In vitro cytotoxicity against breast cancer using biogenically synthesized gold and iron oxide nanoparticles derived from the hydroethanolic extract of Salvia officinalis L

Nanotechnology has a real-world impact on every aspect of life. Many researchers have been drawn to the biosynthesis of gold and iron oxide nanoparticles (Au-NPs and SPIONS) because they have a wide range of life applications. In this work, a single-step environmentally friendly biosynthesis of Au-NPs and SPIONS is reported by reducing solutions of gold aureate and ferric chloride is reported for the first time using the hydroethanolic extract (HEE) of Salvia officinalis (S. officinalis), an edible plant found in Egypt. The phytochemicals present in HEE were responsible for the reduction as well as stabilization of these nanoparticles. Before using the HEE, it was phytochemically screened for its constituents. Qualitatively, the HEE was found to have comparable levels of phenolics, flavonoids, tannins, proteins, carbohydrates, terpenoids, steroids, and polysaccharides. Quantitatively, total phenolics (236.91 ± 2.15 mg GAE/g extract), flavonoids (91.38 ± 0.97 mg QE/g extract), tannins (101.60 ± 1.33 mg/g extract), proteins (284.62 ± 2.65 mg/g extract), carbohydrates (127.73 ± 1.68 mg/g extract), soluble sugars (52.3 ± 0.67 mg/g extract), and polysaccharides (75.43 ± 1.01 mg/g extract) were estimated. In addition, HPLC analysis revealed the identification of seven phenolic compounds [ferulic (67.26%), chlorogenic (3.12%), caffeic (3.11%), p-coumaric (1.13%), protocatechuic (0.65%), catechin (0.69%), rosmarinic (0.53%)] and three flavonoids [apigenin (5.29%), quercetin-7-O-glucoside (3.39%), and luteolin-7-O-rutinose (2.01%)]. The characterization of the biosynthesized NPs was confirmed by Fourier transform infrared (FT-IR) spectroscopy, UV–Vis absorption spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). In vitro cytotoxic studies showed that Au-NPs, SPIONS, and HEE have an inhibitory effect on the growth of human breast cancer (MCF-7) cells at an IC50 of 6.53, 6.97, and 26.12 µg mL−1, respectively, by comparison with the standard drug (Doxorubicin) effect (0.18 µg mL−1).


Introduction
Biosynthesis is one of the most crucial routes for environmentally friendly synthesizing NPs via various biological materials that act as reducing and capping agents for the synthesized NPs. Microorganisms, marine organisms, and plant extracts are among them (Bao and Lan 2019;John Sushma et al. 2016;Priyanka et al. 2016;Premanand et al. 2016;Jafar et al. 2015;Medda et al. 2015;El-Rafie et al. 2013). However, microorganisms require controlled conditions like pH, temperature, and maintenance of culture media and other factors for growth. Plant extracts, on the other hand, are more economical, widely available, safe, and environmentally friendly than other biological methods for the biosynthesis of NPs (Pirtarighat et al. 2017;Ganeshkumar et al. 2013;Kharissova et al. 2013;Cai et al. 2010). Extracts of various plant parts contain natural metabolites such as steroids, saponins, alkaloids, flavonoids, tannins, and other nutritional compounds, retaining the unique properties of NPs as well as the therapeutic potency of the plant extract.
Salvia, the largest genus in the Lamiaceae family, is endemic to the Mediterranean region and the Middle East (Walker et al. 2004). Plants in this genus have a wide range of traditional medicinal uses (Ghorbani and Esmaeilizadeh 2017;Adams et al. 2007). Terpenes, alkaloids, fatty acids, carbohydrates, glycosidic derivatives, phenolic compounds, polyacetylenes, steroids, and diterpenoids are the primary phytoconstituents in S. officinalis leaves, stems, and flowers (Ghalem and Ali 2017;Kadhim et al. 2016;Kontogianni et al. 2013). So, extracts from different parts of S. officinalis may serve as efficient biosynthesizers and stabilizers for a variety of metal and metal oxide nanoparticles.
Cancer is now the second leading cause of death, trailing cardiovascular disease. Detection and treatment of cancer in its early stages remains a major challenge, despite rapid advancements in surgery, diagnostic techniques, chemotherapy, and pharmaceutical chemistry. Breast cancer is the leading cause of death in women (Tanih and Ndip 2013). Despite being the least common type of cancer, bioactive metabolites from Salvia species have been reported to have cytotoxic activity, particularly on breast cancer cell lines (Yıldırım and Kutlu, 2015;Kafil et al. 2015;Alzeer et al. 2014;Loizzo et al. 2014;Özer et al. 2013).
In addition, the biosynthesis of Au-NPs and SPIONS was both environmentally and economically friendly. Both have numerous advantages because of their applicable surface chemistry, which exhibits many interesting properties that can be used in a variety of biomedical applications such as tissue repair, drug delivery, hyperthermia, and cell separation (Vita et al. 2018;Siddiqi et al. 2016). They also helped to solve some of the problems that come with current breast cancer treatment methods.
Based on the aforementioned facts, the objectives of this study were to synthesize and characterize Au-NPs and SPIONS, for the first time, using S. officinalis HEE after its phytochemical investigation. Their in vitro cytotoxicity was assessed against human breast cancer cell lines (MCF-7).

Materials
Iron(III) chloride (FeCl 3 , 99.9%; MW = 162.2) and Tetrachloroauric(III) acid trihydrate (HAuCl 4 . 3H 2 O, 99.9%; MW = 393.83) were supplied by Sigma-Aldrich, USA, and used as iron oxide and gold nanoparticle precursors, respectively. Other chemicals were of analytic quality and were used as received, with no further purification. All aqueous solutions were made with double-distilled water. Before use, all glassware was thoroughly cleaned and washed with distilled water before being dried.

Plant extraction, preparation, and characterization
Leaves of S. officinalis were obtained from an honest spice dealer in Cairo, Egypt. The dried, powdered plant of S. officinalis (100 g) was extracted with 70% ethanol using a Soxhlet apparatus until the extraction was complete, and then the ethanol was filtered out. The solvent was removed under reduced pressure at about 45 °C. The residue (2.54%) of the hydroethanolic extract (HEE) was stored until further laboratory analysis.
The quantitative determination of the aforesaid constituents, including total flavonoids, phenolics, tannins, proteins, total carbohydrates, and soluble sugars, was achieved according to the following procedures:

Total flavonoids
A colorimetric method was used to calculate the total flavonoid content (Haq et al. 2019). In a test tube, 4 mL of distilled water (DH 2 O) was added to 1 mL of the HEE, followed by the addition of 0.3 mL of sodium nitrite solution (5%) and 0.3 mL of aluminium chloride solution (10%). The test tube was incubated for five min, and then 2 mL of sodium hydroxide (1 M) was added to the reaction mixture. The reaction volume was increased to 10 mL with DH 2 O. The test tube was placed in the shaker and the absorption of the colour developed was measured at 510 nm. For the calibration, an aqueous solution of known quercetin concentrations was used, and the results were given in milligrams of quercetin equivalents (QE) per gram of extract.

Total phenolic content (TPC)
The colorimetric technique was used to determine the TPC of the examined plant using the Folin-Ciocalteu method with some modification (Haq et al. 2019). According to this technique, 0.5 mL of the extract solution was mixed in a test tube with 0.25 mL of Folin-Ciocalteu and 2.25 mL of methanol and stirred for 1 min before being placed in the dark for 8 min. After that, the mixture was incubated for 120 min at 25 °C with 2.0 mL of sodium carbonate (7.5% w/v). The absorption was measured at 756 nm, relative to the blank (methanol). Concentrations of total phenolic compounds were determined as milligrams of gallic acid equivalent/g extract (mg GAE)/g HEE) using the regression equation from the calibration curve of the gallic acid standard. The experiment was conducted in triplicate.

Total tannin content
The tannins were determined using the Folin-Ciocalteu method with some modifications (Kavitha Chandran and Indira 2016). About 0.1 mL of the plant extract was added to a volumetric flask (10 mL) containing 7.5 mL of distilled water, 0.5 mL of Folin-Ciocalteu phenol reagent, and 1 mL of 35% sodium carbonate solution, and was then diluted to 10 mL with distilled water. The mixture was shaken well and kept at room temperature for 30 min. A set of reference standard solutions of tannic acid was prepared. Absorbance for the test and standard solutions was measured against the blank (distilled water) at 700 nm. The estimation of the total tannin content was carried out in triplicate.

Protein content
The protein content, expressed as the total nitrogen content, was calculated using the micro-Kjeldahl apparatus by the Pearson procedure (Pearson 1970).

Total sugars
The anthrone technique, defined by Umbriet et al. (1959), was used to calculate total sugars. Six mL of anthrone solution (2 g/L of H 2 SO 4 , 95%) were added to a 3 mL sample and placed in a boiling water bath for 3 min. After cooling, the established colour was spectrophotometrically measured at 620 nm.

HPLC conditions of phenolics and flavonoids
A quantitative identification of the HEE phenolic constituents was performed using HPLC analysis according to the method described in detail by El-Rafie et al. (2016). The HPLC analysis was conducted with the Agilent 1260 series. Chromatographic separation was achieved using the Eclipse C-18 4.6 mm × 250 mm column (5 μm particle size). At a flow rate of 1 mL/min, the mobile phase was made up of water (A) and trifluoroacetic acid (0.05%) in acetonitrile (B). The mobile phase was sequentially programmed in a Nitrogen content = (Titration value−Blank) × 0.14 ∕Weight (g).

Biosynthesis and characterization of nanoparticles
Gold nanoparticles (Au-NPs) synthesis 10 mL of HEE of S. officinalis were added to a certain amount of double-distilled water, and then 1 mL of 0.1 M chloroauric acid (HAuCl 4 ) solution was added to this extract solution. An aqueous solution of NaOH (100 mmol/L) was added dropwise until the mixture's pH reached 13, and the total volume was then completed to 100 mL with distilled water. The mixture was continuously stirred with a magnetic stirrer with different time intervals (15, 30, 45, and 60 min) and at a temperature of 60 °C. The formation of Au-NPs is indicated by a colour change from pale yellow to reddish-violet.

Iron oxide nanoparticles (SPIONS) synthesis.
A known weight of ferric chloride (0.93 g) was dissolved in 10 mL of double distilled water for 15 min with continuous stirring. The resultant solution was mixed with 10 mL of the HEE from S. officinalis. The volume was then adjusted to 60 mL with distilled water, and the procedure was then performed as for Au-NPs synthesis. Within 10 min., the colour changes from yellow to deep green, indicating the formation of SPIONS.

Characterization of NPs
The following approaches (González-Ballesteros et al. 2017;Salem et al. 2019) were used to identify the produced nanoparticles: • Visual and UV-Vis: SPIONS at 200-500 nm and Au-NPs at 450-600 nm were measured using a spectrophotometer (Jasco V-670 UV-V), as shown in Figs. 1a and b, respectively. • FT-IR analysis: Au-NPs and SPIONS were singly purified using repeated centrifugation at 8000 rpm for 16 min and redistributed into 10 mL of deionized water. After drying, the synthesized nanoparticles were subjected to FTIR analysis, and the FTIR spectra were performed and recorded with a Fourier transform infrared spectropho-1 3 tometer of type Nicolet 870 between 4000 and 400 cm −1 with a resolution of 4 cm −1 as shown in Fig. 2a, b. • XRD analysis: The oven-dried nanoparticles were coated onto an XRD grid and analyzed by an X-ray diffractometer (6000-shimadzu-Japan) (Fig. 3a, b). • TEM analysis: TEM-2100 (JEOL Japan) was used to determine the particle size, distribution, and morphology of the formed nanoparticles (Fig. 4a, b).

In vitro cytotoxic activity
The anticancer activities of S. officinalis HEE, biosynthesized Au-NPs, and SPIONS against the MCF-7 breast cancer cell line (ATCC® HTB-22™) were investigated using an in vitro MTT assay. Cell lines obtained from the American Type Culture Collection (Manassas, VA, USA) were maintained in the recommended RPMI-1640 and D10 medium consisting of Dulbecco's complete medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated (56 °C) fetal bovine serum, penicillin (100 IU/mL), l-glutamine (3 mM), and streptomycin (100 mg/mL). An incubator of 95% air and 5% CO 2 at 37 °C with an atmosphere was used for cell growth. The MCF-7cell line was exposed to HEE, Au-NPs, SPIONS, and Doxorubicin (standard drug) in various concentrations; 100, 50, 25, 12.5, 6 μg/mL, and 0 concentrations for control wells (only the nutrient medium was added to the cells) for 48 h later. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, according to Van Meerloo et al. (2011). Briefly, cells were allowed to adhere for one day in a CO 2 incubator at 37 °C in 96-well culture plates. After the respective exposures, MTT (5 mg/mL of stock in PBS) was added and the plates were incubated further for 4 h. Then, supernatants were thrown in, each well mixed gently after the addition of 200 μL of DMSO. The intensity of the absorbance of light was proportional to the number of viable cells in each well, which was read in an enzymelinked immunosorbent assay (ELISA) plate reader by Biotek (ELX-800) at 570 nm. All experiments were performed in triplicate and expressed as mean ± SD:
Our findings are consistent with previous research on Salvia species in which HEE (70%) contains more total phenolics than total flavonoid content (Mocan et al. 2020;Veliˇckovi´c et al. 2011). Concerning the phenolic profiles of Salvia species that have been studied before (Dib et al. 2021;Mocan et al. 2020;Afonso et al. 2019), our results agreed with the types of phenolics found but not with the amounts found.
The most widely used technique for estimating phenolic and flavonoid compounds in plants and seaweeds is highperformance liquid chromatography (HPLC) (Aguilar-Hernández 2017). The phenolic contents in the HEE of S. officinalis leaves were then accurately quantified and identified using HPLC analysis. Table 3 depicts the identification of six polyphenol carboxylic acids, which represent a large group of naturally occurring organic compounds having a wide range of medicinal activities, particularly cytotoxic and antioxidant activities. These phenolic acids (mg/g extract) are either cinnamic acid derivatives, the most abundant of which is ferulic (67.26), followed by chlorogenic (3.12), caffeic (3.11), p-coumaric (1.13), and rosmarinic (0.53), or a benzoic acid derivative, protocatechuic (0.65). In addition, one flavan-3-ol, catechin, and one aglycon from the flavone class (apigenin 5.29) were identified, as well as two glycosides, quercetin-7-o-glucoside (3.39) and luteolin-7-orutinose (2.01). These compounds were found to possess potent antioxidant and cytotoxic activities (Umamaheswari et al. 2018;Zduńska et al. 2018;Liu et al. 2017;Maiyo et al. 2016;Shukla and Gupta 2010).
It is worth mentioning that most of the phenols and flavonoids identified in this study are similar to those found in the aqueous, methanolic, and ethanolic extracts of other Salvia species Following characterization, the HEE of S. officinalis leaves was used separately for the biosynthesis of Au-NPs and SPIONS via the reduction of their respective salts, with the process taking just a few minutes to complete. The formation of NPs was initially evidenced by the change in colour of the solution from light-yellow to reddish-violet in the case of Au-NPs and deep green in the case of SPIONS. The intensity of the colour is deepened by time as nanoparticle formation is directly proportional to time. The reducing properties of the hydroxyl (OH) groups in HEE phytochemicals (Tables 1, 2, 3) may be responsible for the formation of these nanoparticles. The presence of these phyto-reductants in an aqueous alkaline solution with HAuCl 4 or FeCl 3 may result in the following redox reactions (Fig. 1): The high alkalinity of the reaction medium neutralizes HCl once it is formed (Eqs. 3 and 5), shifting the reaction direction between the HEE phenolics and HAuCl 4 or FeCl 3 towards the formation of Au-NPs or SPIONS. This could explain why these nanoparticles only form in a highly alkaline solution (i.e., pH13). Also, active HEE phytochemicals like phenolics, flavonoids, terpenoids, polysaccharides,  75.43 ± 1.01 ---Total soluble sugars (mg/g extract) 52.24 ± 0.68 620 nm tannins, and proteins (Tables 1, 2) play an important role in the capping of the Au-NPs and SPIONS (stabilization and protection from aggregation) in the aqueous medium (Fig. 2), as has been reported before (Zayadi and Bakar 2020).
The formation of NPs was also confirmed by UV-visible surface measurements, as shown in Fig. 3, where the typical plasmon peaks for Au-NPs and SPIONS are in the ranges of 275-325 nm and 525-540 nm, respectively. The HEE constituents involved in the reduction and capping of the nanoparticle can be identified by the FTIR technique. Figure 4 shows that the FTIR of the HEE, Au-NPs, and SPIONS has a broad peak at 3464 cm −1 , which corresponds to the O-H stretch, which may be H-bonded to phenolics and alcohols. At 2921 cm −1 , a narrow band was seen, suggesting the existence of C-H stretched alkane groups. Broadbands were also observed at 1629 cm −1 , which corresponds to similar conjugation effects of N-H stretching; 1372 cm −1 corresponds to the N-O stretch of nitro compounds; 1109 cm −1 is characteristic of the N-H stretch of aliphatic or aromatic amines, and a narrow band at 667 cm −1 is due to the bending vibrations of N-H groups in proteins. There have been other studies that say the wavenumber signal of the previous peaks may be caused by phytoconstituents found in plant extracts like proteins, alkaloids, terpenoids, flavonoids, and phenolics. These phytoconstituents may be responsible for reducing and stabilizing the biosynthesized nanoparticles (Singh et al. 2018;El-Rafie et al. 2017;Sengani 2017;Kalpana et al. 2016).
The XRD spectrum identified the crystalline structure of the biosynthesized NPs of gold and iron oxides (Fig. 5) (Parimala and Santhanalakshmi, 2014), respectively. This result confirmed the nanoparticle crystalline structure. The particle size is obtained by taking the average of the sizes at the peaks using the Debye-Scherrer equation (Naganthran et al., 2022), as shown in Tables 4 and 5 for Au-NPs and SPIONS, respectively. They were found to be 9.27 nm and 64.95 nm, respectively.
Where D is the crystal size, λ is the wavelength of X-ray, ẞ is the full-width at half-maximum (FWHM) value of XRD diffraction lines, Ɵ is the half-diffraction value of 2Ɵ and K = 0.9 is a constant.
The biosynthesized Au-NPs and SPIONS were further studied using transmission electron microscopy (TEM). The latter has an average core dimension of 20-25 nm for Au-NPs and 30-35 nm for SPIONS, according to the TEM images and particle size distribution histograms in Fig. 6. As shown in these photos, the particles are about spherical and hexagonal in shape (Fig. 6).    Because Au-NPs are inert, they are non-toxic and safe to use; thus, their biomedical applications, particularly in cancer treatment and diagnostic tools, have been extensively researched (Anik et al. 2021;Nashat and Haider 2021;Sim and Wong 2021). The MTT assay was used to compare the cytotoxic activity of biosynthesized Au-NPs and SPIONS against the human breast cancer (MCF-7) cell line and its related HEE and Doxorubicin as a standard drug at different concentrations (6.25, 12.5, 25, 50, and 100 l/mL). Table 6 and Fig. 7 show the results. Both nanoparticles and their related HEE produced dose-dependent cell inhibition; the inhibitory concentration (IC 50 value) for Au-NPs, SPIONS, and HEE, respectively, was 6.53, 6.97, and 26.12 µg mL −1 .
Because of their thermal ablation, photodynamic property, and angiogenesis, Au-NPs are cytotoxic to cancer cells. On the other hand, the ability of SPIONS to kill tumor cells may be explained by the production of reactive oxygen species or hyperthermia (Aswathanaryan 2018; Silva et al. 2011;Johannsen et al. 2010). It is likely that HEE has cytotoxic effects because it contains phenolics, especially rosmarinic acid, which has been shown to stop the growth of many human cancers (Yesil-Celiktas et al. 2010;Xavier et al. 2009).
The surface area to volume ratio of Au-NPs and SPIONS nanoparticles is higher than that of HEE. This allows more atoms to be on the surface, as well as easier cell membrane penetration and interaction with intracellular materials, which leads to easier cell destruction.

Conclusions
The current work reveals that Au-NPs and SPIONS can be synthesized in an environmentally friendly manner by using the hydroethanolic extract (HEE) of S. officinalis leaves, where the phytochemicals in this extract serve as both reducing and capping agents for the formed nanoparticles. This simple and easy method has various benefits, which include biocompatibility, cost-effectiveness, and ease of scale-up production. The profitable biosynthesis of both Au-NPs and SPIONS was proved by different techniques such as FTIR, UV-visible spectroscopy, TEM, and XRD. The results of this study reveal that both Au-NPs and SPIONS have potent in vitro cytotoxic effects on MCF-7 (a human breast cancer cell line). Future research would include the validation of environmentally friendly manufactured Au-NPs and SPIONS that could be used in cancer therapy. Accordingly to the results of this work, S. officinalis HEE can be used efficiently to synthesize green Au-NPs as well as SPIONS, which can be used as anticancer agents. The NPs generated in this way could also be used in the biomedical field to discover potential therapeutic agents for a range of human diseases.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors received no financial support for the research, authorship, and/or publication of this article.

Conflict of interest The authors declare no conflict of interest.
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