Elsevier

Phytomedicine

Volume 61, August 2019, 152846
Phytomedicine

Supercritical CO2 fluid extraction of croton crassifolius Geisel root: Chemical composition and anti-proliferative, autophagic, apoptosis-inducing, and related molecular effects on A549 tumour cells

https://doi.org/10.1016/j.phymed.2019.152846Get rights and content

Abstract

Background

The use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals.

Purpose

The objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects.

Methods

Supercritical CO2 fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis.

Conclusion

Forty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC50 value of 25.00 ± 1.62 μg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.

Introduction

Essential oils are natural compounds obtained from plants, the components of which are gaining increasing interest because of their relatively safe status, wide acceptance by consumers, and frequent presence in studies investigating their potential functional utility (Babahmad et al., 2018, Brasesco et al., 2017, Rakmai et al., 2018). Essential oils often act as significant indicators in evaluating the efficacy of traditional medicine (Zhang and Wang, 2009). About 300 essential oils produced from plant species are important in agricultural, food, cosmetic, and health industries (Raut and Karuppayil, 2014). As the second-leading global cause of death after cardiovascular diseases, cancer is a major public health problem (Sa et al., 2016). According to the American Cancer Society, there was an estimated 1685 210 new cancer cases diagnosed in the United States and approximately 600,000 deaths caused by the disease in 2016 (Turkez et al., 2018). There is an urgent need to discover new oncotherapy drugs that are highly effective while presenting low toxicity. It has been reported that many essential oils obtained from plants exhibit promising effects in the treatment of cancer (Song and Sun, 2016). Hence, the exploration of the anti-tumour properties of essential oils as a legitimate research focus should receive the same interest as synthetic anticancer agents (Zhang et al., 2018).

The genus Croton, which belongs to the Euphorbiaceae family, consists of approximately 1300 species that are widely distributed in tropical and sub-tropical regions (Salatino et al., 2007). Plants of this genus are rich sources of structurally diverse diterpenoids, including clerodane, trachlobane, kaurane, crotofolane, and pimarane, all of which possess broad biological activities such as cytotoxic (Giang et al., 2005), antiviral (Wang et al., 2012), antiplasmodial (Langat et al., 2011), anti-inflammatory properties (Premprasert et al., 2013), and specifics of health benefits. In addition, triterpenoids (Pan et al., 2014), flavonoids (Morales-Flores et al., 2015, Zou et al., 2012), and alkaloids (Ravanelli et al., 2016) are also occasionally found in the Croton genus.

Croton crassifolius Geisel, belonging to the Croton genus, is an economically important herb cultivated abundantly in Guangdong, Fujian and Hainan Provinces of China for commercial production of its roots in traditional Chinese medicine (Qiu, 1996). Since ancient times, its roots-known as “Ji Gu Xiang” in China-have been used in prescriptions to treat stomach ache, rheumatism, sore throat, and cancer (Committee, 1999). All the uses and treatments of different diseases are justified by the chemical composition of the plant. Previous studies investigating the chemical constituents of C. Crassifolius roots identified diverse types of diterpenes, which can be classified into clerodane, daphnane, and tigliane according to their skeletal types. Many compounds isolated from C. Crassifolius roots exhibited remarkable anticancer activity and inhibited vessel formation in zebrafish (Wang et al., 2015a, Yuan et al., 2017).

Most of studies on C. crassifolius concerned the use of root extracts in medicine, the utilization of its volatile compounds was few investigated. Huang et al. compared the anti-angiogenic activities of the two extracts (Supercritical fluid extract and steam distillation extract) from C. crassifolius. The supercritical fluid extract showed stronger effect between the two and was subject to further isolation of the active compounds in the herb. However, other anti-tumour effects including anti-proliferative, autophagy and apoptosis-inducing of the supercritical fluid extract were not mentioned (Huang et al., 2015). The objective of this study was to explore the chemical composition of CCEO extracted using supercritical CO2 fluid extraction technology, as well as the compound's potential anti-tumour activities and related anti-proliferative and apoptosis-inducing molecular mechanisms.

Section snippets

Plant materials

C. crassifolius roots were purchased in the Chinese Herbal Medicine Market in Bozhou, Anhui, China in October, 2017. The species was identified by Prof. Chenggang Shan, Institute of Agro-Food Science and Technology, Shandong Academy of Agriculture Sciences (IAFST, SAAS), Jinan, China. A voucher specimen (No.17-10-29) (Fig. 1(a)) was deposited at the Laboratory of Bioactive Substances and Functional Foods, IAFST, SAAS.

Essential oil extraction by supercritical fluid extraction

The dried and powdered roots of C. crassifolius (1.0 kg) were extracted with CO

Identification of the chemical composition of CCEO

The CCEO obtained by SFE-CO2 was yellow brown (Fig. 1(b)) frankincense oil with a yield of 3.41 ± 0.03% (w/w) based on the dry weight of C. crassifolius roots. The CCEO was analysed by GC–MS to determine volatile composition. Chemical analyses of the volatile constituents of the essential oil are summarised in Table 1 and a typical GC–MS chromatogram is shown in Fig. S1 (supplementary data). Forty compounds representing 92.90% of the total oil were identified. The main compounds were

Discussion

Abundantly cultivated in South China, C. crassifolius is a commercially-important herb in China due to its content of highly-valued bioactive compounds (e.g., diterpene in its roots). As a traditional Chinese medicine, its roots are often used to prepare many clinical treatments including anticancer drugs. In recent years, the price of C. crassifolius roots has decreased due to higher production resulting from low value-added processing. The development of novel uses for C. crassifolius is

Acknowledgments

This study was supported by the Provincial Key Research and Development Program of Shandong, China (No. 2018GNC110008); the Taishan Scholars's Program of Shandong, China; the Provincial Natural Science Foundation of Shandong of China (No. ZR2016CB51); the Agriculture Scientific and Technological Innovation Program of Shandong Academy of Agriculture Sciences (No. CXGC2018E09).

The authors would like to thank Prof. Chenggang Shan for identifying the species of C. crassifoliu.

Conflict of interest

The authors declare that they have no conflicts of interest.

References (56)

  • WangY. et al.

    Raddeanin A inhibits growth and induces apoptosis in human colorectal cancer through downregulating the Wnt/β-catenin and NF-κB signaling pathway

    Life Sci.

    (2018)
  • YangC.S. et al.

    Lutescins A and B, two new ellagitannins from the twigs of Trigonostemon lutescens and their antiproliferative activity

    Fitoterapia

    (2018)
  • ZhangJ. et al.

    Chemical composition, in vitro anti-tumor activities and related mechanisms of the essential oil from the roots of Potentilla discolor

    Ind. Crops Prod.

    (2018)
  • R.A. Babahmad et al.

    Chemical composition of essential oil of Jatropha curcas L. leaves and its antioxidant and antimicrobial activities

    Ind. Crops Prod.

    (2018)
  • Barkatullah et al.

    Chemical composition and biological activities of the essential oil of Skimmia laureola leaves

    Molecules

    (2015)
  • C. Brasesco et al.

    Assessing in vitro acaricidal effect and joint action of a binary mixture between essential oil compounds (Thymol, Phellandrene, Eucalyptol, Cinnamaldehyde, Myrcene, Carvacrol) over Ectoparasitic Mite Varroa Destructor (Acari: Varroidae)

    J. Apic. Sci.

    (2017)
  • ChenY.N. et al.

    Involvement of intrinsic and extrinsic apoptotic pathways in baicalein-induced apoptosis in human hepatoma Hep3B cells

    J. Cancer Mol.

    (2005)
  • ChouS.T. et al.

    Zanthoxylum ailanthoides Sieb and Zucc. extract inhibits growth and induces cell death through G2/M-phase arrest and activation of apoptotic signals in colo 205 human colon adenocarcinoma cells

    Anticancer Res.

    (2011)
  • C.M.M.E. Committee

    Chinese Materia Medica

    (1999)
  • D.M. Eisenberg et al.

    Trends in alternative medicine use in the united states, 1990–1997: results of a follow-up national survey

    JAMA

    (1998)
  • A. El-Khattouti et al.

    Crosstalk between apoptosis and autophagy: molecular mechanisms and therapeutic strategies in cancer

    J. Cell Death

    (2013)
  • S.C. Erridge et al.

    International comparisons of survival from lung cancer: pitfalls and warnings

    Nat. Clin. Pract. Oncol.

    (2007)
  • I.M. Ghobrial et al.

    Targeting apoptosis pathways in cancer therapy

    CA: Cancer J. Clin.

    (2005)
  • GiangP.M. et al.

    Cytotoxic diterpenoids from Vietnamese medicinal plant Croton tonkinensis GAGNEP

    Chem. Pharm. Bull.

    (2005)
  • GuanC. et al.

    Inhibitory effect of β-elemene on human breast cancer cells

    Int. J. Clin. Exp. Path.

    (2014)
  • I. Guessous et al.

    Lung cancer screening: current situation and perspective

    Swiss Med. Wkly.

    (2007)
  • GuoJ.R. et al.

    Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

    Biol. Res.

    (2015)
  • M.O. Hengartner

    The biochemistry of apoptosis

    Nature

    (2000)
  • Cited by (5)

    • Chemical composition, anticancer activities and related mechanisms of the essential oil from Alpinia coriandriodora rhizome

      2022, Industrial Crops and Products
      Citation Excerpt :

      Hence, in this study, the level of Bax, Bcl-2, mitochondria cyt c, and total cyt c proteins in EO treated A549 cells were investigated and the results suggested that A. coriandriodora EO could increase the ratio of Bax/Bcl-2, and cause Δψm decline, thereby promoting cyt c release. The release of cyt c results in cleavage and activation of caspase-9, and subsequently stimulates the proteolysis of downstream caspase3, which cleaves many proteins including PARP, and finally induces apoptosis (Chen and Wang, 2002; Liu et al., 2019). The results of our current study indicated that A. coriandriodora EO could activate caspase-9 and caspase-3, and lead to PARP cleavage.

    • Research progress on anti-tumor effects of plant essential oils

      2022, Chinese Traditional and Herbal Drugs
    View full text