Abstract
Green tea (Camellia sinensis) has benefits. Its main potential content is epigallocatechin gallate, which has many bioactivity and pharmacological properties. However, herbal medicines have limitations on low solubility and stability. A nanoparticle delivery system is a perfect form of active ingredient development, because it can mediate the increase in solubility, dissolution rate, and strength of a targeted delivery system. This study aimed to make and test the formulation of the ethanol and ethyl acetate fraction from green tea leaves in the form of a nanoparticle delivery system using chitosan biopolymer as the primary carrier polymer combined with sodium tripolyphosphate as a crosslinker and then carried out the tests on the MDA-MB-231 breast cancer cell line. The results showed that the particle size value was 199.7 nm, the zeta potential was-56.7 mV, and the polydispersity index was 0.337. X-ray diffraction and differential scanning calorimetry test results showed that the C. sinensis fraction was perfectly dispersed molecularly in the nanoparticle system. The results of the cytotoxic test on the MDA-MB-231 breast cancer cell line obtained IC50 values for both fractions, namely 10.70 μg/mL (nano ethanol fraction) and 12.72 μg/mL (nano ethyl acetate fraction). This result showed a significant increase in anticancer activity in both fractions compared to those not formulated (P < 0.05). These results also show that the C. sinensis tea fraction formulated in a nanoparticle delivery system has a great potential as a new therapeutic agent for breast cancer.
Keywords: Cytotoxicity test, drug delivery system, ethanol, ethyl acetate fraction of Camellia sinensis green tea leaves, MDA-MB-231 breast cancer cells, nanoparticle technology
INTRODUCTION
The advantage of herbal drugs has increased because they have fewer side impacts than engineered drugs. The obstacle in herbal products is the active substances in herbal medicinal preparations, which are difficult to penetrate the lipid membranes of the body's cells.[1,2]
Nanoparticles are a technology aiming to modulate the size and dosage form in 10–1000 nm.[3] Moreover, nanotechnology can modify surface characteristics and particle size, so that herbal medicines target a particular receptor or part of an organ. In addition, the release of active compounds can be controlled to minimize the side effects of herbal medicines.[3,4]
Cancer is a disease caused by abnormal cells in body tissues that develop rapidly and uncontrollably.[5] The recapitulation of cancer early detection results in Indonesia from 2006 to 2015 show that the number of sufferers is 1.9 million.[6] Breast cancer is one of the most feared types of cancer, especially for women. Breast cancer is the growth of abnormal cells proliferative in breast tissue.[7] Cancer treatment, in this way, has several drawbacks. Surgery is generally ineffective for cells that have undergone metastases, while chemotherapy has not given optimal results, because the action of drugs is not specific and is relatively expensive.[1,8]
Based on this, the use of herbs as an alternative cancer treatment is increasing. The raw materials for traditional medicines are easy to obtain, safe, relatively inexpensive, and have a fewer side effects than synthetic drugs.[9,10] One of the traditional plants with anticancer properties is green tea. Green tea contains various chemical components, including catechins. The catechin compound in tea is epigallocatechin-3-gallate (EGCG). EGCG has been shown to inhibit cancer cell growth and plays an essential role in stimulating apoptosis or programmed cell death.[5]
Thus, it is necessary to formulate green tea leaves (Camellia sinensis) and test their cytotoxicity and proliferative activity on the MDA-MB-132 breast cancer cell line.
MATERIALS AND METHODS
Materials
Green tea leaves (C. sinensis) deionized water, CH3COOH, C2H5OH, C4H8O2, KBr, chitosan, Na5P3O10, carrageenan, PBS (phosphate buffer saline), breast cancer cells in the MDA-MB-231 cell line, RPMI 1640 Gibco penicillin-streptomycin, fetal bovine serum DMSO, RPMI, MTT, SDS, and aluminum foil.
Research methods
This type of research is an experimental research (laboratory scale) with a randomized posttest-only control group design. Five main steps were carried out in this research. Then proceeded with the manufacture of nanoparticles using a spray pyrolysis tool, the nanoparticle characterization process, and in the final stage, in vitro testing of the cytotoxicity activity of the test preparation nanoparticles against the MDA-MB-231 breast cancer cell line.[7,11]
RESULTS
Molecular interaction results between chitosan compound and sodium tripolyphosphate as polymeric nanoparticle careers.
The bond distance (interaction between chitosan and sodium tripolyphosphate) value is 2.22 A, and the bond energy is −0.081 kcal/mol.
The molecular ratio is 1:1. An illustration of the molecular interaction between chitosan compounds and sodium tripolyphosphate is shown in Figure 1.
Figure 1.
Molecular interaction of chitosan and sodium tripolyphosphate
Measurement of particles, polydispersity index, and zeta potential of ethanol fraction polymeric nanoparticles of green tea leaves (C. sinensis).
From the test results, the particle measurements in each formulation (F1, F2, and F3) were 270.1, 247.7, and 199.7 nm, respectively. Formulation F3 has the smallest particle size, because the ratio of chitosan and sodium tripolyphosphate is in the equimolar equilibrium of 4:1, as shown in [Table 1 and Figure 2].
Table 1.
Measurement of nanoparticles from green tea leaves (Camellia sinensis)
| Parameter | Formula |
||
|---|---|---|---|
| F1 | F2 | F3 | |
| Particle size (nm) | 270.1 | 247.7 | 199.7 |
| Polydispersity | 0.602 | 0.380 | 0.337 |
| Index zeta potential (mV) | −37.2 | −50.5 | −56.4 |
Figure 2.
Particle size distribution on the three formulations, namely: (a). F1, (b). F2, and (c). F3
From this test, the zeta potential value is also obtained, which is the value that becomes the stability parameter of the dispersion system. Furthermore, the F3 formulation had a higher zeta potential value than F2 and F1.
X-ray diffraction (XRD) characterization on ethanol fraction polymeric nanoparticles of green tea leaves.
The diffractogram pattern shows the results of 2Θ angle and intensity values. It depends on the strength of the intermolecular electron bonds in the molecule. It is shown in Figure 3.
Figure 3.
X-ray diffraction characterization results
Characterization of differential scanning calorimetry (DSC) on ethanol fraction polymeric nanoparticles of green tea leaves (C. sinensis).
In the DSC curve, sodium tripolyphosphate shows the peak of the endothermic phase at a temperature of 120.06°C, which is indicated as the material's melting point. This pattern also illustrates that sodium tripolyphosphate is in a crystalline form. Meanwhile, chitosan and carrageenan polymers demonstrated a glass-transition pattern, which indicated an amorphous form.
Cytotoxicity test results of green tea leaf (C. sinensis) on breast cancer cell line.
The results of the two fractions formulated in the form of nanoparticles could significantly increase breast cancer's effectiveness. The test data for the ethanol fraction have an IC50 value of 10.70 μg/mL and the ethyl acetate fraction have an IC50 value of 12.72 μg/mL.
DISCUSSION
The interaction between chitosan and Na5P3O10 (the bond distance) value is 2.22 A, the bond energy is − 0.081 kcal/mol, and the molecular ratio is 1:1. This result is also the basis for choosing the mass balance formula for chitosan molecules and Na5P3O10 molecules. The ratio of 1:1 mole for the chitosan and Na5P3O10 has an equivalent mass ratio of 4:1 in the optimization results of the best formula.[12,13]
The smallest particle size is found in the F3. The proper ratio will make chitosan and Na5P3O10 completely react in the ionic gelation process and create a perfect form and stable nanoparticle system. In contrast, the F2 and F1 ratios have a more significant ratio. The result of this comparison will be the presence of free chitosan molecules that do not completely react with Na5P3O10. Chitosan in this free state has absorption capability, and selectivity.[14] The polydispersity index value describes the uniformity of particle size of the nanoparticle system. The small polydispersity index value represents that the nanoparticle system obtained is monodisperse. Moreover, the large polydispersity index value describes the polydispersity system.
The result shows that the lower the chitosan-to-Na5P3O10 ratio, the lower the polydispersity index.[15] Then, the potential zeta value test was carried out to represent the accumulation of charges that form an electrical double layer on the surface of each nanoparticle. It shows that the higher the potential zeta value, the better stability. In Table 1, F3 has the biggest zeta potential value; as a result, the chitosan-to-Na5P3O10 ratio significantly affects the value of particle size (P < 0.05), polydispersity index, and zeta potential. Finally, F3 has the best characteristics.[16]
The test samples C. sinensis and chitosan both showed a semicrystalline pattern in the XRD analysis, with diffractogram peaks at angles of 2Θ 25–27o (C. sinensis) and 19.72o (chitosan) [Figure 3]. The diffractogram data for C. sinensis-chitosan-tripolyphosphate-carrageenan nanoparticles showed an amorphous pattern. The crystalline lattice of C. sinensis is no longer visible due to this formula. It indicates that C. sinensis has been molecularly distributed in the nanoparticle matrix. Furthermore, chitosan is a semicrystalline polysaccharide.[17,18]
According to the DSC analysis, chitosan compounds peak at roughly 100°C [Figure 4]. The evaporation phase of H2O bound in the chitosan and alginate molecules causes this response. This formula does not reveal the sodium tripolyphosphate crystalline component's endothermic or exothermic peaks. These results correlate with the XRD results in the previous test, proving that the Cs-Ch-Tpp/Cr NPs formula molecularly has dispersed C. sinensis in the nanoparticle matrix system and encapsulated in the nanoreservoir system.[19]
Figure 4.
Differential scanning calorimetry results
The results of cytotoxicity tests [Figure 5] reveal that the nanoparticle dosage has a higher bioavailability. This result showed a significant increase in anticancer activity in both fractions compared to those not formulated (P < 0.05). Furthermore, using the chitosan biopolymer increases drug penetration across cancer cell membranes.[20] This is due to the chitosan polymer's cationic charge, which strongly favors negatively charged cell membranes.[21]
Figure 5.
Cytotoxicity test results
These impressive results also cannot be separated from the role of carrageenan biopolymers. Carrageenan is known to influence the growth cycle of cancer cells, as in previous studies which showed that carrageenan could inhibit the migration of breast cancer cells MDA-MB-231.[11] The mechanism of action of carrageenan in inhibiting the growth of cancer cells has also been investigated, capable of inducing the process of apoptosis.[7] This caused an increase in the cytotoxicity of the ethanol fraction and the ethyl acetate fraction of C. sinensis green tea leaves encapsulated with carrageenan polymer against the MDA-MB-231 breast cancer cell line.
CONCLUSION
F3 had the best physicochemical characteristics of the other formulas based on the results. XRD and DSC test results have also shown that the ethanol fraction of C. sinensis is perfectly dispersed molecularly in the nanoparticle system. The results of the cytotoxic test on the MDA-MB-231 breast cancer cell line showed that nanoparticles could increase the effectiveness of anticancer breast cancer.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
This research would not be possible without the support of Badan Pengembangan dan Pemberdayaan Sumber Daya Manusia Kesehatan (BPPSDMK) and UPPM Poltekkes Kemenkes Bengkulu to make this research goes well. BPPSDMK and UPPM Poltekkes Kemenkes Bengkulu, Indonesia, have given a monetary support to this research. The facilities and technical support were given by the Pharmaceutical Technology Laboratory, Padjadjaran University.
REFERENCES
- 1.Ikawati M, Armandari I, Khumaira A, Ertanto Y. Effects of peel extract from Citrus reticulata and hesperidin, a citrus flavonoid, on macrophage cell line. Indonesian J Pharm. 2019;30:260–8. [Google Scholar]
- 2.Nurkhasanah M, Novitasari R. Immunomodulatory activity of yogurt fortified with honey and Hibiscus sabdariffa L. on reactive oxygen intermediate and nitric oxide secretion. Indonesian J Pharm. 2019;30:141–6. [Google Scholar]
- 3.Chakraborty K, Shivakumar A, Ramachandran S. Nano-technology in herbal medicines: A review. Int J Herbal Med. 2016;4:21–7. [Google Scholar]
- 4.Abraham CC. A novel computationally designed tool to treat the covid-19 pandemic. ChemRxiv. 2020. Available from: https://doi.org/10.26434/chemrxiv.12056154.v2 .
- 5.Yuan JM. Cancer prevention by green tea: Evidence from epidemiologic studies. Am J Clin Nutr. 2013;98:1676S–81S. doi: 10.3945/ajcn.113.058271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kemenkes RI. Ministry of Health. Indonesia: Pusat Data Dan Informasi Kesehatan; 2015. Situasi Penyakit Kanker. [Google Scholar]
- 7.Murad H, Ghannam A, Al-Ktaifani M, Abbas A, Hawat M. Algal sulfated carrageenan inhibits proliferation of MDA-MB-231 cells via apoptosis regulatory genes. Mol Med Rep. 2015;11:2153–8. doi: 10.3892/mmr.2014.2915. [DOI] [PubMed] [Google Scholar]
- 8.Eckschlager T, Plch J, Stiborova M, Hrabeta J. Histone deacetylase inhibitors as anticancer drugs. Int J Mol Sci. 2017;18:1414. doi: 10.3390/ijms18071414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Maigoda TC, Judiono J, Purkon DB, Haerussana AN, Mulyo GP. Evaluation of Peronema canescens Leaves extract: Fourier transform infrared analysis, total phenolic and flavonoid content, antioxidant capacity, and radical scavenger activity. Macedonian J Med Sci. 2022;10:117–24. [Google Scholar]
- 10.Purkon DB, Kusmiyati M, Trinovani E, Fadhlillah FM. Enhancement of Posbindu understanding and skills in making traditional herbal drink as an immunostimulant. J SOLMA. 2021;10:210–9. [Google Scholar]
- 11.Groult H, Cousin R, Chot-Plassot C, Maura M, Bridiau N, Piot JM, et al. λ-Carrageenan oligosaccharides of distinct anti-heparanase and anticoagulant activities inhibit MDA-MB-231 breast cancer cell migration. Mar Drugs. 2019;17:140. doi: 10.3390/md17030140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Bobbala SK, Veerareddy PR. Formulation, evaluation, and pharmacokinetics of isradipine proliposomes for oral delivery. J Liposome Res. 2012;22:285–94. doi: 10.3109/08982104.2012.697067. [DOI] [PubMed] [Google Scholar]
- 13.Heinz H, Pramanik C, Heinz O, Ding Y, Mishra RK, et al. Nanoparticle decoration with surfactants: Molecular interactions, assembly, and applications. Surface Sci Rep. 2017;72:1–58. [Google Scholar]
- 14.Cheung RC, Ng TB, Wong JH, Chan WY. Chitosan: An update on potential biomedical and pharmaceutical applications. Mar Drugs. 2015;13:5156–86. doi: 10.3390/md13085156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Martin TB, Dodd PM, Jayaraman A. Polydispersity for tuning the potential of mean force between polymer grafted nanoparticles in a polymer matrix. Phys Rev Lett. 2013;110:018301. doi: 10.1103/PhysRevLett.110.018301. [DOI] [PubMed] [Google Scholar]
- 16.Clogston JD, Patri AK. Zeta potential measurement. In: McNeils SL, editor. Characterization on Nanoparticle Intended for Drug Delivery, Methods in Molecular Biology (Methods and Protocols) Vol. 697. Maryland, USA: Human Press; 2011. [DOI] [PubMed] [Google Scholar]
- 17.Sutapa IW, Wahid Wahab A, Taba P, Nafie NL. Dsilocation, crystallite size distribution and lattice strain of magnesium oxide nanoparticles. J Phys Conf Ser. 2018;979:012021. [Google Scholar]
- 18.Croisier F, Jerome C. Chitosan-based biomaterials for tissue engineering. Eur Polym J. 2013;49:780–92. [Google Scholar]
- 19.Höhne GW, Sarge SM, Hemminger W. Singapore: John Wiley and Sons; 2014. Calorimetry: Fundamentals, Instrumentation and Applications. [Google Scholar]
- 20.DeVeaux SD, Gomillion CT. Assessing the potential of chitosan/polylactide nanoparticles for delivery of therapeutics for triple-negative breast cancer treatment. Regen Eng Transl Med. 2019;5:61–73. [Google Scholar]
- 21.Rahmani S, Hakimi S, Esmaeily A, Samadi FY, Mortazavian E, Nazari M, et al. Novel chitosan based nanoparticles as gene delivery systems to cancerous and noncancerous cells. Int J Pharm. 2019;560:306–14. doi: 10.1016/j.ijpharm.2019.02.016. [DOI] [PubMed] [Google Scholar]