Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl

ABSTRACT

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

KEYWORDS:

• Acute toxicity
• cytotoxic activity
• in vitro cultivated anoectochilus roxburghiilind
• phytochemicals
• mitochondria-mediated caspase-dependent apoptosis
• subchronic toxicity

List of abbreviations

ALT: Alanine aminotransferase

AST: Aspartate aminotransferase

CytC: Cytochrome c

DAPI: 4′,6-diamidino-2-phenylindole

DMSO: Dimethyl sulfoxide

FACS: Fluorescence activated cell sorting

FITC: Fluorescein isothiocyanate

HDL: High-density lipoprotein

HPLC: High-performance liquid chromatography

HRP: Horseradish peroxidase

iARE:in vitro cultured A. roxburghii extract

LDH: Lactate dehydrogenase

MCV: Mean corpuscular volume

MCH: Mean corpuscular Hb

MCHC: Mean corpuscular Hb concentration

NAC: N-acetylcysteine

PARP: Poly (ADP-ribose) polymerase

PI: Propidium iodide

qRT-PCR: Real-time quantitative reverse transcription PCR

RLU: Relative light unit

Z-VAD-FMK: Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone

ΔΨm: Mitochondrial transmembrane potential

Acknowledgments

We would like to thank Dr. Van Son Dang, Institute of Tropical Biology, Vietnam Academy of Science and Technology for helping us with the botanical identification of Anoectochilus roxburghii Lind. There is no conflict of interest.

Disclosure Statement

The authors declare no competing interests.

Ethics approval

All animals used and protocols adopted in these studies were approved by the Animal Care and Use Committee of Institute of Tropical Biology, Vietnam Academy of Science and Technology (Ethics No. 180-2019/ITB).

Authors’ contributions

All research is done by the authors. Chinh Chung Doan, Thanh Long Le, Nguyen Quynh Chi Ho, Thi Thuy Nguyen, Do Dang Giap, Duc Thang Do, and Thi Phuong Mai Nguyen performed the experiments. Thanh Long Le, Chinh Chung Doan, Thi Phuong Thao Nguyen and Dang Giap Do designed experiments, analyzed data and prepared manuscript. Chinh Chung Doan and Nghia Son Hoang supervised the studies. Chinh Chung Doan and Thanh Long Le prepared and revised the manuscript. The manuscript was read and approved by all authors.

Submission declaration and verification

The manuscript has not been published in another journal in full or in part. All authors approved the manuscript and this submission.

Additional information

Funding

This research was supported by Vietnam Academy of Science and Technology, Ha Noi, Vietnam under grant number: UDNGDP.02/20-21 and Department of Science and Technology of Gia Lai, Gia Lai province, Vietnam under grant number: KHGL-05-19.