1 Characterization and Application of Nanomaterials (2021) Volume 4 Issue 2 doi:10.24294/can.v4i2.1293 Original Research Article Sapindus emarginatus extract embedded with gold nanoparticles: An antiproliferative agent against MCF7 breast cancer cell line S Vignesh Kumar1*, V Kavimani2 1 Department of Biotechnology, Anna University Regional Centre, Coimbatore, India. E-mail: svigneshkum6@gmail. com 2 Department of Mechanical Engineering, Anna University Regional Centre, Coimbatore, India ABSTRACT There are numerous studies reported on the usage of the sapindus emarginatus (SE) fruit in cancer and other treat- ments in the past few years. In this study, crude SE fruit extract was prepared and it was further used to synthesis gold nanoparticles (Au Nps). The synthesized Au Nps were left embedded in the SE fruit extract. The Au Nps embedded in the SE fruit extract (SE-Au Nps) were characterized using UV-Visiable Spectroscopy, Centrifugal Particle Size analyzer (CPS), Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). MTT assay was carried out for both SE fruit extract and SE-Au Nps on MCF7 breast cancer cell line and thus compared. The UV-Visible Absorbance for the SE-Au Nps was obtained at 543 nm. The centrifugal particle size analysis of the Au Nps embedded in SE fruit extract showed the size of the nanoparticles to be widely varying with higher fraction of particles between the size ranges of 15 to 20 nm. The morphology of the Au Nps embedded in SE fruit extract was observed using SEM. The presence of Au Nps in SE fruit extract was confirmed using FTIR. The results of the MTT assay on MCF7 breast cancer cell line proved that the % cell viability was less for SE-Au Nps than that of the SE fruit extract alone. Thus, the antiproliferative activity of the SE fruit extract was significantly enhanced by embedding it with Au Nps and it can be effectively used in therapeutic applications after further studies. Keywords: Gold Nanoparticles; Sapindus Emarginatus; Antiproliferative; MCF7 ARTICLE INFO Received: 25 March 2021 Accepted: 11 May 2021 Available online: 18 May 2021 COPYRIGHT Copyright © 2021 S Vignesh Kumar, et al. EnPress Publisher LLC. This work is licensed under the Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0). https://creativecommons.org/licenses/by- nc/4.0/ 1. Introduction Cancer is the abnormal growth of cells with uncontrolled division resulting in increased number of cells[1]. Cancer is observed as the most dangerous class of disease categorized by uncontrolled cell growth[2,3]. There is a marginal increase in cancer cases in the last few years, and most of the time, it ends up with taking life[4,5]. Breast cancer is a com- plex and heterogeneous disease[6]. In world, breast cancer represents 9% of the global cancer burden and is the third most common tumour. Hu- man breast cancer MCF7 cells represent one of the most widely used experimental models for in vitro studies on breast carcinoma[7]. A considerable part of the current knowledge on breast carcino- mas is based on in vivo and in vitro studies performed with cell lines derived from breast cancer. In reference to the treatments available for cancer, the characteristics of the cancer determine the treatment, which may include surgery, medications (hormonal therapy and chemothera- py), radiotherapy and immunotherapy. There are currently three main 2 groups of medications for breast cancer, such as hor- mone blocking agents (Tamoxifen, Anastrozole or Letrozole), chemotherapy (cyclophosphamide, meth- otrexate and fluorouracil) and monoclonal antibodies (Trastuzumab). Radiotherapy is given after surgery to the region of the tumor bed and regional lymph nodes, to destroy microscopic tumor cells that may have escaped surgery. Radiation can reduce the risk of recurrence by 50%–60%[8]. Most chemotherapy medications work by destroying fast-growing and/or fast-replicating cancer cells, either by causing DNA damage upon replication or by other mechanisms. Along with the medications chemotherapy and ra- diation can also affect healthy cells. For example, damage to the heart muscle is the most dangerous complication of doxorubicin. Trastuzumab is very expensive and its use may cause serious side effects (approximately 2% of patients who receive it suf- fer from significant heart damage). Due to growing resistance and side effects to these therapies search for new therapeutics for breast cancer has become essential. Scientific interest in medicinal plant has bloomed in recent times due to increased efficiency of new plant derived drugs and wide spread concerns about the side effects of modern medicine[9]. Sapindus emarginatus Vahl found in South India is commonly known as soap nut tree. The tree spe- cies is inadequately distributed in diverse geograph- ical provinces like Gangetic plains, Western Ghats and Deccan Plateau in India[10]. The genus Sapindus possesses tremendous medicinal value. Since past, it is used as emetic, tonic, astringent, anthelmintic, for asthma, colic, diarrhea, cholera, tubercular glands and paralysis of limbs. Methanolic extract of fruit of Sapindus emarginatus (SE) found to produce CNS depressant activity[11]. The fruits are usually used for hair problems and also in preparation of shampoos. Traditionally SE is used as anti-inflammatory and antipruritic medicine. The seed is an intoxicant and the fruit rind has oxytropic action. Its powder is used as nasal insufflations[12]. Saponins isolated from dif- ferent plants and animals have been shown to specif- ically inhibit the growth of cancer cells in vitro[13–18]. The saponins from SE fruit extract found to have significant antihyperlipidemic activity[19]. The combined application of saponins with other antitumor compounds may increase cytotox- ic activity of the latter, which is an interesting new possibility in cancer treatment research[20]. The noble metal nanoparticles like gold nanoparticles represent smart and promising candidates in the drug delivery applications due to their unique dimensions, tunable functionalities on the surface, and controlled drug re- lease[21]. Another essential aspect while working with Au NP in bio-applications is safety and biocompat- ibility (Au NP is already approved by the US Food and Drug Administration.) Biologically synthesized and functionalized, Au NP provides many desirable attributes for use as carriers in drug delivery sys- tems as the functionalized Au NP core is essentially inert and nontoxic reported in recent studies[22]. The labeling of AuNPs with biological ligands to specif- ically bind to desired cancer cells increases the ef- fectiveness of thermal energy transfer to cancer cells without harming non-cancerous cells[23]. After cel- lular uptake, the Au Nps can act as tiny, precise and powerful heaters (thermal scalpels) to kill cancer and they are capable of inducing apoptosis in B-chronic lymphocytic leukemia[24]. It has been found that gold nanoparticles em- bedded in Rubia cordifolia (RC) matrix significantly enhanced anti-inflammatory characteristics by inhib- iting nitric oxide release[25]. It was reported in terms of inhibitory concentration for 50% inhibition com- pared to either RC extract or Au NPs. Hence, in this work the SE fruit extract was embedded with gold nanoparticles with the vision of increasing its anti- proliferative activity. 2. Materials and methods 2.1 Preparation of plant extract Fresh fruits of SE were collected from the nurs- ery of Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, Tamilnadu. Collected SE fruits were dried under shade, mechanically pow- dered and stored in an airtight container. Dried and powdered SE fruits were extracted with 95% meth- anol in a Soxhlet extractor. The methanolic extract was concentrated to give a dark brown residue which 3 was partitioned between water-n-Butanol (1:1). The n-butanolic layer was evaporated to give the crude saponin fraction as a brown residue[26]. 2.2 Synthesis of gold nanoparticles (Au NPs) embedded in SE fruit extract (SE-AU NPs) A solution of 50 ml, 0.001 M (0.39382 mg/ml) gold (III) chloride trihydrate (HAuCl4·3H2O) and 50 ml SE fruit extract, diluted in 50 ml of distilled water, was added together drop by drop and stirred on a magnetic stirrer. The solution was ultrasonicat- ed at a high frequency of 10 KHz for 3 h and then maintained at a stationary position for 2 h at room temperature. 2.3 Characterization of SE-AU NPs SE-Au Nps were characterized by UV-Visible Absorption Spectroscopy, Centrifugal Particle Size analyzer (CPS), Fourier Transform Infrared Spec- troscopy (FTIR) and a Scanning Electron Micro- scope (SEM). 2.4 UV-Visible Absorptance Spectroscopy studies UV-Visible Absorptance Spectroscopy have been proved to be quite sensitive to the formation of gold colloids because gold nanoparticles exhibit an intense absorption peak around 540 nm due to the surface plasmon (it describes the collective exci- tation of conduction electrons in a metal) excitation. The sample was analyzed using UV-9000S Spectro- photometer (Lark, India). Distilled water was used as a blank. CPS The gold nanoparticles embedded in the plant extract were isolated by centrifugation. Diluted sus- pensions of sucrose, on the order of 0.01–1.0 [wt%], were prepared. The sugar solution and our sample solutions were injected in disc centrifuge. Nanopar- ticle size was analyzed by injection of 350 µl of sample into CPS operating at a speed of 20,000 rpm. All analyses were run against a known calibration standard and the particles in the solution were analyzed by size distribution graph. SEM Morphology of the synthesized Au Nps in the SE fruit extract was analyzed using SEM. Few drops of the SE fruit extract embedded with the Au Nps was spread on a cover slip and dried. Then the sam- ple on the cover slip was coated with gold in a sput- ter coating unit for few minutes. FTIR The samples were completely dissolved in re- spective solvents, non-sticky and the pH was found to be less than 8 which are necessary conditions to analyze the liquid samples. The sample analysis was carried out using FTIR (Brucker–Tensor 27) in ATR mode with a range from 500 to 4000 cm-1. The liquid samples are directly placed onto the ZnSe crystal to obtain the spectrum. 3. Cell proliferation assay on MCF7 cell line 3.1 Chemicals and reagents MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) Invitrogen, USA. Acridine or- ange was obtained from Sigma, USA. All other fine chemicals were obtained from Sigma–Aldrich, St. Louis. 3.2 Cell culture MCF7 cells obtained from NCCS (National Centre for Cell Science, Pune) were cultured in Rose well Park Memorial Institute medium (RPMI), sup- plemented with 10% fetal bovine serum, penicillin/ streptomycin (250 U/ml), gentamycin (100 µg/ml) and amphotericin B (1 mg/ml) were obtained from Sigma Chemicals, MO, USA. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cells were allowed to grow to confluence over 24 h before use. 4. Cell growth inhibition studies by MTT assay Cell viability was measured with the conven- tional MTT reduction assay, as described previously with slight modification. Briefly, MCF7 cells were 4 seeded at a density of 5 × 103 cells/well in 96-well plates for 24 h, in 200 µl of RPMI with 10% FBS. Then culture supernatant was removed and RPMI containing various concentrations (0.11–100 µg/ml) of test compound was added and incubated for 48 h. After treatment, cells were incubated with MTT (10 µl, 5 mg/ml) at 37 °C for 4 h and then with DMSO at room temperature for 1 h. The plates were read at 595 nm on a scanning multi-well spectrophotometer. Data represented the mean values for six indepen- dent experiments[27]. Cell viability (%) = (Mean OD/ Control OD) × 100. 5. Results and discussion 5.1 Preparation of SE fruit extract Crude extract of SE fruits was obtained as brown colored mixture from the Soxhlet appara- tus. The mixture was separated into upper phase (butanol and crude saponin) and lower phase. The upper phase was evaporated and the crude saponin obtained was brown in color with a colorless lower phase. 5.2 Synthesis of SE-AU NP The change in the brown color of SE fruit ex- tract into purple color after its treatment with HAu- Cl4·3H2O solution clearly indicated the formation of Au Nps. The color change was observed within 1 h in room temperature. Jannathul Firdhouse and Lalitha have earlier reported the similar color change as the indicator for the formation of Au Nps[28]. The reduction of metal ions was roughly monitored by visual inspection as described earlier[29]. The process performed simply at room temperature is compara- tively free of toxic chemical hazards. Figure 1. Synthesized Au Np. (A) HAuCl4·3H2O; (B) Plant Extract + HAuCl4·3H2O; (C) SE-Au NP. 5.3 Characterization of SE-Au Nps UV-Visi- ble Spectroscopy The UV-Visable Absorbance for the SE-Au Nps was obtained at 543 nm and it is due to the excitation of surface plasmon vibrations in the gold nanoparti- cles. The spectra are consistent with previous experi- mental results. Parida et al. reported the synthesis of gold nanoparticles using Allium cepa extract as the reducing agent and the absorption peak was broad and found at 540 nm which might be due to polydis- persity nature of the nanoparticles[30]. Figure 2. UV-Visible Spectroscopy for SE-Au NPs. CPS The particle size analyzer (Figure 3) showed various diameter ranges of nanoparticles. The diam- eter versus % fraction was plotted. The size of the Au Nps embedded in SE fruit extract was found to be widely varying with higher fraction of particles between the size ranges of 15 to 20 nm. The particle size analyzer displayed a total weight of injected gold sample as 57.26 µg. In the injected sample 20 to 15.9 nm diameter of particles are present in high level whereas the 15.9 to 12.7 nm sized particles were in minimum level. This variation may be due to the aggregation of the nanoparticles in the extract. 5 Figure 3. Particle size distribution of Au NPs. SEM The SEM image (Figure 4) of SE-Au Nps showed the Au Nps distributed over the extract. The Au Nps embedded in the SE fruit extract were found to be aggregated and was not clear. The result obtained was similar to that of Paz Elia et al. who observed the morphology of Au Nps using plant ex- tracts as reducing agents[31]. Figure 4. SEM image of SE-Au Nps. FTIR FTIR spectrum of methanolic fruit extract of SE showed characteristic peaks for several func- tional groups like hydroxyl group in the range 3500 to 3000, C-O-C in the range of 1500 to 1000 and C-Br (alkyl) in the range of 1000 to 500. For SE-Au Nps the peak in the range of 3500 to 3000 indicates the presence of -NH2 (amino) group, C-H group at 2119 and carbonyl group C=O in the range of 2000 to 2500. By comparing the spectrum of SE-Au Nps with the spectrum of SE fruit extract and the stan- dard spectrum of gold nanoparticles[32], it is clear that the spectra of SE embedded with gold nanoparticles indicates the presence of both the extract and the gold nanoparticle in it. 6. Cell proliferative study—MTT as- say The antiproliferative activity of SE fruit extract and SE-Au Nps under in vitro conditions were exam- ined on cell proliferation by the MTT assay. MCF- 7 cells were exposed to the two samples at varying concentrations for 48 h and cytotoxicity was deter- mined using MTT assays. MTT results have shown that as the concentration of the samples increase, increased cytotoxicity was observed in a dose-de- pendent manner. In MTT assay, cell viability was significantly reduced to 47, 60 and 81.9% for the concentrations of 100, 10 and 1 µg respectively for SE-Au Nps and 69.9, 85.76 and 90% for 100, 10 and 1 µg respectively for crude SE fruit extract (Figure 7). The SE-AU Nps was found to be more effective compared to the crude SE fruit extract alone which indicates that Au Nps increases the cytotoxicity and has great potential as conjugate with the fruit extract and can be effectively used as anticancer agent. 6 Table 1. MTT assay for cell viability Concentration Cell viability (%) Sample A Sample B 100 µg 47.38003 69.93933 10 µg 60.28682 85.76944 1 µg 81.9636 90.12686 100 ng 98.78654 95.58742 1 ng 97.90403 98.67632 7. Conclusion The SE fruit extract was prepared using the sox- hlet apparatus. The SE fruit extract was used to syn- thesize the gold nanoparticles. The synthesized gold nanoparticles embedded in the SE fruit extract (SE- Au Nps) were confirmed by its absorbance at 543 nm using the UV Vis spectrophotometer. The size of the Au Nps was found to be widely varying with higher fraction of particles between the size ranges of 15 to 20 nm. This may be due to the agglomeration of the particles. Morphology of the SE-Au Nps was ob- served using SEM. The FTIR analysis of the SE-Au Nps confirmed the presence of gold nanoparticles in the extract. MTT assay was carried out for both SE fruit extract and SE-Au Nps on MCF7 breast cancer cell line and compared. The results of the MTT as- say on MCF7 breast cancer cell line proved that the % cell viability was less for SE-Au Nps than that of the SE fruit extract alone. Thus, the antiproliferative activity of the SE fruit extract was significantly en- hanced by embedding it with Au Nps and it can be effectively used in therapeutic applications after fur- ther studies. 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