Green Biosynthesis, Antioxidant, Antibacterial, and Anticancer Activities of Silver Nanoparticles of Luffa acutangula Leaf Extract

Abstract

Studies on green biosynthesis of newly engineered nanoparticles for their prominent medicinal applications are being the torch-bearing concerns of the state-of-the-art research strategies. In this concern, we have engineered the biosynthesized Luffa acutangula silver nanoparticles of flavonoid O-glycosides in the anisotropic form isolated from aqueous leave extracts of Luffa acutangula, a popular traditional and ayurvedic plant in south-east Asian countries. These were structurally confirmed by Ultraviolet-visible (UV-Vis), Fourier transform infrared spectroscopy accessed with attenuated total reflection (FTIR-ATR) spectral analyses followed by the scanning electron microscopic (SEM) and the X-ray diffraction (XRD) crystallographic studies and found them with the face-centered cubic (fcc) structure. Medicinally, we have explored their significant antioxidant (DPPH and ABTS assays), antibacterial (disc diffusion assay on E. coli, S. aureus, B. subtilis, S. fecilis, and S. boydii), and anticancer (MTT assay on MCF-7, MDA-MB-231, U87, and DBTRG cell lines) potentialities which augmented the present investigation. The molecular docking analysis of title compounds against 3NM8 (DPPH) and 1DNU (ABTS) proteins for antioxidant activity; 5FGK (Gram-Positive Bacteria) and 1AB4 (Gram-Negative Bacteria) proteins for antibacterial activity; and 4GBD (MCF-7), 5FI2 (MDA-MB-231), 1D5R (U87), and 5TIJ (DBTRG) proteins for anticancer activity has affirmed the promising ligand-protein binding interactions among the hydroxy groups of the title compounds and aspartic acid of the concerned enzymatic proteins. The binding energy varying from -9.1645 to -7.7955 for Cosmosioside (1, Apigenin-7-glucoside) and from -9.2690 to -7.8306 for Cynaroside (2, Luteolin-7-glucoside) implies the isolated compounds as potential bioactive compounds. In addition, the performed studies like QSAR, ADMET, bioactivity properties, drug scores, and toxicity risks confirmed them as potential drug candidates and aspartic acid receptor antagonists. This research auxiliary augmented the existing array of phytological nanomedicines with new drug candidates that are credible with multiple bioactivities.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Phytological origin is the main source for many flavonoids and corresponding flavonoid O-glycosides (FOGs) [1]; these FOGs are proven antioxidant [2], antimicrobial [3], anticancer [4], antiobesity [5], and medicinal agents [6]. The collective advances of FOGs concerning nanotechnology have emerged as a new arena that captivating medicinal researchers to pursue research in it [7, 8]. As acclaimed in vivo oxidation being identified as a vital process that spawns the ample energy for the proper execution of biological processes in all organisms, often, it causes the overproduction of free radicals in turn for the cell damage and in turn for the metabolic diseases like diabetes, cardiovascular diseases, cancers, and neurological disorders [9, 10]. In its counter administration, antioxidants inhibit the reactive free radicals by neutralizing and arrest the structural disruption of biomolecules in cells [11].

During so, the distinguished biosynthesized phytological nanoparticles (NPs) are identified more remarkable than their plant extracts in exhibiting potential activity [12]. This has fascinated nanotechnology in embodiment with scientific results that abridged the gap between with atomic/molecular structures and bulk materials and accelerated the chemotherapeutic potency in treating various diseases [13, 14]. Structurally, the surface-to-volume ratio of NPs is contrarily proportional to their sizes, [15] more precisely the inherent potential of silver nanoparticles (AgNPs) grows reciprocally with an escalation in the specific surface area owing to high surface energy and catalytic reactivity ¹⁶. The preference and advancement of green chemistry over conventional are due to eco-friendliness, cost-effectiveness, and feasibility for large-scale synthesis [16, 17]. The extensive array of AgNPs with medicinal efficacies like anticancer, [18, 19], antioxidants [20], and antimicrobial [21] abilities is derived from various plant origins like Rhinacanthus nasutus [22], Trigonella foenum-graecum [23], Ocimum bacillicum [24], Vitex negundo L [25], Hypnea musciformis (Wulfen) JV lamouroux [26], Terminalia chebula [27], Raphanus sativus var. aegyptiacus [28], Citrus sinensis [29], Cassia roxburghii [30], Eurocyma longifolia [31], Annona muricata [32], and Eriobotrya japonica [33].

It is profound that excessive usage of antibiotics results in dissemination and emergence of multidrug-resistant strains of several types of microorganisms [31, 34]. In this scenario, the needs and demands to discover new medicinal agents are increasing, and nanotechnology paves ways to synthesize NPs to substitute current antibiotics and other synthetic agents. In such, Luffa acutangula (LA), a traditional perennial flowering climber plant, ordinarily stated as ridge gourd regards to Cucurbitaceae family and is enriched with medicinal properties [35] like antioxidant, antidiabetic, antiproliferative, antiangiogenic, anticataleptic, analgesic, antiulcer, and antimicrobial activities [36, 37].

As LA plant parts are enriched with a large number of pharmacologically active phytochemicals like flavonoids, proteins, saponin triterpene, anthraquinones, fatty acids, and other phytoconstituents, it is ethnopharmacologically used to treat hemorrhoids, leprosy, splenitis, and ringworm infections by topical administration of pulverized leaves of LA [38]. Hence, we synthesised LAAgNPs from the leaf extract of LA and succeeded in synthesising AgNPs. The flavonoids present in leaves viz., Cosmosioside (1, Apigenin-7-glucoside), Cynaroside (2, Luteolin-7-glucoside) with potential antioxidant, antibacterial, and anticancer activities are FOGs originated from O-glycosidic linkage of (2ξ)-β-D-arabino-Hexopyranose with 7-OH group of Apigenin and O-glycosidic linkage of β-D-glucopyranose with 7-OH group of Luteolin (Figure 1).

[figure omitted; refer to PDF]

In this connection, previous studies showed similar colour changes to form dark brown colour [21, 46, 47]. It was confirmed that concentrations of plant extracts are one of the significant aspects that influence the rate of synthesis of silver nanoparticles. Higher intensity of colour was spotted as the concentrations increased from 1.0, 2.5, and 5.0%. This could be a result of higher content of the biomolecules that reacted as reducing agents in silver reduction process. Uniform results had also been noticed in the leaves of Luffa acutangula in synthesising silver nanoparticles [22].

3.1.2. UV-Vis Spectral Studies of LA Silver Nanoparticles

Time interval to measure the absorption peak was 30-150 sec. At 150 sec, the highest peak was observed for all the different concentrations, and all the high adsorption peaks were in the range of standard adsorption of silver nanoparticles. The spectral peaks were recorded at 417, 432, and 448 nm for different concentrations of biologically synthesised nanoparticles at 1.0, 2.5, and 5.0%, respectively (Figure 3).

[figure omitted; refer to PDF]

There are no qualms that silver nanoparticles achieved the highest peak as cause of Surface Plasmon Resonance (SPR) adsorption band. Free electrons of biologically synthesised silver nanoparticles promote the generation of SPR band through coalescing the vibrations of electrons in resonance with the light wave [48]. The aspects like size and shape of the nanoparticles, type of biomolecules existing in the plant extracts, silver nitrate concentration, and amount of extracts have influenced the SPR banding patterns.

3.1.3. XRD Analysis

X-ray diffraction analysis is an advanced method to figure out the crystalline nature of metallic nanoparticles. As shown in Figure 4, the peaks at $2\theta$ values of 38°, 44°, 64°, and 77° are reflecting (111), (200), (220), and (311) lattice plans for silver, respectively. The present result clearly illustrates that the biologically synthesised silver nanoparticles are in crystalline nature and face-centered cubic (fcc) shape. The studies on carob and olive leaf extract confirmed the shape of the synthesised nanoparticles as fcc [49, 50]. Debye Scherrer’s equation (Equation (3)) was used to calculate the average particular size of the silver nanoparticles synthesised by present biological method, where $\beta$ is the full width at half maximum of the diffraction peak, $t$ is the mean crystalline size, $\theta$ is the centre angle of the peak, and $\lambda$ is the wavelength of X-ray source. The recognized crystalline size of LAAgNPs is 44 nm. This clearly illustrates that LAAgNPs is nanocrystalline shape. Similar results have been reported on the biologically synthesised AgNPs using Pulicaria glutinosa plant extract showed average crystalline size of 42 nm [13]. $$\begin{array}{}\text{(3)}& t=\frac{0.89\lambda }{\beta cos\theta }\end{array}$$

[figure omitted; refer to PDF]

The present data is in accordance with the result reported for the biosynthesised AgNPs from aqueous leaf extracts of Terminalia mellueri, Terminalia catappa, Terminalia bellerica, and Terminalia bentazoe showed high DPPH free radical scavenging activity (more than 80%) compared to leaf extracts in the range of 60%-70% [56]. The values represented are the $\text{mean}\pm \text{S}.\text{D}$ of triplicate sample significant level at ($P<0.05$). The IC₅₀ values of LA and LAAgNPs were 126.29 μg/mL and 96.89 μg/mL. The lower IC₅₀ values indicate the greater tendency for antioxidant activity of the extracts. Similar activity was reported for the lower IC₅₀ value of Psidium guajava extract and AgNPs from Psidium guajava was 110 μg/mL and 80 μg/mL, respectively [57].

3.2.2. ABTS Antioxidant Assay

In the present study, ABTS free scavenging test was analysed on AgNPs and leaf extract of LA. ABTS^+· is considered as protonated radical which could readily accept electron from antioxidant compound and transfer its colour from blue to pink which was detected at 734 nm [58]. The leaf extract of LA showed the potential to scavenge the free radicals was found to be 43.8-82.9% at concentrations from 50-300 μg/mL, respectively, whereas biologically synthesised AgNPs showed the activity as 47.9-85.2% at different concentrations from 50-300 μg/mL, respectively (Figure 8). The values represented are the $\text{mean}\pm \text{S}.\text{D}$ of triplicate sample significant level at ($P<0.05$). The IC₅₀ value of standard AA is 31.42 μg/mL which has proven that AA had higher scavenging activity with the lowest IC₅₀ (μg/mL). The IC₅₀ values of LA and LAAgNPs were found as 100.96 and 76.0 μg/mL, respectively. Similar activity was reported for the IC₅₀ value of Psidium guajava extract (105 μg/mL) and found higher than AgNPs from Psidium guajava (70 μg/mL), respectively [57]. Similar ABTS radical scavenging action of biologically synthesised AgNPs was found in previous studies [59, 60].

[figure omitted; refer to PDF]

The present study proved that gram-negative bacteria, E. coli, were more sensitive to the action of biologically synthesised silver nanoparticles compared to gram-positive bacteria. This is in accordance with the result stated by Kim and the coworkers [63]. Literature denoted the inhibitory effects of silver nanoparticles could be associated with characteristics of specific bacterial species. Naturally, gram-positive and gram-positive grouped bacteria have dissimilar membrane structure, especially the difference in thickness of peptidoglycan layer. The mild antibacterial features of synthesised silver nanoparticles in contradiction of gram-positive bacteria could be due to membrane structure [63]. The antibacterial mechanism of action of metallic nanoparticles is still not exactly explained and being unverified. However, several theories and possible mechanism(s) of actions of biologically and chemically synthesised silver nanoparticles have been reported with basic information [64]. The graphic representation (Figure 10) depicts the penetration of silver nanoparticles (AgNPs) into the cell and their different mode of antibacterial mechanisms. The reactivity begins with synthesis of silver nanoparticles using silver nitrate and selected plant extracts.

[figure omitted; refer to PDF]

3.4. Anticancer Activity

Plants contain several types of bioactive compounds that are ideally favorable for the drug development in anticancer therapy. Nowadays, researchers found that the plant-based medicines or drugs are safer and cost-effective when compared to the synthetic drugs [65]. LA is one of the herbal plants which belongs to a family of Cucurbitaceae and widely cultivated in Asia, India, Brazil, and USA [66]. Previously, itself isolated five major components of LA, a bioactive component among them named 1,8 dihydroxy-4-methylanthracene 9,10-dione (DHMA) was reported as potential antiproliferative agent against nonsmall cell lung cancer cells (NCI-H460). DHMA showed promising anticancer activities through inhibition of cell growth, generation of reactive oxygen species (ROS), and induction of p53-mediated apoptotic pathway against human nonsmall cell lung cancer cell line (NCI-H460) [67, 68]. Another study reported on the potential anticancer effect of LA on human colon cancer cell line HT29 cells [69]. LA seeds consist of ribosome inactivating proteins which were reported, and the study revealed the potential anticancer activity of luffaculin 1 and luffaculinin in human leukemia K562 cells [70]. Anticancer effects of AgNPs have been demonstrated in various cell models. It observed a dose-dependent cytotoxic effect of biosynthesized AgNPs from Piper longum extract in MCF-7 breast cancer cells [71]. Cytotoxic effects of AgNPs from other plant extracts such as Iresine herbstii and Vitex negundo Linn were demonstrated in HeLa (cervical) and HCT15 (colorectal) cancer cells, respectively [9, 25]. In the present study, LAAgNPs were tested against four human cancer cell lines, MCF-7, MDA-MB-231, DBTRG, and U87. The synthesised silver nanoparticles by LA leaf extract triggered a dose-dependent reduction in the cell proliferation with IC₅₀ values ranging from 35-42 μg/ml (Figure 11). There are several anticancer mechanisms that have been suggested based on previous studies. AgNPs tend to generate reactive oxidative species (ROS) intracellularly that results excess oxidative stress [72]. High oxidative stress inhibits chromosome inhibition and eventually damage cell cycle of tumor cells [73, 74]. Size independent property of AgNPs enhances cytotoxic effect against drug-resistant cancer cells [75]. In addition, cytotoxic effect can be as the result of poor angiogenesis and programmed cell death by AgNPs [76]. Further studies are needed to interpret the anticancer mechanism(s) of the biosynthesized AgNPs.

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3.5. Molecular Docking Studies

The obtained in vitro antioxidant, antibacterial, and anticancer activity of FOGs have been additionally supported by investigating of ligand-protein binding interactions against the selected enzymatic proteins viz., 3NM8-Chain A (DPPH radical scavenging activity), 1DNU-Chain A (ABTS radical scavenging activity), 5FGK-Chain A (gram-positive bacterial activity), 1AB4-Chain A (gram-negative bacterial activity), 4GBD-Chain A (MCF-7 anticancer activity), 5FI2-Chain C (MDA-MB-231 anticancer activity), 1D5R-Chain A (U87 anticancer activity), and 5TIJ-Chain B (DBTRG anticancer activity); and docking postures and binding interactions were bestowed in Tables 1–12.

Table 1

Potential ligand-protein molecular docking bindings of Cosmosioside (1) with identified proteins.

Cosmosioside (1, Apigenin-7-glucoside, C21H20O10)	3NM8 (chain A)-oxidoreductase-tyrosinase	1DNU (chain A)-oxidoreductase-myeloperoxidase-thiocyanate complex
5FGK (chain A)-A Transferase Enzymatic Protein-CDK8-associated CycC	1AB4 (chain A)-A Topoisomerase Enzymatic Protein-59 KDA fragment of gyrase A	4GBD (chain A)–Lyase-adenosine deaminase
5FI2 (chain C)-Hydrolase Inhibitor Enzymatic Protein -GAC in complex UPGL 00009 inhibitor	1D5R (chain A)-A Hydrolase Enzymatic Protein-PTEN tumor suppressor	5TIJ (chain B)-A Lyase Enzymatic Protein-human enolase 2

Table 2

Potential ligand-protein molecular docking interactions of Cosmosioside (1) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	HB	HBL atoms	HBR atoms	Binding interactions	BL (A°)	BA (°)	H.B. type
3NM8 (chain A) - oxidoreductase - tyrosinase	0	7	-8.2618	3	2	3	Ligand[O(4)-H(10)]-------ASP55(OCOH)	2.0704	54.6126##	Acceptor
							Ligand[O(4)-H(10)]-----ASP55[O(H)CO]	2.2912		Acceptor
							Ligand[C(2)-O(1)]-----------ARG209(HN)	2.8998	—	Donor
1DNU (chain A) - oxidoreductase - myeloperoxidase-thiocyanate complex	30	7	-7.9463	2	2	2	Ligand[O(7)-H(13)]-----------ASP172(OC)	2.4855	—	Acceptor
							Ligand[O(8)-H(18)]----------TYR316(OH)	2.2322	—	Acceptor
5FGK (chain A) - transferase - CDK8 associated CycC	0	0	-9.1645	3	3	3	Ligand[O(4)-H(10)]------ASP103(OCOH)	1.9270	—	Acceptor
							Ligand[O(6)-H(12)]------------VAL27(OC)	2.4636	—	Acceptor
							Ligand[O(8)-H(18)]-------GLU66(OCOH)	2.0588	—	Acceptor
1AB4 (chain A) - topoisomerase -59KDA fragment of gyrase A	0	0	-8.2294	4	4	4	Ligand[O(4)-H(10)]-------ASP87(OCOH)	2.5627	—	Acceptor
							Ligand[O(7)-H(13)]-----GLN94(OCNH2)	2.2952	—	Acceptor
							Ligand[O(8)-H(18)]------------PHE96(OC)	1.9022	—	Acceptor
							Ligand[H(18)-O(8)]----------GLN267(HN)	2.1500	—	Donor
4GBD (chain A) - Lyase - adenosine deaminase	8	1	-7.7955	3	3	3	Ligand[O(4)-H(10)]-------ASP36(OCOH)	2.1348	—	Acceptor
							Ligand[H(13)-O(7)]---------ARG411(HN)	2.0486	—	Donor
							Ligand[C(2)-O(1)]-----------ARG149(HN)	2.5263	—	Donor
5FI2 (chain C) - hydrolase inhibitor - GAC in complex UPGL 00009 inhibitor	0	0	-8.8921	7	6	7	Ligand[O(8)-H(18)]------ASP446(OCOH)	1.8467	—	Acceptor
							Ligand[O(4)-H(10)]----------TYR248(OH)	2.1513	—	Acceptor
							Ligand[O(6)-H(12)]----------GLU380(OH)	1.7125	—	Acceptor
							Ligand[H(18)-O(8)]------ASN318(HNOC)	2.2940	—	Donor
							Ligand[C(9)-O(3)-C(13)]-ASN334(HNOC)	2.6215	—	Donor
							Ligand[H(11)-O(5)]----------ASN387(HN)	2.1575	106.6382@	Donor
							Ligand[H(11)-O(5)]----------TYR413(HO)	2.4479		Donor
1D5R (chain A) - hydrolase - PTEN tumor suppressor	0	0	-8.7975	4	4	3	Ligand[O(4)-H(10)]------ASP324(OCOH)	2.2363	102.4531$	Acceptor
							Ligand[O(6)-H(12)]------ASP324(OCOH)	2.1290		Acceptor
							Ligand[H(13)-O(7)]----------ARG172(HN)	2.2084	—	Donor
							Ligand[H(19)-O(9)]----------ARG173(HN)	2.1463	—	Donor
5TIJ (chain B) - lyase - human enolase 2	5	7	-7.9044	4	3	3	Ligand[O(8)-H(18)]------ASP282(OCOH)	1.7981	—	Acceptor
							Ligand[O(7)-H(13)]---GLU219(O(H)OC)	2.5990	56.2469#	Acceptor
							Ligand[O(7)-H(13)]-----GLU219(OCOH)	1.9638		Acceptor
									69.7145${}^{\ast }$
							Ligand[O(6)-H(12)]-----GLU219(OCOH)	1.7856		Acceptor

^$Ligand H₍₁₀₎-ASP324(OCOH)-Ligand H₍₁₂₎; ${}^{\ast }$Ligand H₍₁₃₎-GLU219(HOCO)- Ligand H₍₁₂₎; ^##ASP55[OC(H)O]-Ligand H₍₁₀₎-ASP55(OCOH); HBL atoms: number of hydrogen bond ligand atoms; ^#GLU219(HOCO)-Ligand H₍₁₃₎-GLU219(O(H)OC); ^@ASN387(NH)-Ligand O₍₅₎-TYR413(HO); HB: number of hydrogen bonds; HBR Atoms: number of hydrogen bond receptor atoms; BE: binding energy; BL: bond length; BA: bond angle.

Table 3

Potential ligand-protein molecular docking bindings of Cynaroside (2) with identified proteins.

Cynaroside (2, luteolin-7-glucoside), C21H20O11)	3NM8 (chain A) - oxidoreductase - tyrosinase	1DNU (chain A) - oxidoreductase - myeloperoxiase-thiocyanate complex
5FGK (chain A) - A Transferase Enzymatic Protein - CDK8 associated CycC	1AB4 (chain A) - A Topoisomerase Enzymatic Protein -59KDA fragment of gyrase A	4GBD (chain A) – lyase - adenosine deaminase
5FI2 (chain C) - Hydrolase Inhibitor Enzymatic Protein - GAC in complex UPGL 00009 inhibitor	1D5R (chain A) - A Hydrolase Enzymatic Protein - PTEN tumor suppressor	5TIJ (chain B) - A Lyase Enzymatic Protein -human enolase 2

Table 4

Potential ligand-protein molecular docking interactions of Cynaroside (2) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	HB	HBL atoms	HBR atoms	Binding interactions	BL(A°)	BA (°)	HB type
3NM8 (chain A) - oxidoreductase - tyrosinase	19	3	-7.9386	2	2	2	Ligand[O(7)-H(13)]----ASP324(OCOH)	2.1516	—	Acceptor
							Ligand[C(2)-O(1)]--------ARG209(HN)	2.3601	—	Donor
1DNU (chain A) - oxidoreductase - myeloperoxidase-thiocyanate complex	0	3	-8.3238	3	3	3	Ligand[O(10)-H(19)]---ASP324(OCOH)	1.9449	—	Acceptor
							Ligand[O(7)-H(13)]---------ARG27(HN)	2.0962	—	Acceptor
							Ligand[O(6)-H(12)]----------ALA24(OC)	2.2144	—	Acceptor
5FGK (chain A) - transferase - CDK8 associated CycC	18	12	-8.8514	5	5	5	Ligand[O(5)-H(11)]----ASP173(OCOH)	2.0107	—	Acceptor
							Ligand[C(14)-O(4)]--------ASP173(HN)	2.2884	—	Donor
							Ligand[C(10)-O(7)]----------LYS52(HN)	2.2874	—	Donor
							Ligand[H(14)-O(8)]---------LYS52(HN)	2.4930		Donor
							Ligand[O(8)-H(14)]---------ASP98(OC)	2.7157		Acceptor
1AB4 (chain A) - topoisomerase -59KDA fragment of gyrase A	1	3	-8.2753	7	7	6	Ligand[O(5)-H(11)]----ASP113(OCOH)	2.0095	49.2269${}^{\ast }$	Acceptor
							Ligand[O(7)-H(13)]- --ASP113(OCOH)	2.6325		Acceptor
							Ligand[O(4)-H(10)]----ASP115(OCOH)	2.0341	—	Acceptor
							Ligand[O(10)-H(19)]-------ASP299(OC)	2.7050	—	Acceptor
							Ligand[C(10)-O(7)]--------LYS270(HN)	2.0314	—	Donor
							Ligand[C(6)-O(2)]---------LYS298(HN)	2.4233	—	Donor
							Ligand[O(9)-H(18)]-------HSD262(OC)	2.3361	—	Acceptor
4GBD (chain A) – lyase - adenosine deaminase	4	4	-7.8306	4	4	4	Ligand[O(10)-H(19)]--ASP215(OCOH)	2.1032	—	Acceptor
							Ligand[O(6)-H(12)]------LYS182(OC)	2.5865	—	Acceptor
							Ligand[O(4)-H(10)]--------LYS184(OC)	1.8875	—	Acceptor
							Ligand[H(19)-O(9)]---------ILE189(HN)	2.2567	—	Donor
5FI2 (Chain C) - hydrolase inhibitor - GAC in complex UPGL 00009 inhibitor	0	0	-9.1290	5	5	5	Ligand[O(10)-H(19)]---ASP446(OCOH)	1.9065	—	Acceptor
							Ligand[C(11)-O(6)]-------ASN330(HN)	2.0675	—	Donor
							Ligand[O(5)-H(11)]--------CYS462(O’C)	2.5840	—	Acceptor
							Ligand[O(7)-H(13)]---------CYS462(OC)	1.9953	—	Acceptor
							Ligand[O(4)-H(10)]---------SER313(OC)	2.5769	—	Acceptor
1D5R (chain A) - hydrolase - PTEN tumor suppressor	0	0	-9.2690	7	7	5	Ligand[O(4)-H(10)]----ASP324(OCOH)	1.9343	90.6238$	Acceptor
							Ligand[O(6)-H(12)]----ASP324(OCOH)	1.8831		Acceptor
							Ligand[C(18)-O(9)]---------LYS183(HN)	2.4417	74.4086#	Donor
							Ligand[C(17)-O(10)]--------LYS183(HN)	2.2624		Donor
							Ligand[O(10)-H(19)]--------TYR176(HN)	2.3215	—	Acceptor
							Ligand[C(4)-O(8)]---------ARG173(HN)	2.2435	—	Donor
							Ligand[O(7)-H(13)]--------ARG172(HN)	2.2234	—	Donor
5TIJ (chain B) -lyase - human enolase 2	24	1	-8.7736	5	4	5	Ligand[O(5)-H(11)]----ASP142(OCOH)	1.9126	—	Acceptor
							Ligand[O(7)-H(13)]---------ASP142(OC)	2.4464	—	Acceptor
							Ligand[C(10)-O(7)]---------ASP142(HN)	2.1296	48.5632@	Donor
							Ligand[C(10)-O(7)]---------SER141(HN)	2.3549		Donor
							Ligand[C(14)-O(4)]---------SER432(HO)	2.2870	—	Donor

Table 5

Potential ligand-protein molecular docking bindings of ascorbic acid (3) with identified proteins.

Ascorbic acid (3, hex-1-enofuranos-3-ulose, C6H8O6)	3NM8 (chain A) - oxidoreductase - tyrosinase	1DNU (chain A) - oxidoreductase - myeloperoxidase-thiocyanate complex

Table 6

Potential ligand-protein molecular docking interactions of ascorbic acid (3) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	No. of HB	No. of HBL atoms	No. of HBR atoms	Binding interactions	BL (A°)	BA (°)	HB type
3NM8 (chain A) - oxidoreductase - tyrosinase	1	4	-6.4837	3	3	3	Ligand[O(2)-H(4)]---ASP140(C=O)	2.1328	—	Acceptor
							Ligand[C-O(1)]--------LYS47(NH)	2.8116	—	Donor
							Ligand[O(5)-H(3)]---PRO219(C=O)	1.9298	—	Acceptor
1DNU (chain A) - oxidoreductase - myeloperoxidase-thiocyanate complex	31	2	-6.6112	4	3	3	Ligand[O(4)-H(6)]------ASP5(O=C)	2.3826	78.7795${}^{\ast }$	Acceptor
							Ligand[O(4)-H(6)]----ASP5(OCOH)	1.9709		Acceptor
									61.7009#
							Ligand[O(5)-H(7)]---ASP5(OCOH)	1.7722		Acceptor
							Ligand[H(7)-O(5)]-----ARG17(NH)	2.7187	—	Donor

${}^{\ast }$ASP5(CO)-Ligand H₍₆₎-ASP5(OCOH); ^#Ligand H₍₆₎-ASP5(OCOH)-Ligand H₍₇₎.

Table 7

Potential ligand-protein molecular docking bindings of ampicillin (4) with identified proteins.

Ampicillin (4, aminobenzylpenicillin, C16H19N3O4S)	5FGK (chain A) - A Transferase Enzymatic Protein - CDK8 associated CycC	1AB4 (chain A) - A Topoisomerase Enzymatic Protein -59KDA fragment of gyrase A

Table 8

Potential ligand-protein molecular docking interactions of ampicillin (4) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	No. of HB	No. of HBL atoms	No. of HBR atoms	Binding interactions	BL (A°)	BA (°)	HB type
5FGK (chain A) - transferase - CDK8 associated CycC	5	0	-8.4767	3	3	3	Ligand[N(2)-H(17)]--ASP173(OCOH)	2.1290	—	Acceptor
							Ligand[C(8)-O(3)]----LYS52(HNCH2)	2.1803	—	Donor
							Ligand[O(1)-H(3)]--------VAL27(OC)	2.3334	—	Acceptor
1AB4 (chain A) - topoisomerase -59KDA fragment of gyrase A	1	4	-7.8486	3	3	3	Ligand[O(1)-H(3)]---ASP104(OCOH)	2.1401	—	Acceptor
							Ligand[N(2)-H(18)]--ASP515(OCOH)	2.4497	—	Acceptor
							Ligand[C(5)-O]-----------LYS129(HN)	2.1821	—	Donor

Table 9

Potential ligand-protein molecular docking bindings of tamoxifen (5) with identified proteins.

Tamoxifen (5, 1-p-β-dimethylaminoethoxyphenyl-trans-1,2-diphenylbut-1-ene, C26H29NO)	4GBD (chain A) – lyase - adenosine deaminase	5FI2 (chain C) - Hydrolase Inhibitor Enzymatic Protein -GAC in complex UPGL 00009 inhibitor

Table 10

Potential ligand-protein molecular docking interactions of tamoxifen (5) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	HB	HBL atoms	HBR atoms	Binding interactions	BL (A°)	BA (°)	HB type
4GBD (chain A) - lyase - adenosine deaminase	0	3	-7.7235	3	1	3	Ligand[C-N]-------ASP316(OCOH)	4.2864	39.9133${}^{\ast }$	Acceptor
							Ligand[C-N]----------ASN314(O=C)	4.4192		Acceptor
									40.7597$
							Ligand[C-N]----------GLU282(OCOH)	3.2491		Acceptor
5FI2 (Chain C) - hydrolase inhibitor - GAC in complex UPGL 00009 inhibitor	4	0	-7.5313	2	2	2	Ligand[C(2)Ph-O]----------ASP466(HN)	4.0253	104. 5635#	Donor
							Ligand[C(2)Ph-O]---------ASN318(HN)	3.5771		Donor

${}^{\ast }$ASP316(HOCO)-Ligand[CN]-ASN314(OC); ^$ASN314(CO)-Ligand[CN]-GLU282(OCOH); ^#ASP466(NH)-Ligand[O]-ASN318(HN).

Table 11

Potential ligand-protein molecular docking bindings of gefitinib (6) with identified proteins.

Gefitinib (6, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy) quinazolin-4-amine, C22H24ClFN4O3)	1D5R (chain A) - A Hydrolase Enzymatic Protein - PTEN tumor suppressor	5TIJ (chain B) - A Lyase Enzymatic Protein - human enolase 2

Table 12

Potential ligand-protein molecular docking interactions of gefitinib (6) with identified proteins.

Enzymatic protein	Cluster number	Cluster element	BE (KCal/mol)	HB	HBL atoms	HBR atoms	Binding interactions	BL (A°)	BA (°)	HB type
1D5R (chain A) - hydrolase - PTEN tumor suppressor	0	0	-8.0338	5	3	4	Ligand[N(2)-H(3)]------ASP324(OCOH)	2.3863	54.6314$	Acceptor
							Ligand[N(2)-H(3)]------ASP324(OCOH)	2.1188		Acceptor
							Ligand[C(5)-O(2)]-------ARG173(HN=C)	2.2728	71.0798#	Donor
							Ligand[C(4)-O]---------ARG173(HN=C)	2.3931		Donor
									50.4066${}^{\ast }$
							Ligand[C(4)-O]--------ARG173(HN-CH)	2.8653		Donor
5TIJ (chain B) -lyase - human enolase 2	13	3	-7.8985	2	1	2	Ligand[C(12)-O(1)]----------ASP142(HN)	2.4308	52.5638$	Donor
							Ligand[C(12)-O(1)]----------SER141(HN)	2.4781		Donor

^$ASP324(HOCO)-Ligand [H₍₃₎]-ASP324(O(H)OC); ${}^{\ast }$ARG173(C = HN)-Ligand[O]-ARG173(HN=CH); ^#Ligand[O₍₂₎]-ARG173(HN=C)-Ligand[O]; ASP142(NH)-Ligand[O₍₁₎]-SER141(HN).

In view of antioxidant activity, the hydroxy groups (-OH) of FOGs bound with carbonyl groups (O=C) of aspartic acid and amino groups (-NH) of arginine in 3NM8 (Chain A) are responsible for DPPH radical scavenging activity; and binding of -OH of FOGs bound with O=C of aspartic acid and alanine, -NH of arginine, and -OH of tyrosine in 1DNU (Chain A) is responsible for ABTS radical scavenging activity. Concerning the antibacterial activity, the -OH of FOGs bound with C=O of aspartic acid, valine and glutamic acid, and -NH of aspartic acid and lysine in 5FGK (Chain A) is responsible for gram-positive bacterial activity; and binding of -OH in FOGs with C=O of aspartic acid, glutamine, histidine and phenyl alanine, and -NH of glutamine and lysine in 1AB4 (Chain A) is responsible for gram-negative bacterial activity. In relation to MCF-7 anticancer inhibition, the -OH of FOGs bound with C=O of aspartic acid and lysine, and -NH of arginine and isoleucine in 4GBD (Chain A) were identified as responsible. Coming to MDA-MB-231 anticancer inhibition, the -OH of FOGs bound with C=O of aspartic acid, cysteine and serine, -OH of tyrosine and glutamic acid, and -NH of asparagine in 5FI2 (Chain C) was identified as responsible.

Concerning the U87 anticancer inhibition, the -OH of FOGs bound with C=O of aspartic acid, and -NH of lysine, and tyrosine and arginine in 1D5R (chain A was identified as responsible). In aspects of DBTRG anticancer inhibition, the -OH of FOGs bound with C=O of aspartic acid and glutamic acid, -OH of glutamic acid and serine, and -NH of aspartic acid and serine in 5TIJ (chain B) was identified as responsible. The binding specificity studies have affirmed the promising ligand-protein binding interactions in between the hydroxy groups of the FOGs and aspartic acid of the concerned enzymatic proteins with a binding energy in the range of -9.2690 to -7.7955 KCal/mol.

3.6. ADMET Properties

The study of ADMET properties of the interested analytes under investigation helps to realize their physicochemical interactions [77]. The potentiality of a drug depends on its degree of absorption and in turn on its inherent bioavailability properties. Once a potential drug be absorbed and self-distributed in to muscles and organs by circulation through extracellular sites and hence lowers its plasma concentration individually, therefore, metabolizes in vivo, then, such metabolites will be distributed by the action of reduction and oxidation reactions by the enzymatic action and work potentially on cellular systems, and ultimately the inert metabolites will be automatically excreted from kidneys. Such analysis of ADMET properties (Table 13) inferred us that the two FOGs are with 0.0373 and 0.0336 of BBB penetration potentiality confirms their CNS significance and esteems their superior permeability and their in vivo distribution. Further, it is supported on the ground of the in vitro Caco-2 cell permeability held with 7.2167 and 4.8722 nm/sec, respectively, which enables their robust permeability to bind to plasma proteins and to penetrate in to the BBB system. The in vitro PPB efficiency with 73.43 and 73.27 respective percentages approves their robust binding capability to plasma proteins. The in vitro MDCK cell permeability with 0.6424 and 0.7567 nm/sec empowers their strong permeability. The %HIA with 47.1059 and 25.1651 supports their interactions with targeted domains of the cells. The negative magnitudes of the toxicity calculations that designate FOGs are nontoxic and with safer drug properties. In ultimate, ADMET analysis of the two FOGs has greatly manifested their potential physicochemical interactions and drug-likeness.

Table 13

ADMET properties the compounds 1-6.

Compounds	In vivo blood-brain barrier penetration (C. brain/C. blood)a	In vitro Caco-2 cell permeability(nm/sec)b	In vitro plasma protein binding(%)c	In vitro MDCK cell permeability(nm/sec)d	Human intestinal absorption (HIA, %)e	Toxicityf
Apigenin-7-glucoside (1)	0.0373	7.2167	73.4332	0.6424	47.1059	Negative
Luteolin-7-glucoside (2)	0.0336	4.8722	73.2796	0.7567	25.1651	Negative
Ampicillin (3)	0.0588	0.6307	36.1547	0.9376	81.4785	Negative
Ascorbic acid (4)	0.1173	2.4836	5.3035	0.8819	33.1572	Negative
Gefitinib (5)	0.0476	54.1474	80.7309	0.07737	96.6375	Negative
Tamoxifen (6)	14.1639	49.5448	94.7448	69.8462	100	Negative

^a$\text{Blood}-\text{brain}\text{barrier}\text{BBB}\text{penetration}=\text{brain}/\text{blood}$; ^bCaco-2 cells are derived from human colon adenocarcinoma and possess multiple drug transport pathways through intestinal epithelium; ^c% of drug binds to plasma protein; ^dMDCK cell system used as tool for rapid permeability screening; ^ehuman intestinal absorption is the sum of bioavailability and absorption evaluated from ratio of excretion or cumulative excretion in urine, bile, and feces; ^fin vitro Ames test by metabolic and nonmetabolic activated TA100 and TA1535 strains collected from rat liver homogenate.

3.7. QSAR Studies

QSAR results (Table 14) indicate that FOGs under study with molecular weights 432.38 and 448.38 (less than 500 Daltons) have confirmed their greater permeability via cell membranes with log $P$ values of 0.68 and 0.19 (less than 5). Correspondingly, the numbers of hydrogen bond acceptors and donors have also obeyed the limitations. The molecular refractivity values with 107.46 and 109.27 cm³/mol have aligned in the standard range (40-130 cm³/mol) and confirmed that the two FOGs are obeying the Lipinski rule of five and are designated as significant oral active drugs. On the other hand, the total polar surface area bankrolled by the addition of polar surface area of the atoms like oxygen, nitrogen, and hydrogen [78], for the two FOGs are with 170.05 and 190.28 A°² obeying the limitations; and the number of total rotatable bonds in FOGs are 4 in number and obeying its potential boundaries; in complementary to obeying of the Lipinski rule, these two accounts for the validation of the Veber’s rule pertaining to the two FOGs of the study. Henceforth, these FOGs are admired to be absorbed, diffused, and transported certainly and ascertained as oral administrable drugs. The TPSA is greatly correlated with the hydrogen bonding of a molecule and is complemented with transport properties of a drug through the membranes, and hence, also accounts for the BBB penetrability [79]. Furthermore, density with 1.642 and 1.713 gm/cc, solubility with -2.74 and -2.45, and Van der Waals volume with 356.17 and 364.19ų, respectively, ascertain the safer and potential drug-likeness of the FOGs. In ultimate, this study greatly helped in accepting the physicochemical interactions of FOGs with the anticipated targets; and in defining their drug properties by complementing with bioactivity and toxicity risks studies, where the ligand interactions and enzyme inhibition properties along with the like drug-likeness and drug scores will be evaluated.

Table 14

QSAR properties of the compounds 1-6.

Entry	Lipinski parameters						Veber parameters			Other parameters
	MW	HB Don	HBAcc	logP (o/w)	MR	Lip. Vio.	TPSA	No. ofRB	Veb.Vio.	No. of H	V. Volume	$\rho$	Solubility
Apigenin-7-glucoside (1)	432.38	10	6	0.68	107.46	0	170.05	4	0	20	356.17	1.642	-2.74
Luteolin-7-glucoside (2)	448.38	11	7	0.19	109.27	0	190.28	4	0	20	364.19	1.713	-2.45
Ampicillin (3)	349.41	7	4	-0.87	89.37	0	112.73	4	0	19	298.87	1.453	-1.57
Ascorbic acid (4)	176.12	6	4	-1.40	36.61	0	107.22	2	0	8	139.71	1.954	-0.35
Gefitinib (5)	446.91	7	1	4.19	118.38	0	68.75	8	0	24	385.07	1.322	-5.06
Tamoxifen (6)	371.52	2	0	6.06	119.87	0	12.47	8	0	29	376.13	1.042	-4.40

MW: molecular weight; HB Don: hydrogen bond donors; HB Acc: hydrogen bond acceptors; logP: octanol to water partition coefficient; MR: molecular refractivity (cm³/mol); Lip Vio: Lipinski violations; TPSA: total polar surface area; No. of RB: number of rotatable bonds; Veb Vio: Veber violations; No. of “H”: number of hydrophobic atoms; V. Volume: Van der Waals volume; $\rho$: density (gm/cc).

3.8. Bioactivity and Toxicity Risk Studies

The bioactivity and toxicity risk exploration studies of the FOGs have shown their bioactivity properties viz., GPCR ligand property, ion channel modulator, kinase inhibitor, nuclear receptor ligand interactions, protease inhibitor, and enzyme inhibitor interactions; and the drug properties like drug-likeness and drug score and established as potential nontoxic drugs (Table 15). This molinspiration exploration comprehensively assists us to explore the cheminformatics of the molecules under investigation by correlating with the in vitro and in vivo results database of the recognized drugs basing on the functional group similarities in mutual. The drug property exploration of the two FOGs has evidenced for their safer drug properties as they are with no risks of tumorigenicity, irritant effects, mutagenicity, and shown no effect on reproductive system. The positive magnitude of the drug-likeness value represents that the scrutinized molecule comprises the significant fragments that are present in the established commercial drugs [45]. Drug-likeness is an significant factor which helps in understanding the kinesis of a molecule from the site of administration to the bloodstream, hence, its good solubility accounts for good absorption and assures the drug-likeness [80]. Similarly, drug score is also a complementary parameter of the drug-likeness and helps to assure to decide molecule’s drug potentiality. Hence, the present investigation reveals that all the properties of the bioactivity and toxicity risk studies are up to the potential limits of the safe drugs and ascertains the FOGs as the drug-like compounds.

Table 15

Bioactivity scores, drug properties, and toxicity risks of the compounds 1-6.

Compounds	Structure	Bioactivity						Drug properties
		GPCRL	ICM	KI	NRL	PI	EI	Drug-likeness	Toxicity risks
Apigenin-7-glucoside (1)		0.10	-0.01	0.14	0.31	0.02	0.43	2.29	0.44
Luteolin-7-glucoside (2)		0.09	-0.02	0.15	0.27	-0.01	0.42	1.79	0.45
Ampicillin (3)		0.04	-0.47	-0.71	-0.61	0.87	0.25	10.72	0.91
Ascorbic acid (4)		-0.53	-0.24	-1.09	-1.01	-0.81	0.20	0.02	0.74
Gefitinib (5)		0.12	-0.04	0.66	-0.21	0.30	0.03	-2.62	0.28
Tamoxifen (6)		0.30	0.002	-0.01	0.57	0.04	0.32	6.3	0.35

GPCRL: G protein-coupled receptor ligand; ICM: ion channel modulator; KI: kinase inhibitor; NRL: nuclear receptor ligand; PI: protease inhibitor; EI: enzyme inhibitor.

4. Conclusions

Luffa acutangula is one of the regularly used plants with various secondary metabolites such as polyphenols and flavonoids, which possesses biological and pharmacological activities. Here in this study, the aim is to test the biogenically synthesised nanoparticles for their biological activities including antibacterial, antioxidant, and anticancer activities. The results revealed that the silver nanoparticles of Luffa acutangula leaf extract enriched with its inherent flavonoid O-glycosides (FOGs, viz., Cosmosioside (1, Apigenin-7-glucoside) and from -9.2690 to -7.8306 for Cynaroside (2, Luteolin-7-glucoside)) prepared by green biosynthetic approach. The biogenically synthesised silver nanoparticles found to be significant against bacteria and cancer cell lines which clearly show antibacterial and anticancer activities. Antioxidants play an important in reducing the oxidative stress and diminishing the growth of the cancerous cell. The results showed that AgNPs showed potential antioxidant activity. The profound studies performed based on the molecular docking analysis have revealed that the FOGs are identified as antagonists of aspartic acid receptor of enzymatic proteins referenced based on the microorganisms, cell lines, and oxidizing agents considered for the in vitro studies. Furthermore, QSAR, ADMET properties showed them as prospective drugs. The results validated that AgNPs could be potential agents to treat various types of cancers and boosting the immune system functions. Nevertheless, future studies with in vivo toxicological studies with clear mechanism of action and the pharmacodynamics studies of LAAgNPs would shed the light more thoroughly to show the possible mechanisms for anticancer activities.

Acknowledgments

The corresponding author Dr. P. V. R. acknowledges Universiti Malaysia Kelantan and Universiti Sains Malaysia for providing the facilities. This research was funded by Fundamental Research Grant Scheme (FRGS), Malaysia (Grant number: R/FRGS/A07.00/00295A/002/2014/000183), Research Acculturation Collaborative Effort (RACE), Malaysia (Grant number: R/RACE/A07.00/01147A/001/2015/000237), and Transdisciplinary Research University (RUT) of Universiti Sains Malaysia (USM), Malaysia (Grant number: 1001/PPSP/853002). The corresponding author thanks Universiti Malaysia Sabah for providing the funding support.

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