Biofabricated Fatty Acids-Capped Silver Nanoparticles as Potential Antibacterial, Antifungal, Antibiofilm and Anticancer Agents

The current study demonstrates the synthesis of fatty acids (FAs) capped silver nanoparticles (AgNPs) using aqueous poly-herbal drug Liv52 extract (PLE) as a reducing, dispersing and stabilizing agent. The NPs were characterized by various techniques and used to investigate their potent antibacterial, antibiofilm, antifungal and anticancer activities. GC-MS analysis of PLE shows a total of 37 peaks for a variety of bio-actives compounds. Amongst them, n-hexadecanoic acid (21.95%), linoleic acid (20.45%), oleic acid (18.01%) and stearic acid (13.99%) were found predominately and most likely acted as reducing, stabilizing and encapsulation FAs in LIV-AgNPs formation. FTIR analysis of LIV-AgNPs shows some other functional bio-actives like proteins, sugars and alkenes in the soft PLE corona. The zone of inhibition was 10.0 ± 2.2–18.5 ± 1.0 mm, 10.5 ± 2.5–22.5 ± 1.5 mm and 13.7 ± 1.0–16.5 ± 1.2 against P. aeruginosa, S. aureus and C. albicans, respectively. LIV-AgNPs inhibit biofilm formation in a dose-dependent manner i.e., 54.4 ± 3.1%—10.12 ± 2.3% (S. aureus), 72.7 ± 2.2%–23.3 ± 5.2% (P. aeruginosa) and 85.4 ± 3.3%–25.6 ± 2.2% (C. albicans), and SEM analysis of treated planktonic cells and their biofilm biomass validated the fitness of LIV-AgNPs in future nanoantibiotics. In addition, as prepared FAs rich PLE capped AgNPs have also exhibited significant (p < 0.05 *) antiproliferative activity against cultured HCT-116 cells. Overall, this is a very first demonstration on employment of FAs rich PLE for the synthesis of highly dispersible, stable and uniform sized AgNPs and their antibacterial, antifungal, antibiofilm and anticancer efficacy.


Introduction
The growing pursuits in metal-based nanomaterials synthesis are hotly debated in several fields while acknowledging their unique physico-chemical and biomedical properties with specific advocacy for fitness in clinical settings as fascinating treatment modality, worldwide [1]. Considering that there is a wide scope to achieve desired properties in synthesized nanoparticles (NPs) including shape, size and stability by manipulating reaction conditions such as pH, temperature, concentration of metal precursors and concentration and nature of bio-reducing agents [2][3][4][5][6][7][8]. Besides, surface capping or encapsulation material of NPs deserves special importance due to being directly or indirectly concerned with

Synthesis and UV-Vis Analysis of LIV-AgNPs
Briefly, an apparent color change in the reaction mixture containing the aqueous solutions of PLE and AgNO 3 in 1:3 ratios (v/v), from pale yellow to light brown indicated the PLE bio-actives meditated bio-reduction of Ag + to LIV-AgNPs after 20 min at 25 ± 5 • C. The color of reaction mixture tuned into intense brown after 24 h. The appearance of a sharp UV-Vis band at λ max 428 nm was observed which is likely due to the surface plasmon resonance (SPR) of nascent LIV-AgNPs in colloidal solution (Figure 1a). The UV-Vis absorption peak position (400-500 nm) and formation of characteristic brown color LIV-AgNPs were found concordant with the reports published on plant mediated green synthesis of AgNPs [6]. Besides, UV-Vis absorption (λ max 428 nm) analysis of colloidal LIV-AgNPs up to six months revealed that the NPs were highly stable as the experiments showed no significant change in SPR peak (Figure 1b).

Assessment of Bio-Actives in Pristine PLE and LIV-AgNPs by GC-MS and FTIR
Before, synthesis of LIV-AgNPs, the pristine PLE was put through to GC-MS analysis [24] in order to presume plausible bio-active compounds that may acted as, (i) reducing agent for free metal cations (Ag + → Ag 0 ), (ii) stabilizing agent while growth on nascent NPs in progress during nucleation phase and (iii) capping of fully grown or stabilized NPs as described in our previous study [24] was illustrated in schematic mechanism of LIV-AgNPs formation ( Figure 2). The GC-MS spectrum of pristine PLE ( Figure 3) reflected a total of 37 peaks (P) for a variety of bio-actives were described in our previous study [24]. Based on their peak area, four major bio-actives in PLE were found to be long/short chained hydrocarbon fatty acids containing terminal -OH and -COOH groups, viz. nhexadecanoic acid (P15-21.95%), linoleic acid (P19-20.45%), oleic acid (P20-18.01%) and stearic acid (P21-13.99%) [24]. Besides, two polyphenolic bio-actives were also detected namely cardanol monoene (P27-11.92%) and piperine (P31-1.83%) [24] likely play axillary role in bio-reduction and capping of NPs (Table S1) [24]. Next, the FTIR-based assessment of as-prepared LIV-AgNPs also demonstrated the presence of PLE bio-actives that can be argued being responsible for bio-reduction of metal cations into nascent NPs, stabilization and capping of AgNPs. The FTIR spectrum in Figure 4a-c, demonstrates a variety of molecular signatures of PLE bio-actives adsorbed on AgNPs, which in fact appeared as sharp, broad, strong and weak signals pertaining to their band behavior such as stretching, banding and vibrations. In Figure 4a, a dense area of FTIR spectrum ranged between 3500 cm −1 and 3700 cm −1 indicated the presence a majority of PLE bio-actives associated to AgNPs surface and hence we analyzed this area at a high resolution. The observations of this section suggest the presence of medium and sharp stretching were assigned to the free -OH groups of alcohols [25]. Whereas, strong and broad stretching around 3236 cm −1 confirmed the presence of intermolecular bonded -OH and -NH groups of carbohydrates/lipids and primary amines, respectively, as depicted in Figure 4b, signify the reduction of Ag + to Ag 0 and capping of AgNPs [26]. The weak vibrations between 2926 and 2850 cm −1 , and 2135 cm −1 were assigned to stretching of C-H and C≡C groups of lipids and alkyne, respectively (Figure 4b). The peak at 1737 cm −1 is likely due to the presence of carbonyl (C-O) group of FAs, whereas peak at 1645 cm −1 represent carboxylic groups (C=O) of FAs and amine group (N-H) of protein ( Figure 4c) [24,26]. Indeed, the appearance of C-O and C=O signals strongly advocate the involvement of FAs and proteins in bio-reduction and PLE bio-actives corona likely physisorbed on the surface of LIV-AgNPs. Besides, the peak at 1456 cm −1 can be ascribed to CH 2 deformation or due to C-O-H bending, 1373 cm −1 represents O-H groups of phenolic compounds, signal at 1153 cm −1 was taken as C-O-C stretching which signified the presence of carbohydrates, peak around 1026 cm −1 was assigned to O-H stretching of polyphenols ( Figure 4c) [26]). Overall, our GC-MS and FTIR results strongly suggest an active role of PLE attributed FAs and polyphenolic in the synthesis of LIV-AgNPs. In same line, Rao and Trivedi [27] have also demonstrated formation of FAs encapsulated AgNPs using stearic, palmitic and lauric acids as bio-reducing and stabilizing agents. Recently, the study of Gnanakani et al. [26] exhibited the FAs namely octadecanoic, hexadecanoic and octadecanoic acids in microalgae Nannochloropsis extract as potential bio-reducing and stabilizing agents in synthesis of AgNPs. Beyond the abundance of FAs, auxiliary phenolics, proteins, carbohydrates and enzymes bio-moieties in the benign milieu of PLE can be argued to play both key roles in plant extract mediated bio-fabrication of nanomaterials [25].

Electron Microscopic Properties of LIV-AgNPs
The SEM micrographs in Figure 5a demonstrated a significant level of agglomerations in LIV-AgNPs when allowed to dry to solid powder. Besides, the elemental composition of PLE-AgNPs obtained by using EDS showed prominent peaks for carbon (30.6%), oxygen (44.85%) and silicon (9.43%) along with the characteristic peak of Ag (11.39%) at approximate 3 keV (Figure 5b). Contrarily to powdered LIV-AgNPs (Figure 5a), the TEM analysis of colloidal LIV-AgNPs solutions witnessed a great level of dispersity in aqueous environment, which was likely attributed by repulsion forces existed between two O-H groups hanging out from the soft PLE corona of AgNPs (Figure 5c). At the same time, the ImageJ software-based size determination on TEM micrographs revealed the sized of LIV-AgNPs was ranged between 1-10 nm with an average diameter of 5.37 ± 1.09 nm (Figure 5d).

SEM Based Analysis of LIV-AgNPs Interaction and Cellular Damage
To validate the antibacterial and antifungal activities of LIV-AgNPs, the treated and untreated cells of test strains were compared under SEM visualization. The results in Figure 8b-c exhibited significantly ruptured cell wall with deep pits and cavities formation in MDR-PA cells treated with 100 µg/mL of LIV-AgNPs, which were likely due to internalization and surface contact killing or on-site augmented cations mediated toxicity, as described elsewhere [25]. Under identical conditions, Gram-positive MRSA cells were observed with significant structural damage along with tremendous bulging and deep cuts in cell membrane (Figure 8e,f), which indicated increased cytoplasmic granularity likely due to prompted interaction and internalization of LIV-AgNPs as compared to untreated cells (Figure 8d) [5]. Similarly, in the case of fungi, the LIV-AgNPs exposed C. albicans cells showed significant changes in native morphology such as deep pits in cells compared to untreated control (Figure 8h,i) as reported elsewhere [32]. Besides, Anuj et al. [33] have demonstrated a steady release of Ag + from AgNPs and thus accumulated cations can destabilize cell membrane to combat with efflux-mediated drug resistance in Gram-negative bacteria. Recent study of Al-Kadmy [34] has also suggested that coating of AgNPs had enhanced penetrative ability through the cell wall and kills the E. coli, S. aureus and vancomycin resistant Enteroccci cells on banknote currency effectively under tentative conditions, as compared to AgNO 3 .   Antibiofilm Studies of LIV-AgNPs Both, bacterial cells; Gram-negative MDR-PA and Gram-positive MRSA, and C. albicans fungi are well known for their biofilm producing ability and chronic nosocomial infections spread in hospital and associated settings [35,36]. Although, several metallic nanoantibi-otics were found having great potential either to cease or eradicate biofilm adherence [37]. Whereas, the propensity of nanoantibiotics to readily diffuse through the biofilm biomass in order to reach microbial cells seemed to be compromised due to enzymatic, non-enzymatic and pH mediated degradations [38]. Interestingly, the evidence suggests that FAs, either free or physisorbed on to surface of NPs can (i) suppress the regulation of quorum-sensing (QS) genes, (ii) quenched the diffusible QS signal factors such as acyl-homoserine lactones and autoinducer-2 (AI-2) and (iii) dysregulate the associated non-QS targets like efflux pumps, oxidative stress and ergosterol synthesis [39][40][41]. Taken together the antimicrobial potential of FAs and AgNPs, we tested LIV-AgNPs for their antibiofilm activities. In, fact, our GC-MS results prompted us to consider the LIV-AgNPs as encapsulated by PLE bio-active FAs viz. n-hexadecanoic acid (P15-21.95%), linoleic acid (P19-20.45%), oleic acid (P20-18.01%) and stearic acid (P21-13.99%) (Figure 3, Table S1) [24] and hence responsible for significant anti-biofilm activities against MDR-PA, MRSA and C. albicans. The data in Figure 9 revealed the inhibition of biofilm formation by MDR-PA cells as 23.31 ± 5.2%, 31.17 ± 3.2%, 40.16 ± 5.5%, 53.37 ± 4.2% and 72.75 ± 2.2%, at 31.25, 62.50, 125, 250 and 500 µg/mL of LIV-AgNPs, respectively, versus untreated control (100%). Under identical conditions, MRSA cells could limit the accumulate biofilm mass as 10.17 ± 2.3%, 15.06 ± 2.5%, 27.00 ± 2.9%, 49.70 ± 3.9% and 54.40 ± 3.1%, respectively. Besides bacterial cells, the biofilm formed by C. albicans was also found declined significantly (p < 0.05 *) as 25.60 ± 2.2%, 35.60 ± 1.3%, 41.65 ± 1.7%, 59.9 ± 3.2% and 85.44 ± 3.3%, respectively. In parallel, the SEM based comparative analyses of untreated controls ( Figure  10a,c,e) and LIV-AgNPs (100 µg/mL) treated MDR-PA (Figure 10b), MRSA ( Figure 10d) and C. albicans (Figure 10f) cells were resulted in significant disruption in their biofilm architectures. Overall, the obtained trends in biofilm formation suggest that FAs hold a great potential to inhibit or disrupt biofilm formation against several microbial pathogens, including S. aureus [42], P. aeruginosa [43] and C. albicans [39,44]. Beyond the proven antibacterial and antibiofilm track record of AgNPs [45][46][47], a variety of FAs have earlier been warranted as potential antimicrobial agent. For instance, study of Santhakumari et al. [48] demonstrated hexadecanoic acid (100 µg/mL) could interrupted the QS by loosening of biofilm architecture (>60%) of vibrios spp. like Vibrio harveyi, V. parahaemolyticus, V. vulnificus and V. alginolyticus without affecting their planktonic growth. Besides, 12.8 µg/mL of hexadecanoic acid alone could inhibit the biofilm formation in P. aeruginosa and E.coli as 64% and 81%, respectively [43]. In the same context, Soni et al. [49] also demonstrated that palmitic acid (hexadecanoic acid), stearic acid, oleic acid and linoleic acid present in extract of ground beef inhibit the auto-inducer signals activity of the reporter strain (Vibrio harveyi) and reduced E. coli biofilm formation.

Antiproliferative Properties of LIV-AgNPs on Human Colon Cancer Cells (HCT-116) Cell Viability Assay by MTT and Microscopic Analysis of HCT-116 Cells
In addition to antimicrobial activities, PLE-capped AgNPs were also assessed for their anticancer potential. For this, human colon cancer cells were-cultured with colloidal LIV-AgNPs (10-100 µg/mL) for 24 and the nano-toxicity of LIV-AgNPs against HCT-116 cells was measured by employing colorimetric MTT assay. Precisely, compared untreated control cells (100 ± 2.5%), there is an apparent decline trend in cell viability as 86.10 ± 5.9%, 81.5 ± 8.2% and 46.75 ± 7.9% at 10, 50 and 100 µg/mL of LIV-AgNPs, respectively ( Figure  11). At about 100 µg/mL, we observed a ca. 50% inhibition of the cell proliferation after 24 h. In parallel, HCT-116 cells exposed to LIV-AgNPs (10, 50 and 100 µg/mL) were also investigated for NPs induced morphological changes. The representative micrographs of HCT-116 cells clearly demonstrate that treatment of LIV-AgNPs caused significant morphological changes (Figure 12 b-d) as compared to untreated cells (Figure 12a). Our results were strongly supported by the findings of Kuppusamy et al. [50] who determined the IC 50 value of their Commelina nudiflora capped-AgNPs as 100 µg/mL against cultured HCT-116 after 24 h. Besides, as compared to a single extract like Chlorophytum borivilianum extract functionalized AgNPs, which showed IC 50 value of 254 µg/mL [51], the as prepared poly-herbal encapsulated LIV-AgNPs can act as much effective anticancer nanomedicine against human colon cancer cells. In this context, linolenic acid polymers impregnated to AgNPs have also been reported to show 82.3% inhibition rate against the rat pheochromocytoma PC 12 tumor cell line [52]. Similarly, fatty acids rich Argemone mexicana extract encapsulated AgNPs (100 µg/mL) were found to inhibit 80% human cervical cancer cell line (SiHa) proliferation [53]. The AgNPs have also been reported disrupting respiratory chain and cell division while releasing Ag+ in order to augment enhanced bacterial killing. It has reported that coating of AgNPs can result in improved functionality and corrosion resistance of magnesium structures in biomedical settings [54]. With the widespread application and inevitable environmental exposure, AgNPs can be accumulated in various organs. More serious concerns are raised on the biological safety and potential toxicity of AgNPs in the central nervous system (CNS), especially in the hippocampus. Further, Chang et al. [54] investigated the biological effects and the role of PI3K/AKT/mTOR signaling pathway in AgNPs mediated cytotoxicity using the mouse hippocampal neuronal cell line (HT22 cells). They found that AgNPs reduced cell viability and induced membrane leakage in a dose-dependent manner and AgNPs also promoted the excessive production of reactive oxygen species (ROS) and caused the oxidative stress in HT22 cells [54].

Preparations of Aqueous Extract of Liv52 Drug
To prepare the fatty acids rich poly-herbal Liv52 drug extract, Liv52 tablets (Himalaya Global Holdings Ltd., Bangalore, India), were crushed to fine powder and 5 g was then dissolved in 100 mL of ultra-pure water. After 1 h, the PLE solution was centrifuged at 12,000 rpm for 10 min and so collected supernatant was additionally filtered through the Wattman paper No. 1 [24]. Thus, obtained aqueous PLE was stored at 4 • C for the green synthesis of LIV-AgNPs.

GC-MS Based Assessment of Bio-Actives in Poly-Herbal Liv52 Drug Extract (PLE)
Considering the fact that Liv52 is a poly-herbal composition of C. spinosa, C. intybus, S. nigrum, T. arjuna and A. millefolium extracts [23], the gas chromatography massspectroscopy (GC-MS) based analysis on methanolic extract of PLE was performed to ascertain the bio-actives compounds that plausible involved in reduction, capping and stabilization of LIV-AgNPs, following the method described elsewhere [24,31].

Nanofabrication of Poly-Herbal liv52 Drug Extract Capped AgNPs (LIV-AgNPs)
For the synthesis of LIV-AgNPs, PLE (25 mL) was mixed into 75 mL of 0.1 mM AgNO 3 solution. The reaction mixture was then kept in dark at room temperature (30 ± 5 • C). The color of reaction mixture was changed from pale yellow to brown after 20 min and became even dark brown within 24 h, which indeed indicated the reduction of Ag + to Ag 0 NPs [8].

UV-Vis Spectroscopy and FTIR Analysis
Formation of LIV-AgNPs was monitored by using UV-Vis spectroscopy in range of 300-800 nm as described recently elsewhere [55]. The Fourier-transform infrared spectroscopy (FTIR) was performed to ascertain the presence of PLE bio-actives that have likely played either key or auxiliary role in the reduction Ag + to Ag 0 , stabilization of nano silver and capping of nascent LIV-AgNPs during synthesis [8].

Electron Microscopic and EDS Analysis of LIV-AgNPs
The shape, size and elemental composition of LIV-mediated synthesized AgNPs was carried out by scanning electron microscope (SEM), transmission electron microscope (TEM) and energy dispersive spectroscopy (EDS) following the methods described in our previous study [56].

XRD Analysis of LIV-AgNPs
The crystallinity and size of bio-synthesized LIV-AgNPs was analyzed by XRD machine as protocol described recently [57].

Microbial and Human Carcinoma Cell Cultures
In this study, multi-drug resistant Pseudomonas aeruginosa (laboratory strain), methicillinresistant Staphylococcus aureus (ATCC 33591) and Candida albicans (ATCC 14053) were used to investigate the antibacterial, anticandidal and antibiofilm activities of synthesized PLE-AgNPs. For anticancer efficacy assessment, the human colon cancer (ATCC No. CCL-247) cell line was used. Both, the microbial and human carcinoma cell cultures were maintained as described in earlier studies [9,58]. The antibacterial and antifungal activity of synthesized LIV-AgNPs was carried out using two-fold micro broth dilution method in the range of 62.5 to 2000 µg/mL against Gram-negative MDR-PA, Gram-positive MRSA and C. albicans fungal strains as method described by Ansari et al. [59]. The MIC value is defined as the lowest concentration of LIV-AgNPs at which no visible growth of bacteria and Candida was observed. After MIC determination of LIV-AgNPs, aliquots of 100 µl from wells having no visible growth was seen were further spread on MHA and SDA plates for 24 h at 37 • C and 28 • C, respectively, to calculate the MBC and MFC values. The lowest concentration of LIV-AgNPs that kills 100% population of tested bacteria and Candida, is considered as MBC/MFC values [59].
Further, agar well diffusion assay was performed to determine the zone of inhibition (in millimeter) of LIV-AgNPs against Gram-negative MDR-PA, Gram-positive MRSA and C. albicans as method described by Jalal et al. [8].

Ultrastructural Alteration Caused by LIV-AgNPs in Bacterial and Candidal Cells
The morphological changes caused by LIV-AgNPs in bacterial and yeast strains cells were examined by SEM analysis following protocol described in previous reports [60]. Briefly,~10 6 CFU/mL of MDR-PA, MRSA, and C. albicans cells treated with 100 µg/mL of LIV-AgNPs were incubated at 16 h at a recommended temperature. Thereafter, washing of treated and untreated samples were performed using centrifugation and then the pellets was fixed with glutaraldehyde (4% v/v) followed by osmium tetroxide (1%). After fixations, dehydration, drying and gold coating was performed and finally the effects of LIV-AgNPs on test strains of bacteria and Candida was seen under SEM at an accelerated voltage of 20 EV [61].

Inhibition of Biofilm Forming Abilities of MDR-PA, MRSA and C. albicans
The inhibition in biofilm formation after treatment with LIV-AgNPs was quantitated by employing the microtiter crystal violet assay [61]. Briefly, 20 µl of freshly cultured MDR-PA, MRSA and C. albicans were admixed with 180 µl of varying concentrations (31.25, 62.50, 125, 250 and 500 µg/mL) of as prepared LIV-AgNPs and then the plates were kept in incubator for 24 h. The cells without LIV-AgNPs were considered as control group. After incubation, the content from the microtiter wells were decanted and gently washed with PBS and left for drying. The adhered biofilm biomass was then stained with crystal violet solution (0.1% w/v) for 30 min. The excess dyes were decanted and washed again with PBS and dried the wells completely. So stained biofilm was then solubilized with 95% ethyl alcohol and quantitated by optical density at 595 nm [62].

Visualization of Biofilm Architecture by SEM
Besides, the effect of LIV-AgNPs on MDR-PA, MRSA and C. albicans biofilm architecture was investigated by SEM [62]. In brief, 100 µl fresh cultures of tested bacterial and yeast strains with and without LIV-AgNPs were inoculated on a glass coverslip in a 12-wells plate for overnight. After incubation, the glass coverslips were taken off and washed with PBS to remove the unadhered cells. After washing, the coverslips were fixed with glutaraldehyde (2.5% v/v) for 24 h at 4 • C. After fixation, washed the coverslips again and then subjected it to dehydration, drying and gold coating. After that, the effects of LIV-AgNPs on biofilm of tested bacteria and yeast were observed using SEM [61].

MTT Assay
Human colorectal carcinoma cell line was used to investigate the anticancer potential of synthesized LIV-AgNPs at different concentrations (10, 50 and 100 µg/mL) in a 96-well cell culture plates by measuring optical density at 570 nm and the cell viability (%) was estimated using given formula [62].

Statistical Analysis
Statistical analysis of data was done by one-way analysis of variance (ANOVA), Holm-Sidak method, multiple comparisons versus the control group (Sigma Plot 11.0, San Jose, CA, USA). The results indicate mean ± S.D. values determined with three independent experiments done in triplicate. The level of statistical significance chosen was * p < 0.05 unless otherwise stated.

Conclusions
This study demonstrates a simple one-pot procedure for synthesis of fatty acids rich aqueous extract of poly-herbal drug Liv52 stabilized LIV-AgNPs. GC-MS results demonstrated substantial proofs that PLE contributed terminal -OH and -COOH functional groups bearing FAs, namely n-hexadecanoic acid (21.95%), linoleic acid (20.45%), oleic acid (18.01%) and stearic acid (13.99%), that were speculated to reduce Ag + into Ag 0 and followed by stabilization with soft corona formation around the nascent NPs surface during synthesis reaction. Besides, the LIV-AgNPs were found to be potential nano-therapeutics agents in order to control bacterial growth and biofilm formation against Gram-negative MDR-PA, Gram-positive MRSA and C. albicans strains, in vitro. Significant interaction of PLE-AgNPs with both, Gram-negative and Gram-positive bacterial and fungal strains was observed. The propensity of LIV-AgNPs interaction and internalization in planktonic cells as well as biofilm biomass appeared clearly in SEM analysis of treated experimental sets of MDR-PA, MRSA and C. albicans owing to the difference in their cell wall composition. However, the antibacterial and antibiofilm potential of LIV-AgNPs might be due to a swift surface contact through a stubborn biofilm matrix formed around the colonized cells requires further investigations to understand the mechanism of their action mode for nanoantibiotics development. In addition, the dose-dependent cytotoxicity trend of LIV-AgNPs against cultured human colon cancer cells ensured that the FAs-rich PLE capped nanomaterials could act as potential anticancer nanodrugs. However, the anticancer data of LIV-AgNPs here reported are only preliminary and will be successively deeply investigated exploring their cytotoxicity on normal cells as well as the antiproliferative activity of LIV-52 extract alone, as control.

Data Availability Statement:
The data presented in this study are available in this manuscript.