Translate this page into:
Gold nanoparticles (AuNPs) and Rosmarinus officinalis extract and their potentials to prompt apoptosis and arrest cell cycle in HT-29 colon cancer cells
⁎Corresponding author. essamebrahim@hotmail.com (Essam H. Ibrahim)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Background
One of the significant morbidity and mortality causes is the colorectal cancer. Rosmarinus officinalis plant is utilized as food and medicine. Currently, nanoparticles are widely employed in medicinal preparations. This work aimed to explore the potentials of R. officinalis leaves acetone extract, alone or in combination gold nanoparticles (AuNPs), to kill colon cancer cells.
Methods
Fresh leaves of R. officinalis were collected from the Al Soudah, Saudi Arabia and then dried to prepare acetone extract. AuNPs were prepared utilizing the extract and portrayed using UV/Vis spectrophotometry and scanning electron microscopy (SEM). Active ingredients exist in the extract and extract + AuNPs were screened using FT-IR spectroscopy. Biological properties of the extract and extract + AuNPs including anti-cancer activity and apoptotic capacity were studied.
Results
Results of UV/Vis spectrophotometry and SEM demonstrated that AuNPs are of 79 nm in diameter. FTIR analysis revealed the existence of bioactive molecules in the extract. Extract and extract + AuNPs arrested HT-29 colon cancer cells at G2/M phase.
Conclusion
R. officinalis acetone extract and extract + AuNPs could arrest the proliferation of HT-29 colon cancer cells. Extract and extract + AuNPs actuated apoptosis in cancer cells as opposed to necrosis.
Keywords
HT-29 colon cancer cells
Rosmarinus officinalis
Apoptosis
Gold nanoparticles
Aseer
1 Introduction
Normal cells divide under well-controlled manner according to host body needs. Cancer cells, the abnormal version of host cells, are characterized by their powerful capacity of proliferation with reduced capacity of apoptosis. The abnormal cell proliferation of cancer is due to the loss of perfect control mechanisms in the production of growth factors leading to imperfect cellular homeostasis and maintenance causing abnormal tissue architecture (Hanahan and Weinberg, 2011). Several pharmaceutical derivatives have been obtained through the screening of plant natural compounds and showed anticancer properties (Da Rocha et al., 2001).
Rosmarinus officinalis (Rosemary) plant is one of the Lamiaceae (mint) family. It is widely existing in the Mediterranean region and distributed in many other locations through the world. Its leaves are utilized in the treatments of many disorders long time ago as well as food additives. Rosemary has many medicinal activities such as antibacterial (Nieto et al., 2018), the power to treat depressive behavior (MacHado et al., 2012), antitumor (Kontogianni et al., 2013), antioxidant (Nieto et al., 2018), hepato-protective power (Sotelo-Félix et al., 2002; Abdel-Wahhab et al., 2011; Rašković et al., 2014), anti-parasitic, wound-healing agent (Hamidpour, 2017), antispasmodic in renal colic, smooth muscle relaxant (Habtemariam, 2016), gastric ulcerative lesions (Corrêa Dias et al., 2000), control of hypercholesterolemia, relief of physical fatigue (Fernández et al., 2014), lipid peroxidation reduction (Posadas et al., 2009), radio-protective-antimutagenic capacities (Del Baño et al., 2006), treatment for cutaneous allergy (Tabassum and Hamdani, 2014), anti-inflammatory (De Melo et al., 2011), antiviral (Nolkemper et al., 2006), antithrombotic (Yamamoto et al., 2005), and anti-hyperglycemic (Naimi et al., 2017).
Several phytochemicals with biological characteristics were obtained from rosemary oil and extracts. These phytochemicals include acids (rosmarinic, ursolic, oleanolic, caffeic, and carnosic), eucalyptol, camphor, rosmadial, secohinokio, and eugenol/luteolin derivatives (Borges et al., 2019; Einbond et al., 2012; Gonçalves et al., 2019). Rosemary preparations (e.g. carnosic and rosmarinic acids and diterpenes carnosol) are believed to apply strong biological effects (e.g. antitumor, anti-allergic, antibacterial and antioxidant) (González-Vallinas et al., 2015; Moore et al., 2016). Several studies demonstrated that R. officinalis exhibit good anti-cell propagation properties against some cancer cell lines (Kontogianni et al., 2013; Petiwala et al., 2013). Many plants, including edible ones, have medicinal impacts against several diseases including colon cancer (Balakrishna and Kumar, 2015; Xu et al., 2015).
The science concerning with study and manufacturing of nano-dimension materials is called nanotechnology (Rajeshkumar, 2016). Gold nanoparticles (AuNPs) production employing plant extract is clean, ecofriendly, and cost effective method comparing to other methods (chemical and physical). Gold nanoparticles are reported to have anti-bacterial (Mohamed et al., 2017), anticancer (Rajeshkumar, 2016b) and immunomodulatory (Dykman and Khlebtsov, 2017) activities.
In the current work, biological proprieties (anticancer, antibacterial, effects on cell cycle/apoptosis) of R. officinalis leaf acetone extract and its synthesized AuNPS on HT-29 cells were examined. In addition, the power of the extract to produce AuNPs was evaluated.
2 Experimental methods
2.1 Rosemary leaf extract preparation
Leaves of R. officinalis (Fig. 1) were collected in August 2019, from Abha, Aseer, Saudi Arabia. The leaves extract was prepared as described elsewhere (Ibrahim et al., 2021). A stock preparation (1%) was designed in acetone, sterilized (0.45 µm filter, Fisher Scientific) and stocked at −20 °C.
R. officinalis plant collected from Al Soda mountain, Aseer, KSA.
2.2 AuNPS synthesis/characterization and functional group analysis
AuNPs preparation, characterization, size/morphology, and plant extract functional group analysis were done according Ghramh et al. (2019).
2.3 Maintenance, preparation of cells and cytotoxicity tests
HT-29 cancer cell line was maintained utilizing the same methods and reagents described by Ganesan et al. (2020) The investigation of the extract and extract/AuNPs cytotoxicity towards HT-29 cells was done according to Ghramh et al. (2021) at concentration of 0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0 132.0, 264, 528.0 and 1056.0 μg/mL.
2.4 Apoptotic effects of extract and extract/AuNPs
The extract and extract containing AuNPs at IC50 concentrations were separately added to HT-29 cells and incubated for 48 h. The apoptotic effect was tested using similar reagents and methodology described by Ibrahim et al. (2021).
3 Results
3.1 Gold nanoparticles production
Gold nanoparticles synthesis was observed through the color alteration of the mixture (AuCl4/ extract, Fig. 2A). After color alteration (Fig. 2C), AuNPs assembling was reviewed spectrophotometrically (Fig. 2D). Results uncovered the manufacturing of AuNPs at a specific peak (520 nm).UV/Vis light monitoring of AuNPs synthesis by R. officinalis; A: Extract; B: extract light absorbance; C: extract after synthesis of AuNPs; D: extract + AuNPs light absorbance.
3.2 Functional groups
The FTIR spectral analysis of the R. officinalis extract is displayed in Fig. 3. Strong broad band (characteristic to alcoholic) in the range of 3633–3300 cm−1, concerned to stretching vibration of O—H groups. Two bands (2938 and 2851 cm−1) are due to stretching vibrations of the groups CH2 and CH3. Bands present at 1717, 1696 and 1661 cm−1 are due to stretching vibration of C⚌O/C⚌C groups of flavonoids and amino acids. A peak ranged from 1457 to 1276 cm−1 is attributed to aromatic C⚌C and due to the presence of series of small peaks in the area 1400–2000 cm−1. Numerous peaks found at 1030–523 cm−1 represent C—O stretch of acid, anhydride, ester, alcohol, ether, monosubstituted alkene and halo compound.FTIR spectrum of R. officinalis extract.
3.3 Characterization of AuNPs
SEM examination divulged that the manufactured AuNPs are nearly uniform spherical in shape with median size of 79 nm.
3.4 Extract cytotoxicity toward HT-29 cells
R. officinalis extract inhibited HT-29 cell line growth at a significant (p > 0.0001) levels at the concentration 32–1000 μg/mL. Extract containing AuNPs also inhibited HT-29 cells growth at a significant (p > 0.0001) levels but only up to concentrations ranged 32–1000 μg/mL (Fig. 4).Effects of R. officinalis extract and extract + AuNPs on HT-29 cell growth.
3.5 Apoptotic effects of Rosmarinus officinalis acetone extract
Evaluation of the extract and extract containing AuNPs was done using Annexin V staining (Table 1). The extract caused significant (p < 0.05) apoptosis (9.44%) in HT-29 cells, while extract + AuNPs treated HT-29 cells showed higher effect (16.04%) over the untreated cells (1.92%).
Treatment
Apoptosis (%)
Necrosis (%)
Total
Early
Late
Extract
9.44
1.52
6.31
1.61
Extract + AuNPs
16.04
6.17
8.21
1.66
4 Discussion
The use of therapeutic medicinal plants to fight cancer is a good approach. R. officinalis is an edible and also used in cosmetics. In the current work, we targeted the valuation of the biological effects R. officinalis when combined with gold nanoparticles. In addition, we investigated the impact of the extract on HT-29 cells.
In the current study, we utilized R. officinalis leaf extract to create AuNPs. The biomolecules contained in the extract, and shown by RTIR analysis, could reduce and cap gold ions forming the AuNPs. Many studies also reported the presence of active biomolecules in R. officinalis leaves (Cheung and Tai, 2007; Wang et al., 2012; González-Vallinas et al., 2015; Moore et al., 2016). The created AuNPs appeared as spheres of average size of 79 nm. Many researchers were able to create nanoparticles with diverse sizes utilizing Rosmarinus officinalis leaf extracts (Ghaedi et al., 2015; Soltanabad et al., 2018; Hadi Soltanabad et al., 2020).
Typically, the cell cycle of cells passes through four consecutive stages starting from the quiescence stage (G0) to the propagation (G1, S, G2, and M) stage, and return back to either the G0 or G1 stage (Jingwen et al., 2017). Genes controlling the cell cycle are always mutated in cancers, causing uncontrolled cell division and tumor development (Williams and Stoeber, 2012). Abnormal (cancer) cells start over G1 straightforwardly after M stage leading to abnormal cell division. The target of anticancer medicines is to induce the cell cycle arrest at the M phase (American Cancer Society, 2015).
Extract and extract + AuNPs prepared in the current work demonstrated growth suppressive impacts on human colon cancer HT-29 cells. Vasanth et al. (2014) stated that nanoparticles included in R. officinalis extract had the power to enhance apoptosis in cervical cancer cells through the elevation in ROS levels and its subsequent action. Extract and extract + AuNPs revealed non-toxic potential or cell cycle interference impacts. This distinctly mentions that extract + AuNPs interferes the cell cycle of cancer cells.
Programmed cell death or apoptosis works efficiently in the equilibrium of healthy cells by removing the cancer cells (Levine et al., 2001; Wang et al., 2015). It is documented that the bioactive materials exist in R. officinalis have the power to initiate apoptosis in cells of the tumor. Bioactive materials exist in the plant’s extract (e.g. carnosol, carnosic & rosemarinic acids) have been shown to induce apoptosis in cells of the tumor, possibly through the synthesis of nitric oxide (Đilas et al., 2012; Tai et al., 2012; Kontogianni et al., 2013; Petiwala et al., 2013). p53, a tumor suppressor, exerts a critical part in the apoptosis inducement and affects the mitochondrial intrinsic apoptosis pathway (Vaseva and Moll 2009; Nieminen et al., 2013).
In the current study, HT-29 cells were arrested by the extract and extract + AuNPs at G2/M stages. Other in vitro ivestigations utilized several cancer cell lines (DLD-1, CaCo-2, SW480, and SW620 colon cancer cells) concluded that R. officinalis has anticancer characteristics (Slameová et al., 2002; Yi and Wetzstein 2011; González-Vallinas et al., 2013).
5 Conclusion
R. officinalis leaf acetone extract could synthesize AuNPs with 79 nm diameter. Extract and extract + AuNPs could arrest HT-29 cancer cell proliferation. Extract and extract + AuNPs actuated apoptosis in cancer cells as opposed to necrosis. The edible plant R. officinalis can be utilized in the preparation of anticancer formulas, at least against colon tumors, either alone or in combination with gold nanoparticles.
Acknowledgments
Authors would like to acknowledge the support of the Research Center for Advanced Materials Science (RCAMS), King Khalid University, Abha Saudi Arabia for funding this work through research program KKU/RCAMS/22.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- Protective effect of a natural herb (Rosmarinus officinalis) against hepatotoxicity in male albino rats. Comun. Sci.. 2011;2:9-17.
- [Google Scholar]
- American Cancer Society (2015) Chemotherapy Drugs: How They Work Understanding the life cycle of a cell. Am Cancer Soc 17. 22/10/2015.
- Evaluation of synergetic anticancer activity of berberine and curcumin on different models of A549, Hep-G2, MCF-7, Jurkat, and K562 cell lines. Biomed Res. Int.. 2015;2015:1-7.
- [CrossRef] [Google Scholar]
- Rosmarinus officinalis essential oil: A review of its phytochemistry, anti-inflammatory activity, and mechanisms of action involved. J. Ethnopharmacol.. 2019;229:29-45.
- [Google Scholar]
- Anti-proliferative and antioxidant properties of rosemary Rosmarinus officinalis. Oncol. Rep.. 2007;17:1525-1531.
- [CrossRef] [Google Scholar]
- Antiulcerogenic activity of crude hydroalcoholic extract of Rosmarinus officinalis L. J. Ethnopharmacol.. 2000;69:57-62.
- [CrossRef] [Google Scholar]
- Natural products in anticancer therapy. Curr. Opin. Pharmacol.. 2001;1:364-369.
- [CrossRef] [Google Scholar]
- Rosmarinus officinalis L. essential oil inhibits in vivo and in vitro leukocyte migration. J. Med. Food. 2011;14:944-949.
- [CrossRef] [Google Scholar]
- Radioprotective-antimutagenic effects of rosemary phenolics against chromosomal damage induced in human lymphocytes by γ-rays. J. Agric. Food Chem.. 2006;54(6):2064-2068.
- [Google Scholar]
- In vitro antioxidant and antiproliferative activity of three rosemary (Rosmarinus officinalis L.) extract formulations. Int. J. Food Sci. Technol.. 2012;47(10):2052-2062.
- [Google Scholar]
- Immunological properties of gold nanoparticles. Chem. Sci.. 2017;8:1719-1735.
- [CrossRef] [Google Scholar]
- Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. Fitoterapia. 2012;83(7):1160-1168.
- [Google Scholar]
- Effectiveness of Rosmarinus officinalis essential oil as antihypotensive agent in primary hypotensive patients and its influence on health-related quality of life. J. Ethnopharmacol.. 2014;151:509-516.
- [CrossRef] [Google Scholar]
- Punicalagin regulates apoptosis-autophagy switch via modulation of annexin a1 in colorectal cancer. Nutrients. 2020;12:1-17.
- [CrossRef] [Google Scholar]
- Rosmarinus officinalis leaf extract mediated green synthesis of silver nanoparticles and investigation of its antimicrobial properties. J. Ind. Eng. Chem.. 2015;31:167-172.
- [Google Scholar]
- Synthesis of gold nanoparticles (AuNPs) using ricinus communis leaf ethanol extract, their characterization, and biological applications. Nanomaterials. 2019;9(5):765.
- [Google Scholar]
- Antimicrobial, immunomodulatory and cytotoxic activities of green synthesized nanoparticles from Acacia honey and Calotropis procera. Saudi J. Biol. Sci.. 2021;28:3367-3373.
- [CrossRef] [Google Scholar]
- Effects of in vitro gastrointestinal digestion and colonic fermentation on a rosemary (Rosmarinus officinalis L) extract rich in rosmarinic acid. Food Chem.. 2019;271:393-400.
- [Google Scholar]
- Antitumor effect of 5-fluorouracil is enhanced by rosemary extract in both drug sensitive and resistant colon cancer cells. Pharmacol. Res.. 2013;72:61-68.
- [Google Scholar]
- Rosemary (Rosmarinus officinalis L.) extract as a potential complementary agent in anticancer therapy. Nutr. Cancer. 2015;67:1223-1231.
- [Google Scholar]
- The therapeutic potential of rosemary (Rosmarinus officinalis) Diterpenes for Alzheimer’s disease. Evid.-based Complement Altern. Med.. 2016;2016:1-14.
- [CrossRef] [Google Scholar]
- Carnosic acid content increased by silver nanoparticle treatment in rosemary (Rosmarinus officinalis L.) Appl. Biochem. Biotechnol.. 2020;191:482-495.
- [CrossRef] [Google Scholar]
- Rosmarinus officinalis (Rosemary): A novel therapeutic agent for antioxidant, antimicrobial, anticancer, antidiabetic, antidepressant, neuroprotective, anti-inflammatory, and anti-obesity treatment. Biomed. J. Sci. Tech. Res.. 2017;1(1–6)
- [CrossRef] [Google Scholar]
- Lepidium sativum and its biogenic silver nanoparticles activate immune cells and induce apoptosis and cell cycle arrest in HT-29 colon cancer cells. Biomater. Tissue Eng. 2021;11(2):195-209.
- [Google Scholar]
- Cell cycle regulation and anticancer drug discovery. Cancer Biol Med. 2017;14(348)
- [CrossRef] [Google Scholar]
- Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity. Food Chem.. 2013;136(1):120-129.
- [Google Scholar]
- Vesicle-associated membrane protein of arabidopsis suppresses bax-induced apoptosis in yeast downstream of oxidative burst. J. Biol. Chem.. 2001;276:46284-46289.
- [CrossRef] [Google Scholar]
- Rosmarinus officinalis L. hydroalcoholic extract, similar to fluoxetine, reverses depressive-like behavior without altering learning deficit in olfactory bulbectomized mice. J. Ethnopharmacol.. 2012;143:158-169.
- [CrossRef] [Google Scholar]
- Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int. J. Vet. Sci. Med.. 2017;5(1):23-29.
- [Google Scholar]
- Anticancer effects of rosemary (Rosmarinus officinalis L.) extract and rosemary extract polyphenols. Nutrients. 2016;8(731)
- [CrossRef] [Google Scholar]
- Rosemary extract as a potential anti-hyperglycemic agent: Current evidence and future perspectives. Nutrients. 2017;9:1-19.
- [Google Scholar]
- Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis. Proc. Natl. Acad. Sci. U S A. 2013;110(20)
- [CrossRef] [Google Scholar]
- Antioxidant and antimicrobial properties of rosemary (Rosmarinus officinalis L.): A review. Medicines. 2018;5(98)
- [CrossRef] [Google Scholar]
- Antiviral effect of aqueous extracts from species of the Lamiaceae family against Herpes simplex virus type 1 and type 2 in vitro. Planta Med.. 2006;72(15):1378-1382.
- [Google Scholar]
- Polyphenols from the Mediterranean herb rosemary (Rosmarinus officinalis) for prostate cancer. Front. Pharmacol.. 2013;4:1-4.
- [CrossRef] [Google Scholar]
- Protective effect of supercritical fluid rosemary extract, Rosmarinus officinalis, on antioxidants of major organs of aged rats. Exp. Gerontol.. 2009;44(6-7):383-389.
- [Google Scholar]
- Synthesis of silver nanoparticles using fresh bark of Pongamia pinnata and characterization of its antibacterial activity against gram positive and gram negative pathogens. Resour. Technol.. 2016;2:30-35.
- [CrossRef] [Google Scholar]
- Anticancer activity of eco-friendly gold nanoparticles against lung and liver cancer cells. J. Genet. Eng. Biotechnol.. 2016;14:195-202.
- [CrossRef] [Google Scholar]
- Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. BMC Complement. Altern. Med.. 2014;14(225)
- [CrossRef] [Google Scholar]
- Rosemary-stimulated reduction of DNA strand breaks and FPG-sensitive sites in mammalian cells treated with H2O2 or visible light-excited Methylene Blue. Cancer Lett.. 2002;177:145-153.
- [CrossRef] [Google Scholar]
- Ag-conjugated nanoparticle biosynthesis mediated by Rosemary leaf extracts corre-lates with plant antioxidant activity and pro-tein content. Int. J. Nanosci. Nanotechnol.. 2018;14:319-325.
- [Google Scholar]
- Evaluation of the effectiveness of Rosmarinus officinalis (Lamiaceae) in the alleviation of carbon tetrachloride-induced acute hepatotoxicity in the rat. J. Ethnopharmacol.. 2002;81(2):145-154.
- [Google Scholar]
- Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro. Phytomedicine. 2012;19:436-443.
- [CrossRef] [Google Scholar]
- Anticancer activity of Moringa oleifera mediated silver nanoparticles on human cervical carcinoma cells by apoptosis induction. Colloids Surfaces B Biointerfaces. 2014;117:354-359.
- [Google Scholar]
- The mitochondrial p53 pathway. Biochim. Biophys. Acta – Bioenerg.. 2009;1787:414-420.
- [CrossRef] [Google Scholar]
- Chemotherapy-induced miRNA-29c/catenin-δ signaling suppresses metastasis in gastric cancer. Cancer Res.. 2015;75:1332-1344.
- [CrossRef] [Google Scholar]
- Antibacterial activity and anticancer activity of Rosmarinus officinalis L. essential oil compared to that of its main components. Molecules. 2012;17:2704-2713.
- [CrossRef] [Google Scholar]
- Chemical composition, antioxidative and anticancer activities of the essential oil: Curcumae rhizoma-sparganii rhizoma, a traditional herb pair. Molecules. 2015;20:15781-15796.
- [CrossRef] [Google Scholar]
- Anti-tumorigenic activity of five culinary and medicinal herbs grown under greenhouse conditions and their combination effects. J. Sci. Food Agric.. 2011;91:1849-1854.
- [CrossRef] [Google Scholar]
Appendix A
Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jksus.2022.102304.
Appendix A
Supplementary data
The following are the Supplementary data to this article: