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. 2018 Oct 5;18(1):273.
doi: 10.1186/s12906-018-2303-9.

Comparative cardio and developmental toxicity induced by the popular medicinal extract of Sutherlandia frutescens (L.) R.Br. detected using a zebrafish Tuebingen embryo model

Longsheng Chen  1 , Minjie Xu  2 , Zhunan Gong  2 , Samkele Zonyane  3 , Shuwen Xu  4 , Nokwanda P Makunga  5
Affiliations

Comparative cardio and developmental toxicity induced by the popular medicinal extract of Sutherlandia frutescens (L.) R.Br. detected using a zebrafish Tuebingen embryo model

Longsheng Chen et al. BMC Complement Altern Med. .

Abstract

Background: Sutherlandia frutescens is one of the most promising commercialized, indigenous and medicinal plants of South Africa that is used as an immune-booster, and a traditional treatment for cancer. However, few studies report on its toxicology and dosage in vivo. There is still room to better understand its cytotoxicity effects in animal systems.

Methods: We prepared two extracts, one with 80% (v/v) ethanol, and the other, with water. Both were studied to determine the maximum tolerable concentration when extracts were applied at 0 to 200 μg/ml to a Tuebingen zebrafish embryo line. The development of zebrafish embryos after 24 h post fertilization (hpf) was studied. A concentration range of 5 μg/ml to 50 μg/ml was then chosen to monitor the ontological development of cultured embryos. A liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was used to study the differences of the two experimental extracts. Chemical variation between the extracts was illustrated using chemometrics.

Results: Both extracts led to bleeding and pericardial cyst formation when applied at high concentrations to the zebrafish embryo culture. Chronic teratogenic toxicities, leading to pericardial edema, yolk sac swelling, and other abnormal developmental characteristics, were detected. The aqueous extracts of S. frutescens were less toxic to the larvae than the ethanol extracts, validating preference for aqueous preparations when used in traditional medicine. Chemical differences between the water extracts and alcoholic extracts were analysed using LC-MS/MS. A supervised metabolomics approach, targeting the sutherlandiosides and sutherlandins using orthogonal partial least squares-discriminant analysis (OPLS-DA), illustrated that sutherlandiosides were the main chemical features that can be used to distinguish between the two extracts, despite the extracts being highly similar in their chemical constituents.

Conclusion: The water extract caused less cytotoxic and abnormal developmental effects compared to the ethanolic extract, and, this is likely due to differences in concentrations of extracted chemicals rather than the chemical profile per se. This study provides more evidence of cytotoxicity effects linked to S. frutescens using the zebrafish embryo bioassay as a study tool.

Keywords: Aqueous and ethanol extract; Cardiotoxicity; Cycloartane glycosides; Cytotoxicity; In vivo model; Lessertia; Medicinal plants; Plant metabolomics; Teratogenicity; Terpenoids.

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Conflict of interest statement

Ethics approval

The use of zebrafish embryos was approved by the Institutional Animal Care and Use Committee at Nanjing Normal University (permit number: 2090658). This work was conducted in the laboratories of ZG. The zebrafish bioassay utilized in this study complied with the requirements of the provisions and general recommendations of Chinese experimental animal administration legislation governing the ethics of the use of animals in scientific studies. The methods applied herein where thus carried out in accordance with approved guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest. This work stems from a bilateral research grant awarded to LC and NPM, respectively.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Embryos treated with different concentrations of S.fru-OH or S.fru-H2O at 96 hpf. a An example of a control (untreated) embryo with a normally developed straight spine without any visible signs of bleeding. b The short arrow points to a darkened region where a cyst is forming. The boxed zone highlights the pericardial region. c The short arrow points to a pink discolouration within the pericardial zone (shown using a rectangle). d A distinct pink sphere is visible within the pericardial zone (rectangle) and long arrows point to a curving spine. e A clear mass of tissue and an enlarged abdominal region is highlighted by the rectangle. f Long arrows point to a slightly curving spine. Pericardial cyst formation caused by exposure of zebrafish embryos to S.fru-H2O at 100 and 200 μg/ml was also accompanied by enlarged bellies (shown here by a horizontal arrow)
Fig. 2
Fig. 2
The percentage of morphologically abnormal and normal zebrafish treated with different concentrations of S.fru-OH or S.fru-H2O compared to the control (n = 18 per group)
Fig. 3
Fig. 3
Cardiotoxicity in zebrafish embryos caused by different concentrations of S.fru-OH (5, 10 and 30 μg/ml) or S.fru-H2O (5, 10, 30, 50, 100 and 200 μg/ml). The heart rate was measured at 96 hpf and the values are described as mean ± SD
Fig. 4
Fig. 4
The death rate of zebrafish embryos exposed to various concentrations of S. frutescens. a S.fru-OH extract and b S.fru-H2O were applied at 0–300 μg/ml; and, the hatching rate of zebrafish embryos exposed to various concentrations of S. frutescens. c S.fru-OH extract and d S.fru-H2O were applied at 0–300 μg/ml
Fig. 5
Fig. 5
a Loadings plots based on PCA separation of S.fru-OH extract and S.fru-H2O. b S-Plot of the PLS-DA method used to study the S.fru-OH [= − 1] and S.fru-H2O [=1] extracts of S. frutescens
See this image and copyright information in PMC

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