In vitro role of biosynthesized nanosilver from Allium sativum against helminths

Abstract

The exploration of natural materials for the production of nanoparticles against parasites is currently of particular interest due to its ecofriendly nature. In this study, we described the biosynthesis of silver nanoparticles derived from methanolic garlic extract. Infrared spectroscopy and GC-Mass spectrometry were used to screen the phytochemical composition of garlic and figure out how much of active components. In this study, Allium sativum extract (ASE) and ASE loaded in silver nanoparticles (Bio-AgNPs) were used to treat helminthiasis in vitro. Three doses of ASE (100, 50 and 25 mg/mL) and three doses of Bio-AgNPs (1, 0.5 and 0.25 mg/mL) were used to study the anthelmintic activity using the earthworm, Allolobophora caliginosa. Also, Albendazole was used as a reference drug. The Bio-AgNPs exhibits IC50 of 3.2 µg/mL which indicates their lower toxicity in normal cell lines. The phytochemical screening using GC mass showed the presence of many active compounds with medicinal activities. Both ASE and Bio-AgNPs posses’ anthelmintic activities by inducing paralysis and death in a time dependent manner when compared to the reference drug, Albendazole. Collectively, nanosilver manufactured from A. sativum extract properly functions as an anthelmintic agent and can quickly and dose-dependently kill worms.

Keywords:
nanoparticles; medicinal plants; phytochemistry; Allium sativum; anthelmintic activity; cytotoxicity

1 Introduction

Parasitic infection is common in developing countries due to insufficient control measures. In many countries, parasitic infections caused by protozoa and helminths result in death and economic loss (Ozoliņa et al., 2018). The most common complaints of worm infection are weakness due to malnutrition and anaemia (Jones & Berkley, 2014). The currently prescribed antihelmintic medication causes various problems with the human body, particularly the liver and kidney (Tripathi, 2013). Because they have fewer or no adverse effects, natural products like plants are highly suggested for usage as anthelmintic medicines (Wunderlich et al., 2014).

Garlic, Allium sativum L. is a member of the Alliaceae family, has been widely recognized as a valuable spice and a popular remedy for various ailments and physiological disorders (Londhe et al., 2011; Arslaner, 2020). It is the second largest crop after onion grown all over the world (Vijayakumar et al., 2019). Garlic has been used for spice, food flavoring agent and an ingredient in folk medicine since ancient times (Vijayakumar et al., 2019). Garlic is probably one of the earliest known medicinal plants where garlic cloves are a rich source of vitamins, minerals and trace elements, although most are found in only minute quantities (Lewis & Elvin-Lewis 2003; Yasin et al., 2022).

Nanoparticle biosynthesis is a major research field due to its significant applications in medicine (Pantidos & Horsfall, 2014) . Because of its efficiency and environmental friendliness, synthesis of NPs from plant sources has become increasingly important (Lakshmanan et al., 2018).

The use of silver nanoparticles in the treatment of several parasite diseases has produced encouraging results. These nanoparticles are produced using diverse plant extracts, providing a method that is simple, less expensive, non-toxic, and contain active ingredients that have antiparasitic properties (Dutta et al., 2017; Bajwa et al., 2022). Anthelminthic activity of AgNPs synthesized from various plants against earthworm has previously been reported (Rashid et al., 2016; Shelar et al., 2019; Vijayakumar et al., 2019).

Current control and prevention procedures are frequently insufficient, and more effective controls are required to ensure safe food for human consumption (Zarlenga & Gamble, 2019). Furthermore, the high cost of antihelminth drugs has compelled us to look for medicinal plants as a new source of helminth infection treatment. Here, we used nanosilver synthesized from methanolic A. sativum extract against the earthworm, Allolobophora caliginosa.

2 Materials and methods

2.1 Collection of materials and extracts preparation

Fresh bulbs of garlic, Allium sativum were collected from the local market of Cairo, Egypt. Then the plant’s material for the study was identified and authenticated by the taxonomists at Botany Department in Helwan University.

Dried and ground bulbs (about 100 g) of A. sativum were cut into small pieces then submitted to extraction with 300 mL methanol (70%) for 24 h. After extraction, the solvent was filtered and then evaporated by Rotavapor. The obtained garlic extract was stored at −20 °C until being used (Raju et al., 2008).

2.2 Synthesis of AgNPs

In accordance with Murugan et al. (2016), 5 mL of A. sativum extract was combined with silver nitrate (AgNO₃, 8 × 10^-3 M, ~67.93 mg) in 45 × 10³ µL of methanol to produce nanosilver. With the use of UV-visible spectroscopy, the decreased nanosilver solution was measured. The size and kind of NS are then determined by transmission electron microscopy using a JEOL JEM-2100 (JEOL Ltd., Tokyo, Japan).

2.3 Infrared spectroscopy

For A. sativum extract analysis, we used a Nicolet 6700 Fourier-transform infrared spectroscopy (FT-IR) optical spectrometer from Thermo Scientific (Waltham, MA, United States). We mixed the powder of the extract (10 mg) with 100 mg of potassium bromide powder (1:99 wt%) to obtain a translucent sample disk that we then loaded into an FTIR spectroscope with a scan range of 400-4000 cm−1. The chemical bonds in a molecule can be determined by interpreting the infrared absorption spectra (Pakkirisamy et al., 2017).

2.4 Determination of total phenolics and total flavonoids in Bio-AgNPs

A modified Folin-Ciocalteu technique was used to determine the total phenolic content of Bio-AgNPs. A microplate reader was used to measure the samples (Thermo Fisher Scientific, Waltham, MA, USA). The total phenolic content was determined using a standard curve for gallic acid (Al-Zharani et al., 2019).

By employing an aluminium chloride-based colorimetric assay, the total flavonoid concentration was calculated. At 368 nm, the absorbance was measured. Quercetin, a reference flavonoid, was used as a calibration curve to quantify the flavonoids in the samples (Ghosh et al., 2013).

2.5 Gas chromatography-mass spectrometry analysis for ASE

The phytochemical analysis of Bio-AgNPs was carried out using gas chromatography-mass spectrometry (GC-MS), as recommended by Kanthal et al. (2014). The Agilent Technologies, USA 7000D GC/MS Triple Quad GC-MS unit was used.

2.6 MTT cytotoxicity assay

According to Satyavani et al. (2012), the cytotoxic activity of the biosynthesized AgNPs was tested in Hep-2 cells by using the 3-(4, 5-dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. To assess the half maximal cytotoxic concentration (IC50), different concentrations of biosynthesized AgNPs were prepared in 10% DMSO in ddH₂O. Cell viability was evaluated by the MTT colorimetric technique where the absorbance of formazan solutions was measured at λmax 540 nm with 620 nm as a reference wavelength using a multi-well plate reader (BMGLABTECH®FLUOstar Omega, Germany).

2.7 Experimental worms

Adult earthworms belonging to species of Allolobophora caliginosa were collected from the wet soil of Abo Rawash district of Giza for this study as its anatomical and physiological resemblance with the intestinal roundworm parasite of human beings (Thorn et al., 1987). Because of easy availability, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro (Ajaiyeoba et al., 2001). Worms in distilled water were used as a control. In this experiment, the time to reach paralysis and death state was expressed in minutes (Dkhil, 2013). Three doses were used (100, 50 and 25 mg/mL to study the anthelmintic activity of Allium sativum and three doses of Bio-AgNPs (1, 0.5 and 0.25). We used a reference drug, Albendazole (EIPICO, Tenth of Ramadan City, Egypt) with a concentration of 20 mg/mL (Murugamani et al., 2012).

2.8 DPPH radical scavenging method for antioxidant activity

Using 2,2-diphenyl-1-picrylhydrazyl, the free radical scavenging activity of Bio-AgNPs was determined (DPPH). In a summary, 80 mL of a methanolic solution of DPPH (100 mM) was combined with 20 mL of the biosynthesized nanosilver (1 mg/mL), and the mixture was then incubated for 30 min at 25 °C in the dark. At 517 nm, the absorbance was measured, and the radical scavenging activity was estimated (Ghosh et al., 2013).

2.9 Statistical analysis

All values are expressed as the means and standard deviations. Significance was evaluated using t-test at p ≤ 0.05 using a statistical package program (SPSS version 17.0).

3 Results

Examination of the Bio-AgNPs using transmission electron microscopy (TEM) at showed that the nanoparticles were spherical in shape with a size ranged from 10 to 30 nm. The image also showed that no residues related to the plant extract remain in the prepared product, which indicates that the prepared nanostructure content is highly pure with good morphology (Figure 1).

Figure 1
Transmission electron microscopy image of AgNPs biosynthesized by using Allium stivum extract.

Figure 2 and Table 1 showed the FTIR analysis of the extract with major bands from 524.22 to 3269.78 Cm−1. O-H, N-H, C=C, S=O, C-O, C-N, and C-I stretching were indicated at different bands indicating many different classes of compounds as aliphatic primary alcohol, amine salt, alkene, sulfonyl chloride, alkyl aryl ether, vinyl ether, aliphatic ether, amine, ketone, and halo compounds.

Figure 2
FT-IR spectrum of Allium sativum extracts.

Table 1
IR spectrum of Allium sativum extract.

Absorption (cm-1)	Appearance	Transmittance (%)	Group	Compound Class
3269.78	Strong, broad	20.26	O-H stretching	alcohol
2933.34	Strong broad	18.17	N-Hstretching	amine salt
1635.68	strong	10.13	C=Cstretching	alkene
1400.04	strong	8.67	S=Ostretching	sulfonyl chloride
1271.41	strong	7.87	C-Ostretching	alkyl aryl ether
1218.84	strong	7.55	C-Ostretching	vinyl ether
1125.39	strong	6.97	C-Ostretching	aliphatic ether
1016.71	medium	6.29	C-Nstretching	amine
929.26	strong	5.75	C=Cbending	alkene
814.11	medium	5.04	C=Cbending	alkene
524.22	strong	3.24	C-Istretching	halo compound

Analysis of A. sativum extracts was shown by GC-MS Chromatogram, the presence of 33 active phytochemical compounds appearing at different peak areas. Compounds with elevated peaks are; 4-Methyl-2H-pyran, 1,3-Cyclopentanedione, 2,5-Piperazinedione, 3-methyl-, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- methyl-, 5-Hydroxymethylfurfural, 1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-, and 1-Penten-3-on e (Figure 3, Table 2).

Figure 3
GC-MS chromatogram of Allium sativum extracts.

Table 2
The gas chromatography-mass spectrometry (GC/MS) analysis of Allium sativum extract.

	Component RT	Compound Name	Molecular Wight	[M-H]- (m/z) Molecular Wight -1	Formula	Area	Peak %
1.	1.1043	Dimethylamine	45.0837	44.0837	C2H7N	218294270	0.601165
2.	1.5286	Thioacetic acid	76.118	75.118	C2H4OS	76508376	0.210698
3.	2.6798	4-Methyl-2H-pyran	96.1271	95.1271	C6H8O	1053143381	2.900271
4.	3.2424	1,3-Cyclopentanedione	98.0999	97.0999	C5H6O2	741855116	2.043008
5.	3.7447	2,5-Piperazinedione, 3-methyl-	128.13	127.13	C5H8N2O2	1025267976	2.823504
6.	4.1062	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- methyl-	44.1253	43.1253	C6H8O4	1371735632	3.777648
7.	4.4126	5-Hydroxymethylfurfural	126.1100	125.11	C6H6O3	4200788988	11.56863
8.	5.0346	Phenol, 2-methoxy-6-(2-propenyl)-	164.2011	163.2011	C10H12O2	432154271	1.190118
9.	5.4714	Furane-2-carboxylic acid, 5-(4-chloro-3- methylphenoxymethyl)-	266.67	265.67	C13H11ClO4	62948021	0.173354
10.	5.9350	Pentanedioic acid, (2,4-di-t-butylphenyl) monoester	320.4	319.4	C19H28O4	59332216	0.163396
11.	6.2279	1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-	134.1736	133.1736	C6H14O3	18209937809	50.14868
12.	6.5310	1-Amino-2-methylnaphthalene	157.21	156.21	C11H11N	218031903	0.600442
13.	7.1040	2-Butanone, 4-(4-hydroxy-3-methoxyphenyl)-	194.2271	193.2271	C11H14O3	88287015	0.243135
14.	7.9313	1-Penten-3-one	84.1164	83.1164	C5H8O	8269373041	22.77318
15.	8.3094	Phenylphosphonic acid, dodecyl propyl ester	368.5	367.5	C21H37O3P	14311247	0.039412
16.	8.6755	Isoquinoline, 1-[3-benzyloxy5-hydroxybenzyl]- N-formyl-1,2,3,4-	403.5	402.5	C25H25NO4	93451114	0.257357
17.	9.3342	1,7-Di(3-ethylphenyl)-2,2,4,4,6,6-hexamethyl1,3,5,7-tetraoxa-2,4,6-trisilaheptane	448.8	447.8	C22H36O4Si3	16085205	0.044297
18.	10.2343	1H-Pyrrole-2,5-dione, 1-(4-methylphenyl)-	187.1947	186.1947	C11H9NO2	26713778	0.073568
19.	10.8227	Cyclopentanecarboxylic acid, 4-nitrophenyl ester	235.24	234.24	C12H13NO4	20239894	0.055739
20.	11.5512	2-Phenylindolizine	193.24	192.24	C14H11N	6047924	0.016655
21.	11.9177	2,3-Dihydro-6-methoxyfuro(2,3-b)quinoline	201.22	200.22	C12H11NO2	1767183	0.004867
22.	12.4072	5-Acetamido-1,2,3-trimethoxybenzene	225.24	224.24	C11H15NO4	6321975	0.01741
23.	12.8574	Pyridine, 2-[2-(dibutylphosphino)ethyl]-	251.35	250.35	C15H26NP	9467151	0.026072
24.	13.3328	3,5-Di(2-pyridyl)pyrazole	222.24	221.24	C13H10N4	9994631	0.027524
25.	14.2779	Adipic acid, di(but-2-en-1-yl) ester	254.32	253.32	C14H22O4	13140653	0.036188
26.	15.1261	Carbamic acid, 2,5-dimethoxyphenyl-, butyl ester	253.29	252.29	C13H19NO4	23491091	0.064693
27.	15.7144	Phthalic acid, 4-chlorophenyl phenyl ester	352.8	351.8	C20H13ClO4	1014007	0.002792
28.	16.3797	Terephthalic acid, di(2-fluorophenethyl) ester	410.4	409.4	C24H20F2O4	824346	0.00227
29.	17.2190	4-Hydroxy-N-methylphenylethylamine dipfp	443.24	442.24	C15H11F10NO3	1640911	0.004519
30.	17.9988	2-(Benzthiazol-2-yl)-6-methoxybenzofuran	281.3	280.3	C16H11NO2S	11849044	0.032631
31.	18.3808	Trimetozine	281.3044	280.3044	C14H19NO5	21414146	0.058973
32.	18.8693	Phthalic acid, 3,5-dimethylphenyl 4- formylphenyl ester	374.4	373.4	C23H18O5	680625	0.001874
33.	19.4438	1,3,6,9b-Tetraazaphenalene-4-carbonitrile, 2- methyl-	209.21	208.21	C11H7N5	5784905	0.015931

The total phenolic and flavonoid contents in Bio-AgNPs reached 17.2 ± 0.04 and 3.45 ± 0.03 µg/mL, respectively while the antioxidant activity of the nanomaterial reached 62.8 ± 2.7 (Table 3).

Table 3
Total phenolic, flavonoid, and radical scavenging activity of AgNPs synthesised from Allium sativum extract.

Total phenolics (µg/g)	Total flavonoid (µg/g)	Antioxidant (%)
17.2 ± 0.04	3.45 ± 0.03	62.8 ± 2.7

The cytotoxic effects of Bio-AgNPs synthesized from A. sativum extracts were evaluated against Hep-2 cells under in vitro conditions. In the present cytotoxicity analysis, various concentrations of Bio-AgNPs (3.125, 6.25, 12.5, 25, 50, and 100 μg/mL) was tested for 24 h. The cell viability is concentration dependent with increase in Bio-AgNPs concentration decrease in cell viability was observed. The Bio-AgNPs exhibits IC50 of 3.2 μg/mL which indicates their lower toxicity in normal cell lines. Bio-AgNPs was found to be less toxic and the viability of Hep-2 cells was slightly affected up to 100 μg mL−1 (Figure 4). The viability of Hep-2 cells was reduced to about 10% at 100 μg/mL of AgNO₃.

Figure 4
Cell viability using MTT assay. The viability of Hep-2 cells after exposure to Bio-AgNPs for 24 h. *Significance against control group at p < 0.001.

Compared to the negative control, treating the worms with ASE at dosages of 100, 50, and 25 mg/mL quickly caused worm paralysis and death (Table 4). Furthermore, the duration of paralysis and mortality was shortened as ASE concentration rose. Also, Bio-AgNPs induce the same effect. As an increase in ASE or Bio-AgNPs concentration is inversely related to the duration of paralysis and death, this impact was therefore dose dependent. Interestingly, worm paralysis and death were more strongly induced by larger doses of ASE (100 mg/mL) and Bio-AgNPs (1 mg/mL) than by albendazole (Table 4). Collectively, the schematic Figure 5 summarizes the study findings.

Table 4
Anthelminthic action of Allium sativum extracts (ASE) and AgNPs synthesized from ASE (Bio-AgNPs).

Group	Time taken for paralysis (min)	Time taken for death (min)
Water	-	-
ASE (25 mg/mL)	5.9 ± 1.08	6.4 ± 1.6
ASE (50 mg/mL)	3.8 ± 0.9	4.01 ± 0.7
ASE (100 mg/mL)	1.5 ± 0.3	2.1 ± 0.2
Bio-AgNPs (1 mg/mL)	2.7 ± 0.7	3.7 ± 0.5
Bio-AgNPs (0.5 mg/mL)	3 ± 0.7	5 ± 0.8
Bio-AgNPs (0.25 mg/mL)	4 ± 0.6	6 ± 0.7
Albendazole (20 mg/mL)	3.58 ± 1.1	8.45 ± 1.3

Figure 5
Schematic figures to summarizing the main findings of the study.

4 Discussion

Infections with helminths not only hinder productivity but also impair food quality. The decreased live weight gain, accelerated puberty age, low productivity, and increased vulnerability to various infections in parasitized animals all contribute to significant economic losses for stakeholders (Yadav et al., 2004; Asif Raza et al., 2007).

Livestock production systems are hampered by gastrointestinal parasitism, particularly that caused by helminth species. Animal weight, meat and milk output, and fertility all suffer severe weight loss and health problems as a result of these ailments (Zajac & Garza, 2020).

Anthelmintic drugs are generally effective at controlling parasitic infections, but their overuse has resulted in an increase in the population of resistant parasites (Waller, 1997). Nanoparticles synthesized from medicinal plants are promising agent with reduced or no side effects (Dkhil et al., 2021).

In this study, the change of color of the nanomaterials from yellow to brown indicates the formation of Bio-AgNPs. Mulvaney (1996) reported that, the colour change could be due to the excitation of the surface plasmon resonance effect and the reduction of silver nitrate.

Spherical particles with sizes ranging from 10 to 50 nm were identified by TEM. The findings are in line with earlier research that indicated the coated AgNPs' garlic extract particles ranged in size from 10 to 50 nm (Rastogi & Arunachalam, 2011).

Silver nanoparticles are used often in medicine and medication delivery and show great promise as anticancer agents (Gurunathan et al., 2009). The MTT assay has been widely used to measure the cell proliferation rate based on the fact that live cells reduce yellow MTT to blue formazan products. After 24 hours, the viability of cancer cells decreased with an increase in the concentration of Bio-AgNPs. Furthermore, the reduction in Hep-2 cell viability demonstrates the anti-cancer effects of Bio-AgNPs. When evaluating a compound's usefulness as a pharmacological drug, the balance between its therapeutic potential and toxic side effects is critical. The cytotoxicity of the produced Bio-AgNPs against the epithelioma (Hep-2) cancer cell line was examined in vitro.

AgNPs cause toxicity by interrupting the respiratory chain, releasing reactive oxygen species, and inhibiting ATP synthesis (AshaRani et al., 2009). Moreover, AgNPs' cytotoxic effects are caused by their physiochemical interaction with intracellular genetic materials. According to earlier studies, AgNPs' anticancer effect may also result from the caspase-3-activated induction of apoptosis (Sriram et al., 2012).

According to reports, the positive charge on the silver ion is what causes its antibacterial effects since it may draw in negatively charged microbe cell membranes via electrostatic interaction (Hamouda et al., 2001; Dibrov et al., 2002). A. sativum contains phenolic and flavonoid compounds; these phytochemicals can attach with free proteins in the parasite cuticle and cause deaths (Rashid et al., 2016). A. sativum and AgNPs had stronger anthelmintic effects when used together than when used separately. Bio-AgNPs' wormicidal activity against earthworms suggests that they are also effective against parasitic infections in humans (Rashid et al., 2016).

5 Conclusion

Collectively, nanosilver manufactured from A. sativum extract properly functions as an anthelmintic agent and can quickly and dose-dependently kill worms. To create safe diagnostic and therapeutic solutions, additional study is necessary to understand the mechanisms of action of nanoparticles.

Acknowledgements

This study was supported by the Researchers Supporting Project (RSP2023R25), King Saud University, Riyadh, Saudi Arabia, and also was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R96), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

• Practical Application: Nanosilver from garlic as a fight against helminths.

• Availability of data and material

  The data used to support the findings of this study are included within the article.

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Publication Dates

• Publication in this collection
  17 Mar 2023
• Date of issue
  2023

History

• Received
  02 Nov 2022
• Accepted
  28 Dec 2022