Cytotoxic activity of silver nanoparticles prepared from Psidium guajava L. (Myrtaceae) and Lawsonia inermis L. (Lythraceae) extracts

Purpose: To biosynthesize silver nanoparticles (AgNPs) using Psidium guajava L. and Lawsonia inermis L. leaf extracts, and investigate their antioxidant and cytotoxic activities. Methods: The aqueous extracts were prepared by maceration in distilled H2O followed by partitioning with EtOAc. AgNPs were prepared by treating the extracts with 1 mM AgNO3 and then were characterized by UV-vis and FTIR analyses, and transmission electron microscopy (TEM). MTT cytotoxicity and 2,2`-azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) antioxidant assays were used to assess their cytotoxic and antioxidant properties, respectively. Results: AgNPs from P. guajava and L. inermis extracts exhibited good morphological stability and showed moderate antioxidant activity (68.1 and 71.9%, respectively) compared to their extracts. Equipotent cytotoxicity against HCT-116 and MCF-7 cells was observed for AgNPs derived from P. guajava, while AgNPs derived from L. inermis possessed two-fold cytotoxicity compared to their corresponding extracts. Phytochemical analysis of P. guajava afforded pyrogallol, quercetin, quercetin3-O-β-xylopyranoside, quercetin-3-O-β-arabinopyranoside, and quercetin-3-O-α-arabinofuranoside, while L. inermis afforded lawsone and luteolin. Conclusion: Flavonoids and phenolics play a major role in reducing Ag+ ions, surface coating, antioxidant, and cytotoxic activities of AgNPs. The biocompatible AgNPs produced by L. inermis demonstrate promising cytotoxic activity that could contribute to new cancer treatments.


INTRODUCTION
Over the past few years, there has been an upsurge in the synthesis of metallic nanoparticles (NPs) due to their powerful applications in different areas, including medicine [1]. Methods used for synthesizing AgNPs include chemical and physical techniques, such as ion-sputtering, thermal production, reduction, inert gas condensation, and sol-gel methods which unfortunately use high energy requirements, hazardous chemicals, difficult wasteful purification processes, and high cost [2]. So, there is a rising demand to develop environmentally safe processes, such as green syntheses/biological syntheses either by microorganisms or plant extracts [3,4]. The leaves of Psidium guajava L., the common guava tree, Myrtaceae, contains flavonoids, which are responsible for many pharmacological activities, including antioxidant, anti-inflammatory, antitumor, and antiviral activities [5]. Lawsonia inermis L. (Lythraceae), commonly known as Henna or Mehndi, has many benefits, including cosmetic, psychological, and medicinal applications with lawsone (2-hydroxy-1,4 naphthoquinone) as the main biomolecule, in addition to diverse groups of compounds, such as simple phenolics, coumarins, flavonoids, tannins, naphthalene derivatives, lignans, alkylphenones, triterpenes, steroids, alkaloids, and volatile oils [6].
Breast and colon cancers are common causes of death worldwide [7]. Several cancers respond to chemotherapy initially but finally, develop resistance. Thus, discovering a safe and costeffective drug for cancer treatment is vital. AgNPs were reported to inhibit human glioblastoma cells' proliferation, impair normal cellular functions, disrupt membrane integrity and induce several apoptotic signaling genes, causing programmed cell death [8] [9].
Hence, this study aimed to investigate forty-one plant extracts for their ability to synthesize AgNPs, and also, to evaluate the antioxidant and cytotoxic activities of the latter and to isolate the bioactive molecules.

EXPERIMENTAL Plant materials
The specified plant parts (Table 1) were either harvested in February 2014 from the farm of Pharmacognosy Dept., Faculty of Pharmacy, Mansoura, or purchased from a specialized herbal store in Mansoura, Egypt. The taxonomical identities of the obtained plants were confirmed by Prof. Farid A. Badria, Professor of Pharmacognosy, Faculty of Pharmacy, Mansoura, and according to Boulos (2005) [10]. The collected plant parts were dried and ground to a fine powder before their extraction. Voucher specimens were deposited in the herbarium of Pharmacognosy Dept., Faculty of Pharmacy, Mansoura, Egypt under the successive codes (from 014-Mansoura-5 to 014-Mansoura-46).

Plant extraction
The dried powders of the investigated plants (100 g) were extracted by maceration with dist. H2O for 24 h. (3 x 150 mL) and kept in a refrigerator for further processing. The combined aqueous extracts, in each case, were filtered using a Büchner funnel and partitioned by shaking with EtOAc (3 x 450 mL) in a separating funnel. The combined EtOAc extracts, in each case, were then evaporated to dryness using a rotary evaporator at 45 ˚C and stored at 4 ˚C for further investigation using AgNPs synthesis assay. For phytochemical investigation of the most active extracts, the above extraction procedure was repeated using 1000 g of powdered plant materials.

Synthesis of AgNPs
Aqueous silver nitrate solution (AgNO3, 1 mM) was prepared in a dark brown bottle to be used in the green synthesis of AgNPs [11]. Serial dilutions (0.1, 0.05, 0.025, and 0.0125 mg/mL) of each plant extract were prepared in dist. H2O to investigate the suitable concentrations that cause rapid synthesis of stable AgNPs. Typically, in a Wassermann tube, 2 mL of 1 mM AgNO3 solution were added dropwise to 1 mL of each plant concentration at 50 ˚C. The change in color in each case was monitored at several time intervals for one hour by measuring the absorbance of the reaction mixture at 450 nm. The produced AgNPs were collected by an ultracentrifuge (MIKRO 220 R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) at 15,000 r.p.m. for 30 min followed by re-dispersion of the obtained pellets in DMSO.

UV-visible spectroscopy
The process of reducing silver ions (Ag + ) from solution to AgNPs was observed by recording the UV-vis spectra of the reaction mixture at 10, 20, 30, and 60 min [12,13]. The spectra were measured at the UV-vis range from 200 to 800 nm using a SHIMADZU, UV-1601PC spectrophotometer. Silver nanoparticles were analyzed using quartz cuvettes (1 cm path) at room temperature. The corresponding dilution of untreated aqueous plant extract was used as the blank.

Fourier transform infrared spectroscopy (FTIR)
The FTIR analysis for each plant extract before and after AgNPs production was achieved to characterize and compare the diminished peaks of the functional groups of the biomolecules that are possibly involved in the green synthesis process [14]. FTIR measurements were performed by blending the dry synthesized AgNPs with KBr pellets (IR grade) and analyzing with an FTIR spectrometer (Nicolet IS10 of Thermo Fisher Scientific Inc., USA) from 400-4000 cm -1 , using OMNIC 8.0.380 software, at a resolution of 8 cm -1 , and at room temperature.

Transmission electron microscopy (TEM)
Imaging and analytical identification of the synthesized AgNPs were performed by TEM to evaluate their morphological properties, including shape and size [15]. A JEOL TEM-1230 instrument attached to a CCD camera, at an accelerating voltage of 120 kV was used for this purpose. The sample was prepared by adding a few drops of AgNPs, dispersed in dist. H2O on a carbon-coated copper grid, and was left to evaporate before imaging.

ABTS antioxidant assay
The antioxidant activity of the investigated materials was assessed using ABTS radical scavenging assay according to the reported data [16]. The inhibition (%) was calculated based on the reduction in absorbance (OD734) of the reaction mixture and according to the equation: Ascorbic acid (100 μL, 2 mM) was used as a positive control. Blank was prepared from a methanol-phosphate buffer (1:1 v/v) with no ABTS added. Negative control was run with ABTS with no sample added.

Cell lines
Two human cell lines; the colon cancer (HCT-116) and the breast cancer (MCF-7) cell lines (Holding company for biological products and vaccines, VACSERA, Cairo, Egypt), were used to evaluate the cytotoxic activity.

Cytotoxicity assay procedure
The investigated cells were seeded (5 x 10 4 cells/mL) in 96-well plates (100 µL/well). After overnight incubation, different samples (6 concentrations, in triplicates) were added and further incubated at 37°C and in a 5% CO2 atmosphere for 48 hours. DMSO (0.5%) was utilized as a negative control. Subsequently, aliquots of MTT solution (15 µL, 5 mg/mL in PBS) were added to each treated well and incubated for another 4 hours. The produced formazan metabolite was dissolved by the addition of aliquots of acidified sodium dodecyl sulfate solution, SDS (100 µL, 10% SDS in 0.01 M HCl). The absorbance (OD570) was recorded by a Biotek® microplate reader after incubation for 14 hours [17]. Standard deviation (SD) and IC50 were calculated. The anticancer drug, cisplatin was utilized as the positive control.

Purification of the EtOAc extract of Psidium guajava
The EtOAc extract of P. guajava leaves (2.

Purification of EtOAc extract of Lawsonia inermis
The EtOAc extract of L. inermis leaves (2 g), was purified using a silica gel cc. (

Statistical analysis
Data are presented as mean with SD. Statistical significance was set at p < 0.05. Data were subjected to One-Way ANOVA followed by a post-hoc Tukey's test. All statistical analyses were carried out using GraphPad Prism software.

UV/Vis spectra
The color change obtained after adding AgNO3 solution is a quick indicator for monitoring AgNP synthesis [12]. A stable yellowish-brown color was produced due to the excitation of surface plasmon resonance (SPR).  Therefore, the different dilutions for each plant extract were used as blanks to compare the produced color. Forty-one plant extracts were evaluated for their capacity towards the production of AgNPs by measuring the UV-vis absorbance of the reaction mixture at λ450, and the results are displayed in Table 1.
Furthermore, the reduction process for the most active extracts (viz., P. guajava and L. inermis leaves) was monitored by scanning the UV-vis spectra of their reaction mixtures at the wavelength range from 200 -800 nm, at 0 -60 min after treatment, Figure 1 (a & b). The recorded spectra showed absorption maxima at 440 to 460 nm, corresponding to the characteristic SPR of the resulting AgNPs [18]. Figure 1: Overlay of the UV-vis absorbance spectra (from λ200-λ800 nm) of the reaction mixtures after addition of 1 mM AgNO3 solution for the production of AgNPs, recorded as a function of time; a) Psidium guajava leaf extract with a marked peak at λ462 nm, and b) Lawsonia inermis leaf extract with a marked peak at λl447 nm, at 10, 20, 30, and 60 min

Fourier transform infrared (FTIR) spectra
FTIR spectroscopy was used to recognize the potential functional groups of the biomolecules in the active extracts, which may be accountable for the reduction of silver ions and the stabilization of the produced AgNPs [14]. FTIR absorption spectra of P. guajava and L. inermis leaves extracts, before and after reduction of silver ions, are displayed in Figures 2a & 2b, respectively.

Transmission electron (TEM) micrographs
TEM was used to identify the size and shape of AgNPs formed by the tested plant extracts [15]. Representative TEM micrographs of the developed AgNPs by P. guajava and L. inermis leaf extracts after 60 min using the optimum concentration in each case is displayed in Figure  3. It revealed the production of nanoparticles of a size range from 5 to 30 nm, mostly spherical, well-dispersed, and capped with an organic matter from the plant constituents.

ABTS antioxidant activity
The potential antioxidant activity of AgNPs synthesized and capped by P. guajava and L. inermis leaves extracts, was evaluated using ABTS radical cation antioxidant assay. Additionally, the antioxidant effect of their corresponding extracts was tested so it can be compared with the produced AgNPs. The antioxidant capacity is represented as a percentage of inhibition in the color intensity of the preformed blue ABTS radical cation at λ734 nm, Table 2. 90.7 ± 0.7 * a G = Psidium guajava and H = Lawsonia inermis, and ascorbic acid was used as a standard. b Data presented as mean ± standard deviation (SD), n = 3; * Compared to control and statistically significant at p < 0.05

Cytotoxic activity of AgNPs
Cell-based in vitro MTT colorimetric assay is a very important and simple method for the evaluation of cytotoxicity [17]. In our study, two human cancer cells; HCT-116, the colorectal carcinoma, and MCF-7, the breast cancer cells, were used to evaluate the cytotoxic activity of the synthesized AgNPs and their corresponding extracts by MTT assay. Cisplatin (nM) was used as a positive control and IC50 for the investigated samples was calculated in mg/mL, Table 3. Cisplatin was used as a positive control. b IC50 values are represented as mean ± SD, n = 3. * Compared to their corresponding extract and at statistical significance, p < 0.05

AgNPs synthetic capacity of the isolated compounds
To evaluate the green synthetic capacity of AgNPs by the isolated compounds, the UV-vis absorption spectral patterns after treatment with AgNO3 solution were studied. The obtained results are presented in Table 4.

DISCUSSION
The current study investigated the capacity of several plant extracts towards the green synthesis of AgNPs. Also, it aimed at the characterization and cytotoxic evaluation of the produced nanoparticles by the most active plant extracts. In addition, identification of the biomolecules and related functional groups responsible for the green synthesis process. The size-based optical characteristics of the produced AgNPs is a fast indicator, and is initially indicated by the color change of the reaction mixture [12,18]. UV-vis absorbance at λ450 indicated that only two extracts viz., P. guajava and L. inermis leaf extracts, out of forty-one investigated plant extracts, showed high AgNPs synthetic activity, Table 1. The optimum concentration for producing AgNPs by P. guajava and L. inermis leaf extracts was 0.05 mg/mL. The obtained absorbance was stable and increased by time (from 0 -60 min after adding AgNO3 solution), where typical absorption maxima were obtained in the case of P. guajava and L. inermis leaf extracts at λ462 and λ447, respectively.
FTIR spectral evaluation showed absorption bands at 1034 -1061 cm -1 (C-O stretching), while the presence of absorption bands at 382 -3421 cm -1 (O-H stretching) indicated phenolic/alcoholic groups that are dominant in tannins and flavonoids. On the other hand, peaks that arose around 1651 -1680 cm -1 (C=O) indicated the presence of carbonyl groups which may be ketones as in flavonoids, or carboxylic groups as in tannins. Clearly, the FTIR spectra of the extracts of P. guajava and L. inermis leaves before and after reduction of Ag + to Ag º ( Figure  2a & 2b, respectively) showed that the carbonyl peaks were considerably attenuated, indicating its contribution in the AgNPs-synthesis process. Such compounds were participating in both synthesis and capping (coating) of the produced silver nanoparticles [23,24].
The TEM micrographs of AgNPs formed by the leaves extracts of P. guajava and L. inermis, Figure 3a & 3b, respectively, showed typical morphological characteristics including, the shape and size of the prepared AgNPs, indicating a good stabilization effect of the investigated plant extracts. This was further confirmed by monitoring their physical properties, where no visible changes were observed over the period of a few months [15].
The antioxidant activity of photosynthesized AgNPs by DPPH assay was previously reported, and it was concluded that AgNPs can be utilized as potential free radical scavengers [14]. In the current study, the synthesized AgNPs by P. guajava and L. inermis showed moderate antioxidant activity with 68.1 and 71.9% inhibition, respectively (Table 2). However, significant antioxidant activities (p < 0.05) were obtained for P. guajava and L. inermis extracts (89.7 and 90.3%, respectively) which is comparable to L-ascorbic acid (90.7% inhibition). Therefore, it is worth noting that the obtained antioxidant activity in the first case is mostly due to AgNPs and is not attributable to the capping compounds, as the antioxidant polyphenolics were involved in the reduction of Ag + to Agº and become deactivated. This further explains the role of polyphenolics in the green synthesis of AgNPs, and why the antioxidant activity of the synthesized AgNP is lower than that of the extract. These results are in full agreement with the previously published review that correlated the antioxidant activity of green synthesized AgNPs to both the surface coating material as well as the silver nano-sized particles [25].
Previous studies reported the potential use of silver nanoparticles as cytotoxic agents [8,9]. The cytotoxic activity of P. guajava-synthesized AgNPs showed no significant difference from that of the corresponding plant extract (p < 0.05). However, L. inermis-synthesized AgNPs showed a significant increase (p < 0.05) in the cytotoxic activity towards both MCF-7 and HCT-116 (IC50 55 and 56.6 µg/mL, respectively) compared to the leaves extract of this plant which showed higher IC50 values against the investigated cell lines (>100 and 80 µg/mL, respectively). Consequently, the above findings suggested that the cytotoxic activity is attributed to both the AgNPs and the surface capping material formed by the plant phytoconstituents. In other words, the obtained cytotoxic activity is a result of a synergetic action between AgNPs and the plant phytoconstituents, as in the case of L. inermis.
Phytochemical investigation of the active extracts of P. guajava and L. inermis afforded eight known compounds categorized as simple phenols (G1, H1, and H2) and flavonoids (G2-G5, and H-3), Figure 4. Generally, pyrogallol G1 was more active than flavonoids (G2-G5, and H-3) in the green synthesis of AgNPs, Table 4. For pyrogallol, it can be concluded that the occurrence of ortho-dihydroxy phenolic function is accountable for reducing Ag + and stabilizing the synthesized AgNPs according to the outlined mechanism, Figure 5.
Meanwhile, for flavonoids, the 3-OH is important for the activity, as in the flavonol G2 (quercetin) that showed higher activity compared to the flavone H3 (luteolin), Table 4. However, luteolin is still active due to the presence of 3`,4`-ortho dihydroxyl groups of B-ring, indicating their contribution to the activity. The glycosidic linkage present in the 3-O-pentosides series did not influence the activity, as deduced from the results of compounds G3 -G5 that exhibited nearly the same activity as their aglycone, quercetin G2. The suggested mechanism of reduction of Ag + and stabilization of the produced AgNPs by biomolecules having the flavonoid structure is demonstrated in Figure 6. Regarding lawsone H2, it almost has the same activity as flavonoids. Regarding the aromatic acid, pcoumaric acid H1, it showed no significant activity due to the lack of the ortho-dihydroxyl groups, which is shown to be important for the AgNPs synthesis activity.

CONCLUSION
AgNPs with good morphological stability have been prepared from the leaf extracts of Psidium guajava and Lawsonia inermis. Furthermore, the extracts afforded seven compounds and revealed that simple phenols and flavonoids, especially ortho-dihydroxy bearing structures, may have a major role in the reduction of Ag + and coating of the synthesized AgNPs. The findings further reveal that while the synthesized AgNPs forthrightly contributed to the antioxidant effect, their cytotoxic activity may involve both AgNPs and the surface capping material formed by the plant phytoconstituents. Therefore, more research is needed to elucidate the anticancer mechanism of AgNPs synthesized by these natural products.

Conflict of interest
No conflict of interest is associated with this work.