficult to attribute cytotoxicity effect to a specific phytochemical. However, further study is required to determine the exact toxicity markers responsible for activity. Active constituents could be isolated and further studied as antiviral, anticancer, antimicrobial and anti-fungal properties.

CONCLUSION

The Euphorbia species investigated in this study had a similar composition of phytochemicals, (phytosterols, glycosides, triterpenoids and flavonoids). Phytochemicals present in the species are known to possess various pharmacological activities, which support the use of Euphorbia species to treat various health conditions. The cytotoxicity exhibited by hexane and DCM extracts of E. arabica, E. bupleurifolia, E. enopla and E. horrida var. provide scientific preliminary evidence for their use in treatment of cancer. The clear differences in the TLC chemical profiles of the toxic and non-toxic species show the effectiveness and reliability of methods for application as a quick screening to either verify the species or determine the toxicity of the species.

SIGNIFICANCE STATEMENT

This study discovers the different levels of cytotoxicity of six Euphorbia species (E. arabica, E. bupleurifolia, E. enopla, E. gorgonis, E. horrida indigenous and E. horrida var.) that can be beneficial for fingerprinting of medicinal plants for use to distinguish and confirm the presence of secondary metabolites of interest. This study will help the researcher to uncover the critical areas of analytic chromatographic techniques used to screen species used for medicinal purposes, as the presence of chemical constituents such as secondary metabolites (phytochemicals) affect the efficacy and safety of the outcome of treatment.

ACKNOWLEDGMENTS

The authors express sincere thanks to Central University of Technology and University of the Free State, Bloemfontein, South Africa, for providing facilities to conduct these studies and National Research Foundation of South Africa for funding.

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